Introduction to Fluorescence Sensing: Volume 1: Materials and Devices [3rd ed.] 9783030601546, 9783030601553

Table of contents :
Front Matter ....Pages i-xxii
Principles and Techniques in Chemical and Biological Sensing (Alexander P. Demchenko)....Pages 1-29
Overview of Strategies in Fluorescence Sensing (Alexander P. Demchenko)....Pages 31-54
Fluorescence Detection in Sensor Technologies (Alexander P. Demchenko)....Pages 55-110
Photophysical Mechanisms of Signal Transduction in Sensing (Alexander P. Demchenko)....Pages 111-166
Organic Dyes and Visible Fluorescent Proteins as Fluorescence Reporters (Alexander P. Demchenko)....Pages 167-236
Small Luminescent Associates Based on Inorganic Atoms and Ions (Alexander P. Demchenko)....Pages 237-266
Fluorescent Inorganic Particles in Nanoscale World (Alexander P. Demchenko)....Pages 267-306
Fluorescent Organic Dyes and Conjugated Polymers in Nanoscale Ensembles (Alexander P. Demchenko)....Pages 307-355
Fluorescent Carbon Nanostructures (Alexander P. Demchenko)....Pages 357-399
Smart Luminescent Nanocomposites (Alexander P. Demchenko)....Pages 401-438
Passive Support Materials for Fluorescence Sensors (Alexander P. Demchenko)....Pages 439-482
Fluorescent Composites Combining Multiple Sensing and Imaging Modalities (Alexander P. Demchenko)....Pages 483-502
Evanescent Field Effects and Plasmonic Enhancement of Luminescence in Sensing Technologies (Alexander P. Demchenko)....Pages 503-529
Non-conventional Generation and Transformation of Sensor Response (Alexander P. Demchenko)....Pages 531-566
Techniques and Devices Used in Fluorescence Sensing (Alexander P. Demchenko)....Pages 567-611
Frontiers for Future Research. Two-Photonic, Highly Excited and Single-Molecular Sensors (Alexander P. Demchenko)....Pages 613-646
Back Matter ....Pages 647-657

Citation preview

Alexander P. Demchenko

Introduction to Fluorescence Sensing Volume 1: Materials and Devices Third Edition

Introduction to Fluorescence Sensing

Alexander P. Demchenko

Introduction to Fluorescence Sensing Volume 1: Materials and Devices Third Edition

123

Alexander P. Demchenko Palladin Institute of Biochemistry National Academy of Sciences of Ukraine Kyiv, Ukraine Yuriy Fedkovych National University Chernivtsi, Ukraine

ISBN 978-3-030-60154-6 ISBN 978-3-030-60155-3 https://doi.org/10.1007/978-3-030-60155-3

(eBook)

1st edition: © 2009 Springer Science + Business Media B.V. 2nd edition: © Springer International Publishing Switzerland 2015 3rd edition: © Springer Nature Switzerland AG 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Preface

The field of chemical and biological sensing is immense. It is nearly the whole world of natural and synthetic compounds that have to be analyzed in a broad variety of conditions and for a broad variety of purposes. In the human body, we need to detect and quantify virtually all the genes (genomics) and the products of these genes (proteomics). We need to know how they interact performing their functions (interactomics). In our surrounding there is a need to analyze a huge number of compounds including millions of newly synthesized products. Among them, we have to select potentially useful compounds (e.g. drugs) and discriminate those that are inefficient and harmful. No less important is to control agricultural production and food processing. There is also a practical necessity to provide control in industrial product technologies, especially in those that produce pollution. Permanent monitoring is needed to maintain safety of our environment. Protection from harmful microbes, clinical diagnostics and control of patient treatment are the key issues of modern medicine. New problems and challenges may appear with the advancement of human society in the XXI century. We have to be ready to meet them. Modern society needs the solution of these problems on the highest possible scientific and technological level. The science of intermolecular interactions is traditionally a part of physical chemistry and molecular physics. Now it becomes a strongly requested background for modern sensing technologies. The most specific and efficient sensors are found in the biological world, and the sensors based on biomolecular recognition (biosensors) have gotten a strong impulse for development and application. A strong move is observed for improving them by endowing new features or even by making their fully synthetic analogs. Modern optics and electronics make their own advance in providing the most efficient means for supplying the input and output signals and now become oriented at satisfying the needs of not only researchers but also a broad community of users. This book is focused on one sensing technology, which is based on fluorescence. This is not only because of limited space or limited expertise of the present author. Indeed, fluorescence techniques are the most sensitive; their sensitivity reached the absolute limit of single molecules. They offer very high spatial resolution; that with v

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overcoming the light diffraction limit has reached molecular scale. They are also the fastest; their response develops on the scale of fluorescence lifetime and can be as short as 10−8–10−10 s. But their greatest advantage is versatility. Fluorescence sensing can be provided in solid, liquid and gas media and at all kinds of interfaces between these phases. It is because the fluorescence reporter and the detecting instrument are connected via light emission, fluorescence detection can be made non-invasive and equally well suited for remote industrial control and for sensing different targets within the living cells. All these features explain the high popularity of fluorescence sensing. The fascinating field of fluorescence sensing needs new brains. Therefore the book is primarily addressed to students and young scientists. Together with basic knowledge, they will get an overview of different ideas in research and technology and guide to their own creative activity. Providing a link between basic sciences needed to understand the sensor performance and frontier of research, where new ideas are explored and new products developed, the book will make a strong link between research and education. For the active researcher it will also be a source of useful information in nearly all areas where fluorescence sensing is used. After the publication of previous edition of this book, a tremendous progress is observed in all fields of research, development and application related to its subject. In order to follow this progress, in a new 3rd edition a number of changes and additions has been made. The present edition is separated into two volumes. The first volume is focused on basic principles of operation of fluorescence sensors and on technologies that were developed to use them. In comparison with previous edition, the correspondent chapters were upgraded and new sections introduced. Particularly, new sections appear describing the dye-doped nanostructures and nanocrystals with new properties. Special chapters are devoted to carbonic nanostructures, plasmonic effects and multimodal nanocomposites. Prospects for applications of novel methods and materials, particularly for single-molecular sensing, are discussed in the last chapter. All other chapters were revised with the addition of new information, new illustrations and new points of discussion. Each chapter is terminated by the section “Sensing and thinking”, in which, after a short summary, a series of questions and exercises is suggested to the reader. The second volume overviews the molecular and supramolecular receptors in sensor design and various applications, including biological imaging. Thus, this book is organized with the aim to satisfy both curious student and busy researcher. Enjoy your reading. Kyiv, Ukraine July 2020

Alexander P. Demchenko

Contents

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Principles and Techniques in Chemical and Biological Sensing 1.1 General Definitions and Formulation of Key Problems . . . . 1.1.1 Sensors or Assays? . . . . . . . . . . . . . . . . . . . . . . . . 1.1.2 Homogeneous and Heterogeneous Formats . . . . . . 1.2 Elements of a Typical Sensor . . . . . . . . . . . . . . . . . . . . . . 1.2.1 The Recognition Element . . . . . . . . . . . . . . . . . . . 1.2.2 The Reporter Element . . . . . . . . . . . . . . . . . . . . . . 1.2.3 The Transducer . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Diversity of Sensing Techniques and Their Operation Principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.1 The Label-Free Techniques . . . . . . . . . . . . . . . . . . 1.3.2 The Label-Based Approach . . . . . . . . . . . . . . . . . . 1.4 Distinguishing Features of Fluorescence Technology . . . . . . 1.4.1 Fluorescence as the Technique of Choice . . . . . . . . 1.4.2 Large-Scale Manipulation with Emission Colors and Lifetimes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4.3 Broad Diversity of Molecular and Nanoscale Emitters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4.4 Possibilities for Enhancement of Reporting Signal . 1.4.5 Catalytic Enhancement in Sensors and Biosensors . 1.4.6 Single-Analyte Sensors and Multi-analyte Arrays . . 1.5 Trends for Future Development . . . . . . . . . . . . . . . . . . . . . 1.6 Sensing and Thinking. High Time for New Endeavor . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Overview of Strategies in Fluorescence Sensing . . . . . . . . . . 2.1 Labeling Targets in Fluorescence Assays . . . . . . . . . . . . 2.1.1 Arrays for DNA Hybridization . . . . . . . . . . . . . 2.1.2 Labeling in Protein-Protein and Protein-Nucleic Acid Interactions . . . . . . . . . . . . . . . . . . . . . . .

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2.1.3

Advantages and Limitations of the Approach Based on Pool Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Sandwich Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1 Sensing the Antigens and Antibodies . . . . . . . . . . . . 2.2.2 Labeling with Catalytic Amplification . . . . . . . . . . . 2.2.3 Ultrasensitive DNA Hybridization Assays . . . . . . . . 2.2.4 Advantages and Limitations of the Approach . . . . . . 2.3 Competitor Displacement Assays . . . . . . . . . . . . . . . . . . . . . 2.3.1 Unlabeled Sensor and Labeled Competitor in Homogeneous Assay . . . . . . . . . . . . . . . . . . . . . 2.3.2 Labeling of Both Receptor and Competitor . . . . . . . 2.3.3 Advantages and Limitations of the Approach . . . . . . 2.4 Direct Reagent-Independent Sensing . . . . . . . . . . . . . . . . . . 2.4.1 The Principle of Direct ‘Mix-and-Read’ Sensing . . . 2.4.2 Contact and Remote Sensors . . . . . . . . . . . . . . . . . . 2.4.3 Advantages and Limitations of the Approach . . . . . . 2.5 Sensing and Thinking. The Cost of Brightness and Amplification in Sensor Response . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Fluorescence Detection in Sensor Technologies . . . . . . . . . . . . 3.1 Fluorescence Fundamentals . . . . . . . . . . . . . . . . . . . . . . . 3.1.1 The Light Emission Phenomenon . . . . . . . . . . . . 3.1.2 Fluorescence Parameters Used in Sensing . . . . . . 3.1.3 Parameters Characterizing the Properties of Fluorescence Emitters . . . . . . . . . . . . . . . . . . . 3.1.4 Fluorophore Emissive Power: The Brightness and Quantum Yield . . . . . . . . . . . . . . . . . . . . . . 3.2 Intensity-Based Sensing . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1 Peculiarities of Fluorescence Intensity Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2 How to Make Use of Quenching Effects? . . . . . . 3.2.3 Quenching: Static and Dynamic . . . . . . . . . . . . . 3.2.4 Non-linearity Effects . . . . . . . . . . . . . . . . . . . . . . 3.2.5 Internal Calibration in Intensity Sensing . . . . . . . 3.2.6 Intensity Change as a Choice for Fluorescence Sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Anisotropy-Based Sensing and Polarization Assays . . . . . . 3.3.1 Background of the Method . . . . . . . . . . . . . . . . . 3.3.2 Practical Considerations . . . . . . . . . . . . . . . . . . . 3.3.3 Applications of Fluorescence Anisotropy in Sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.4 Comparisons with Other Methods of Fluorescence Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Lifetime-Based Fluorescence/Luminescence Response . . . . . . 3.4.1 Physical Background of Time-Resolved Detection . . 3.4.2 Technique Using the Time Resolution . . . . . . . . . . . 3.4.3 Time-Resolved Anisotropy . . . . . . . . . . . . . . . . . . . 3.4.4 Applications of Time-Resolved Fluorescence . . . . . . 3.4.5 Phosphorescence and Long-Lasting Luminescence . . 3.4.6 Comparison with Other Fluorescence Detection Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Wavelength-Shifting Sensing . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1 The Physical Background Under the Wavelength Shifts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2 The Measurements of Wavelength Shifts in Excitation and Emission . . . . . . . . . . . . . . . . . . . 3.5.3 Wavelength-Ratiometric Measurements . . . . . . . . . . 3.5.4 Application of k-Ratiometry in Sensing . . . . . . . . . . 3.5.5 Comparison with Other Fluorescence Reporting Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6 Two-Band k-Ratiometry with Single Fluorophore . . . . . . . . . 3.6.1 Generation of Two-Band k-Ratiometric Response by Ground-State Isoforms . . . . . . . . . . . . . . . . . . . . 3.6.2 Excited-State Reactions Generating Two-Band Response Within Single Dye in Emission . . . . . . . . 3.6.3 Coupling a Reporting Dye with the Reference . . . . . 3.6.4 Fluorescent Excimers . . . . . . . . . . . . . . . . . . . . . . . 3.6.5 The Systems with Excitation Energy Transfer (EET) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7 Sensing and Thinking: The Optimal Choice of Fluorescence Detection Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Photophysical Mechanisms of Signal Transduction in Sensing 4.1 Basic Signal Transduction Mechanisms: Electron, Charge and Proton Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1 Photoinduced Electron Transfer (PET) . . . . . . . . . 4.1.2 Intramolecular Charge Transfer (ICT) . . . . . . . . . 4.1.3 Twisted Intramolecular Charge Transfer (TICT) . . 4.1.4 Intramolecular Excited-State Proton Transfer . . . . 4.1.5 Excited-State Intramolecular Proton Transfer (ESIPT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.6 Future Directions . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Excimer and Exciplex Formation . . . . . . . . . . . . . . . . . . . 4.2.1 The Dyes Forming Excimers and Exciplexes . . . . 4.2.2 Nonemissive Dimers . . . . . . . . . . . . . . . . . . . . . .

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4.2.3 4.2.4

Application in Sensing Technologies . . . . . . . . . . Comparison with Other Fluorescence Reporter Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Excitation Energy Transfer (EET) . . . . . . . . . . . . . . . . . . 4.3.1 Physical Background of the Method . . . . . . . . . . 4.3.2 Applications of EET Technology . . . . . . . . . . . . 4.3.3 Observing New Developments . . . . . . . . . . . . . . 4.4 Signal Transduction Via Conformational Changes . . . . . . . 4.4.1 Excited-State Isomerism in Small Molecular Reporter Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4.2 Conformational Changes in Conjugated Polymers 4.4.3 Conformational Changes in Peptide Sensors and Nucleic Acid Aptamers . . . . . . . . . . . . . . . . . . . . 4.4.4 Molecular Beacons (Hairpin-Like Structures) . . . . 4.4.5 Proteins Exhibiting Conformational Changes . . . . 4.4.6 Prospects in Exploration of Conformational Changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 Smart Sensing with Logic Operations . . . . . . . . . . . . . . . 4.5.1 Why Logic Operations in Fluorescence Sensing? . 4.5.2 Logic Operations on Molecular Scale . . . . . . . . . 4.5.3 Exploration of Basic Signal Transduction Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5.4 Prospective Applications in Sensing and Imaging Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6 Sensing and Thinking. How to Optimize Photophysical Transformations for Generating the Output Signal? . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

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Organic Dyes and Visible Fluorescent Proteins as Fluorescence Reporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1 Fluorescent Organic Molecules and Their Characteristics . . 5.1.1 General Properties of Organic Dyes . . . . . . . . . . . . 5.1.2 The Photostability of Organic Dyes . . . . . . . . . . . . 5.1.3 Protection of Dyes with Macrocyclic Hosts . . . . . . 5.1.4 New Discoveries and Improvements . . . . . . . . . . . 5.1.5 Labeling and Sensing—Two Basic Methodologies . 5.2 Organic Dyes Optimal as Labels and Tags . . . . . . . . . . . . . 5.3 The Dyes Providing Fluorescence Response . . . . . . . . . . . . 5.3.1 Special Requirements for Fluorescence Reporters . . 5.3.2 Fluorophores in Protic Equilibrium. pH-Reporting Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3.3 Hydrogen Bond Responsive Dyes . . . . . . . . . . . . . 5.3.4 The Environment-Sensitive (Solvatochromic and Solvatofluorochromic) Dyes . . . . . . . . . . . . . .

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5.3.5 5.3.6 5.3.7

Electric Field Sensing (Electrochromic) Dyes . . . . . Supersensitive Multicolor k-Ratiometric Dyes . . . . Prospects in Improving the Target-Responsive Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4 Organic Dyes Optimal for Particular Applications . . . . . . . . 5.4.1 Near-IR Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.2 Two-Photonic Dyes . . . . . . . . . . . . . . . . . . . . . . . 5.4.3 Dyes Demonstrating Phosphorescence and Delayed Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.4 Organic Photochromic Compounds . . . . . . . . . . . . 5.4.5 Dyes for Sensing in Protein and Nucleic Acid Science . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5 Visible Fluorescent Proteins . . . . . . . . . . . . . . . . . . . . . . . 5.5.1 Green Fluorescent Protein (GFP) and Its Fluorophore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.2 Proteins of GFP Family . . . . . . . . . . . . . . . . . . . . 5.5.3 Excited-State Reactions in Fluorescent Proteins . . . 5.5.4 Labeling and Sensing Applications of Fluorescent Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.5 Other Proteins with Visible Fluorescence Emission 5.5.6 Finding Analogs of Fluorescent Protein Fluorophores . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5.7 Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6 Sensing and Thinking. The Search for Novel Dyes and for New Mechanisms of Their Response . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Small Luminescent Associates Based on Inorganic Atoms and Ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1 Fluorescent Few-Atom Clusters of Noble Metals . . . . . . 6.1.1 The Cluster Structures and Their Stability . . . . . 6.1.2 The Mechanisms of Light Absorption and Emission in Noble Metal Clusters . . . . . . . . 6.1.3 Spectroscopic Properties . . . . . . . . . . . . . . . . . . 6.1.4 Formation, Stabilization and Protection of Metal Clusters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1.5 Applications in Sensing and Imaging . . . . . . . . . 6.2 Metal Complexes that Exhibit Phosphorescence . . . . . . . 6.2.1 Metal-to-Ligand Charge Transfer (MLCT) Phosphorescence . . . . . . . . . . . . . . . . . . . . . . . 6.2.2 Phosphorescent Metal-Chelating Porphyrins . . . . 6.2.3 Upconversion by Triplet-Triplet Annihilation . . .

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6.3

Lanthanides, Their Complexes and Conjugates . . . . . . . . . . . 6.3.1 Photophysics of Lanthanide Chelates . . . . . . . . . . . . 6.3.2 Luminescence Spectra of Lanthanides . . . . . . . . . . . 6.3.3 Lanthanide Chelates as Labels and Reference Emitters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.4 Lanthanide-Based Heterogeneous and Homogeneous Immunoassays . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3.5 Switchable Lanthanide Chelates in Sensing . . . . . . . 6.4 Sensing and Thinking. Three Roads to Most Efficient Use of Smallest Metal-Based Emitters . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Fluorescent Inorganic Particles in Nanoscale World . . . . . . . . 7.1 Introduction to Light-Emitting Nano-world . . . . . . . . . . . . 7.1.1 Size, Shape and Dimensions of Nanomaterials . . . 7.1.2 Variations in Nanoparticles Composition, Crystallinity and Order . . . . . . . . . . . . . . . . . . . . 7.1.3 Interactions at Nanoscale Surfaces . . . . . . . . . . . . 7.1.4 Excitation Energy Transfer with and between Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1.5 Design of Functional Nanomaterials . . . . . . . . . . 7.2 Semiconductor Quantum Dots . . . . . . . . . . . . . . . . . . . . . 7.2.1 The Composition of Quantum Dots . . . . . . . . . . . 7.2.2 The Origin of Emission of Semiconductor Nanomaterials. The Loosely Bound Excitons . . . . 7.2.3 The Spectroscopic Properties of Quantum Dots . . 7.2.4 Stabilization and Functionalization of Quantum Dots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.5 Applications of Quantum Dots in Sensing . . . . . . 7.2.6 Semiconductor Nanocrystals of Different Shapes and Dimensions . . . . . . . . . . . . . . . . . . . . . . . . . 7.2.7 Magic-Sized Quantum Dots . . . . . . . . . . . . . . . . 7.2.8 Porous Silicon and Silicon Nanoparticles . . . . . . . 7.2.9 Prospects in QDs Development and Applications . 7.3 Nanoparticles with Long Persistent Luminescence . . . . . . 7.4 Lead Halide Perovskite Nanocrystals . . . . . . . . . . . . . . . . 7.5 Upconverting Nanocrystals . . . . . . . . . . . . . . . . . . . . . . . 7.5.1 The Photophysical Mechanism of Upconversion . . 7.5.2 The Spectroscopic Properties of Upconversion Nanomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . 7.5.3 Modulation of Fluorescent Signal . . . . . . . . . . . . 7.5.4 Present and Prospective Applications . . . . . . . . . .

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Sensing and Thinking. Small Things Bright and Beautiful, What is Their Advantage? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300

8

Fluorescent Organic Dyes and Conjugated Polymers in Nanoscale Ensembles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1 General Effects Observed on Dye-Dye Association . . . . . . . . 8.1.1 Excitonic Behavior of Organized Dye Assemblies . . 8.1.2 Modulation of Photoreactions Valuable for Sensing . 8.1.3 Exchange of Excitation Energies . . . . . . . . . . . . . . . 8.1.4 Concentration-Dependent Quenching . . . . . . . . . . . . 8.1.5 The Effect of External Quenchers, “Superquenching” . . . . . . . . . . . . . . . . . . . . . . . . . . 8.1.6 Variations of Anisotropy in Excitation Energy Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2 Highly Ordered Nanoscale Associates of Organic Dyes (H- and J-Aggregates) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.2.1 Excitonic J-Aggregates . . . . . . . . . . . . . . . . . . . . . . 8.2.2 Emissive and Quenched H-Aggregates . . . . . . . . . . . 8.3 Aggregation-Protected and Aggregation-Induced Emission . . 8.3.1 Dye Aggregates: Emissive and Quenched . . . . . . . . 8.3.2 Modifying Intermolecular Interactions on Dye Aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.3.3 Aggregation-Induced Emission (AIE) . . . . . . . . . . . 8.4 Nanoscale Assemblies Supported by Organic Polymers and Silica Matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4.1 Compositions of Organic Dyes Based on Organic Polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4.2 Organic Dyes Incorporated into Dendrimers . . . . . . . 8.4.3 DNA-Based Fluorescent Nanostructures . . . . . . . . . . 8.4.4 Organic Dyes in Silica-Based Fluorescent Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8.4.5 Summarizing: Dye-Doped Nanoparticles in Sensing . 8.5 Light-Emitting Conjugated Polymers . . . . . . . . . . . . . . . . . . 8.5.1 Structure and Spectroscopic Properties . . . . . . . . . . . 8.5.2 Conjugated Polymer Nanoparticles (P-Dots) . . . . . . . 8.5.3 Conjugated Polymers as Fluorescence Reporters in Designed Sensors . . . . . . . . . . . . . . . . . . . . . . . . 8.6 Sensing and Thinking. The Struggle for Brightness and Signal Amplification in Sensor Design . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Fluorescent Carbon Nanostructures . . . . . . . . . . . . . . . . . . . . 9.1 Light-Emissive Nanodiamonds . . . . . . . . . . . . . . . . . . . . . 9.2 The sp2 Forming Structures: Graphene Sheets, Nanotubes, Fullerenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.3 Fluorescent Graphene and Graphene Oxide Nanoparticles . 9.4 Fluorescent Carbon Dots (C-dots) . . . . . . . . . . . . . . . . . . 9.4.1 Carbon Dots in the Family of Light-Emissive Nanomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . 9.4.2 The Spectroscopic Properties of C-dots . . . . . . . . 9.5 The Origin of Fluorescence Emission of Carbon Nanostructures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9.5.1 Arising Questions to Be Answered . . . . . . . . . . . 9.5.2 Carbon Nanoparticles as Collective Emitters . . . . 9.5.3 The Exciton Model . . . . . . . . . . . . . . . . . . . . . . . 9.6 Analytical, Bioanalytical and Imaging Applications of Carbon Nanomaterials . . . . . . . . . . . . . . . . . . . . . . . . . 9.6.1 General Strategy . . . . . . . . . . . . . . . . . . . . . . . . . 9.6.2 Chemical Sensing with Carbon Nanostructures . . . 9.6.3 Applications in Biotechnologies . . . . . . . . . . . . . 9.6.4 Cellular Imaging Applications . . . . . . . . . . . . . . . 9.7 Sensing and Thinking: The Values of New Discoveries in the Science of Carbon . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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10 Smart Luminescent Nanocomposites . . . . . . . . . . . . . . . . . . . . . 10.1 Broad-Scale Manipulation with Fluorescence Parameters in Nanocomposites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.1 Assembling, Screening, Immobilization . . . . . . . . . 10.1.2 Plasmonic Enhancement . . . . . . . . . . . . . . . . . . . . 10.1.3 Excitonic Effects. Coherent and Incoherent Energy Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.1.4 Exciton-Exciton and Plasmon-Exciton Interactions . 10.2 Realization of Electronic Energy Transfer (EET) in Nanocomposites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.2.1 Homo-Transfer and Hetero-Transfer . . . . . . . . . . . 10.2.2 General Rules for Collective Effects in EET . . . . . 10.2.3 Light-Harvesting (Antenna) Effect . . . . . . . . . . . . . 10.2.4 Wavelength Converting. Cascade Energy Transfer . 10.2.5 Extending the Emission Lifetimes . . . . . . . . . . . . . 10.2.6 Variations of Anisotropy . . . . . . . . . . . . . . . . . . . . 10.2.7 Modulation of Luminescence by Excitation Light . . 10.3 Superenhancement and Superquenching . . . . . . . . . . . . . . . 10.4 Optical Choice of EET Donors and Acceptors . . . . . . . . . . 10.4.1 Lanthanide Chelates and Other Metal-Chelating Luminophores . . . . . . . . . . . . . . . . . . . . . . . . . . .

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10.4.2 Semiconductor Quantum Dots (QDs) . . . . . . . . 10.4.3 Upconverting Nanomaterials . . . . . . . . . . . . . . 10.4.4 Conjugated Polymers . . . . . . . . . . . . . . . . . . . 10.4.5 Organic Dyes and Visible Fluorescent Proteins 10.4.6 Noble Metal Nanoparticles . . . . . . . . . . . . . . . 10.4.7 Fluorescent Carbon Nanoparticles . . . . . . . . . . 10.5 Wavelength Referencing, Multiplexing and Multicolor Coding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10.5.1 Wavelength Referencing and k-Ratiometry . . . 10.5.2 Multiplex Assays and Suspension Arrays . . . . . 10.5.3 Materials for Multicolor Coding . . . . . . . . . . . 10.6 Sensing and Thinking: Multiple Functions of Small Nanoparticles, How to Achieve that? . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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11 Passive Support Materials for Fluorescence Sensors . . . . . . . . . 11.1 Self-assembly on the Surface . . . . . . . . . . . . . . . . . . . . . . . 11.1.1 Template-Assisted Assembly . . . . . . . . . . . . . . . . . 11.1.2 Formation of Supporting and Transducing Surfaces 11.1.3 Self-assembled Monolayers . . . . . . . . . . . . . . . . . . 11.1.4 Langmuir-Blodgett Films . . . . . . . . . . . . . . . . . . . 11.1.5 Layer-by-Layer Approach . . . . . . . . . . . . . . . . . . . 11.1.6 Prospects for Designing Smart Surfaces . . . . . . . . . 11.2 Operating with Building Blocks for Nanoscale Sensors . . . . 11.2.1 Inorganic Colloidal Scaffolds . . . . . . . . . . . . . . . . 11.2.2 Graphene and Graphene Oxide . . . . . . . . . . . . . . . 11.2.3 Carbon Nanotubes . . . . . . . . . . . . . . . . . . . . . . . . 11.2.4 Fullerenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3 Organic and Biological Structures as Scaffolds . . . . . . . . . . 11.3.1 Two-Dimensional Self-assembling of S-Layer Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.3.2 Peptide Scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . 11.3.3 DNA Templates. Origamis . . . . . . . . . . . . . . . . . . 11.4 Functional Lipid and Polymer Bilayers . . . . . . . . . . . . . . . 11.4.1 Liposomes as Integrated Sensors . . . . . . . . . . . . . . 11.4.2 Stabilized Phospholipid Bilayers . . . . . . . . . . . . . . 11.4.3 Polymersomes . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.4.4 Formation of Protein Layers Over Lipid Bilayers . . 11.5 The Principles of Formation of Assembled Structures . . . . . 11.5.1 Affinity Coupling . . . . . . . . . . . . . . . . . . . . . . . . . 11.5.2 Self-assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.6 Conjugation, Labeling and Cross-Linking . . . . . . . . . . . . . . 11.6.1 Nano-Bio Conjugation . . . . . . . . . . . . . . . . . . . . . 11.6.2 Techniques of Conjugation and Labeling . . . . . . . .

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11.6.3 Co-synthetic Modifications . . . . . . . . . . . . . . 11.6.4 Chemical and Photochemical Cross-Linking . . 11.7 Sensing and Thinking. Extending Sensing Possibilities with Smart Decorated Surfaces and Nano-Ensembles . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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12 Fluorescent Composites Combining Multiple Sensing and Imaging Modalities . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.1 Multimodal Approach in Sensing and Imaging . . . . . . . 12.2 Design and Functioning of Multimodal Supramolecular Structures and Nanoparticles . . . . . . . . . . . . . . . . . . . . 12.2.1 The Principle of Formation of Materials with Multimodal Properties . . . . . . . . . . . . . . . . . . 12.2.2 Fluorescence Combined with X-Ray Computed Tomography . . . . . . . . . . . . . . . . . . . . . . . . . 12.2.3 Combining Fluorescence with NMR Imaging . . 12.2.4 Nanocomposites Demonstrating Triple-Modal Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.3 Magnetic-Fluorescent Nanoparticles with Different Functionalities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.4 Sensing and Thinking. Composites Assembling Several Modalities in Sensing and Imaging. What for? . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Evanescent Field Effects and Plasmonic Enhancement of Luminescence in Sensing Technologies . . . . . . . . . . . . . . 13.1 Evanescent-Wave Fluorescence Sensing . . . . . . . . . . . . 13.1.1 Excitation of Dyes by Evanescent Field . . . . . . 13.1.2 Evanescent Wave Sensing Technology . . . . . . 13.2 Plasmonic Effects in Fluorescence . . . . . . . . . . . . . . . . 13.2.1 General Character of Plasmonic Effects . . . . . . 13.2.2 Fluorescence Enhancement in Surface Plasmon Resonance Conditions . . . . . . . . . . . . . . . . . . . 13.2.3 Modulation of Light Emission by Plasmonic Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . 13.2.4 Exciton-Plasmon Interactions . . . . . . . . . . . . . 13.3 Light Absorption and Scattering by Plasmonic Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.3.1 Going Deeper into the Properties of Localized Plasmons . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.3.2 The Effects of Particle Shape and Hetero-Composition . . . . . . . . . . . . . . . . . . . . 13.4 Broad-Scale Applications of Plasmonic Sensors . . . . . . 13.5 Microwave Acceleration of Metal-Enhanced Emission .

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13.6 Sensing and Thinking. Plasmonics, Is that Sole Search for Brightness? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 524 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 525 14 Non-conventional Generation and Transformation of Sensor Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.1 Chemiluminescence and Electrochemiluminescence . . . . . . 14.1.1 Realization of Chemiluminescence . . . . . . . . . . . . 14.1.2 Catalytic Generation and Enhancement of Chemiluminescence . . . . . . . . . . . . . . . . . . . . . 14.1.3 Nanoparticle-Based Platforms in Chemiluminescence . . . . . . . . . . . . . . . . . . . . . 14.2 Electrogenerated Chemiluminescence . . . . . . . . . . . . . . . . . 14.2.1 Cathodic Generation of Luminescence . . . . . . . . . . 14.2.2 Essentials of Chemical Sensing Techniques and Their Prospects . . . . . . . . . . . . . . . . . . . . . . . 14.3 Bioluminescence. The Luciferin—Luciferase Reactions . . . . 14.3.1 The Origin of Bioluminescence . . . . . . . . . . . . . . . 14.3.2 Genetic Manipulations with Luciferase . . . . . . . . . 14.3.3 Bioluminescence Producing Excitation Energy Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.3.4 Attractive Features of Bioluminescence . . . . . . . . . 14.4 Radioluminescence and Cherenkov Effect . . . . . . . . . . . . . . 14.4.1 Self-illumination by Radioactive Decay . . . . . . . . . 14.4.2 Optical Luminescence Excited by X-Rays . . . . . . . 14.4.3 Emission Tomography Based on Cherenkov Effect . 14.4.4 Light-Addressable Potentiometric Sensors . . . . . . . 14.4.5 Sensor Operation Based on Photoelectrochemistry . 14.4.6 Photocurrent Generation as Informative Signal . . . . 14.4.7 Applications of Photoelectrochemical Sensors . . . . 14.4.8 Photovoltaic Cell as Sensor Device . . . . . . . . . . . . 14.5 Photoacoustic Spectroscopy and Imaging . . . . . . . . . . . . . . 14.5.1 Generation of Photoacoustic Waves by Absorbing Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.5.2 Photoacoustic Tomography in Life Sciences . . . . . 14.5.3 Contrast Agents for Photoacoustic Imaging . . . . . . 14.6 Sensing and Thinking. Coupling the Seemingly Uncoupled in Approaches to Sensing . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Techniques and Devices Used in Fluorescence Sensing 15.1 Instrumentation for Fluorescence Spectroscopy . . . 15.1.1 Standard Spectrofluorimeter . . . . . . . . . . 15.1.2 The Light Sources . . . . . . . . . . . . . . . . .

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15.1.3 Passive Optical Elements . . . . . . . . . . . . . . . . . . 15.1.4 The Light Detectors and Imagers . . . . . . . . . . . . . 15.1.5 Practical Considerations . . . . . . . . . . . . . . . . . . . 15.2 Miniaturized Integrated Instrumentation and Imaging with Smartphone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.2.1 Miniaturized Integrated Optical Systems . . . . . . . 15.2.2 Sensing and Imaging Using Lab-On-Smartphone Platforms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.3 Optical Waveguides and Optodes . . . . . . . . . . . . . . . . . . . 15.3.1 Optical Fiber Sensors with Optode Tips . . . . . . . . 15.3.2 Evanescent-Field Waveguides . . . . . . . . . . . . . . . 15.4 Multi-analyte Spotted Microarrays . . . . . . . . . . . . . . . . . . 15.4.1 Fabrication of Biochips . . . . . . . . . . . . . . . . . . . . 15.4.2 Success and Problems with Microarray Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.4.3 Read-Out and Data Analysis . . . . . . . . . . . . . . . . 15.4.4 Applications of Spotted Microarrays . . . . . . . . . . 15.4.5 Prospects of Spotted Microarray Technology . . . . 15.5 Suspension Arrays and Barcoding . . . . . . . . . . . . . . . . . . 15.5.1 Construction of Suspension Arrays . . . . . . . . . . . 15.5.2 Reading the Information from Encoded Microspheres . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.5.3 Present and Future Applications . . . . . . . . . . . . . 15.6 Microfluidic Devices . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15.6.1 Fabrication and Operation of a Lab-On-A-Chip . . 15.6.2 Microfluidic Devices as Microscopic Reactors and Analytical Tools . . . . . . . . . . . . . . . . . . . . . 15.6.3 Fluorescence Detection in Microfluidic Devices . . 15.6.4 Prospects for Broad-Range Practical Applications . 15.7 Sensing and Thinking. Optimizing Convenience, Sensitivity and Precision . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Frontiers for Future Research. Two-Photonic, Highly Excited and Single-Molecular Sensors . . . . . . . . . . . . . . . . . . . . . . . . . 16.1 Two-Photon Excitation, Stimulated Emission, Lasers as Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.1.1 Two-Photon and Multi-photon Excited Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.1.2 Amplified Stimulated Emission . . . . . . . . . . . . . . 16.1.3 Laser as Prospective Sensor . . . . . . . . . . . . . . . . 16.2 Light Emission and Photoreactions from High-Energy Electronic States . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contents

16.3 Near-Field Photonics: From Imaging to Nanoplasmonics and Nanolasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.4 Far-Field Visualization and Sensing of Single Molecules . . 16.5 Single Molecular Dynamic Studies in Ultra-small Volumes . 16.5.1 Operation with Ultra-small Volumes and Single Molecular Detection . . . . . . . . . . . . . . . . . . . . . . . 16.5.2 Fluorescence Correlation Spectroscopy and Its Extensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16.5.3 Kinetic Fingerprinting in Analysis on Single-Molecular Level . . . . . . . . . . . . . . . . . . . . . 16.6 Lighting Up the Behavior of Small Systems . . . . . . . . . . . . 16.7 A Word in Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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About the Author

Alexander P. Demchenko is the scientist well known globally for his fundamental works on the excited-state dynamics of electron, proton and energy transfer and on their coupling with molecular relaxations in dielectric environment. His research interests that extend to a broad interdisciplinary scale involve photophysics and biophysics, photochemistry and biochemistry. Particularly important are the results of his works on internal dynamics in proteins and biomembranes and also on introduction of new generation of fluorescent sensors and probes. Dr. Demchenko graduated from the Department of Physics of the Kyiv State Shevtchenko University in 1967, got Ph.D. degree in Biochemistry in 1972 and Habilitation in Biophysics in 1982. Working in his home institution, Palladin Institute of Biochemistry in Kyiv, Ukraine, he was invited as a visiting professor to different universities and research institutions of USA, Germany, Turkey, Hungary, France and Taiwan. Presently, he is lecturing at the Yuriy Fedkovych National University, Chernivtsi, Ukraine. Dr. Demchenko is the author of two classical monographs “Ultraviolet spectroscopy of proteins” (Springer, 1986) and “Introduction to fluorescence sensing” (Springer, 2009 and 2015) that became popular in teaching on a graduate level. He was also the editor of three books in “Springer Series on Fluorescence”. He is one of the most cited scientists of Ukraine.

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About the Author

His contribution to science was marked by election as a Honorary member to Hungarian Academy of Sciences, by Palladin award in Biochemistry and by Gregorio Weber award for Excellence in Fluorescence.

Chapter 1

Principles and Techniques in Chemical and Biological Sensing

Booming of sensor technologies is a response to a strong demand in society. As a result, almost every physical principle and technique that can detect interactions between the molecules, particles and interfaces was suggested and tested for application in sensing. In this Chapter after the introduction of fundamental concept of molecular recognition we provide a short survey of different technologies in molecular sensing that are based on different physical principles and realized in heterogeneous and homogeneous formats. The basic functional elements in a general sensor design that are the receptor, transducer and emitter are considered. The unique features of fluorescence as detection technique in sensing and imaging are discussed and the range of its applications outlined. A strong move is observed towards the development of multiplexing (many inputs) and multiparametric (many outputs) sensor technologies. No interaction—no information. This principle is clearly seen in the background of all sensing technologies. In every interaction, we have at least two partners (Fig. 1.1). One is, of course, the object that has to be detected, called the target or analyte. It can be a species of any size and complexity, starting from protons, small molecules and ions, up to large particles and living cells. The other partner designed or selected for target detection and for providing this information, is the sensor. Interaction between target and sensor is based on the principle of molecular recognition—strongly selective reversible noncovalent binding. In this Chapter we provide a short survey of basic strategies in molecular sensing and show that almost every physical principle and technique that can detect interactions between molecules, particles and interfaces was suggested and tested for application in sensing. The role of these techniques in response to molecular recognition is different. Some of them are of rather general nature and others are rather specific. Then we outline the distinguishing features of those detection methods that are based on fluorescence. We will see that fluorescence allows achieving the ultimate limit of sensitivity—detection of single molecules. Also, the method is characterized by ultra-high resolution in time and space and the possibility of sensing at a distance. Such versatility allows achieving high-throughput multiplex sensing in different media including living cells and tissues. © Springer Nature Switzerland AG 2020 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-030-60155-3_1

1

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1 Principles and Techniques in Chemical and Biological Sensing

Targets: Gasses Molecules Polymers DNA, RNA Microbes Whole cells, etc.

Molecular recognition

Sensors : Molecules, Nanostructures Performed media Interfaces etc.

Media: Air, Gasses, Solutions Living cells, Whole bodies, etc. Fig. 1.1 Specific interactions (molecular recognition) between various targets and sensors generating the sensor response for target detection

1.1 General Definitions and Formulation of Key Problems The simplest common definition of a sensor is that “a sensor is something that senses”, i.e. receives information and transforms it into a form compatible with our perception, knowledge and understanding. Our body is full of sensors that respond to light, heat, taste, etc. With the development of civilization, they became insufficient for orientation of personality, community or the whole society in new conditions. More and more we need objective knowledge on what is happening inside and outside of our body and what are the benefits and threats to the whole society. There is a necessity to know what compounds are useful and what are harmful, how safe and healthful is our environment and to follow that continuously. Different industrial processes including that of production of agricultural goods, food processing and storage need to be controlled as well. The human genome is a very useful piece of knowledge only when we can analyze gene expression and find its relation with individuality, age and disease. We need to know the distribution inside the living cells of many compounds, including enzymes and their regulators and also substrates and products of these reactions. This information may need to be obtained throughout the whole cell life cycle including its division, differentiation, aging, and apoptosis. All that can be accomplished only with the help of man-made sensors. They will be the subject of the present book. The man-made sensors are often called ‘chemical sensors’ and those of them that involve biology-related compounds and/or biospecific target binding—‘biosensors’. According to the definition approved by IUPAC, ‘a chemical sensor is a device that transforms chemical information, ranging from the concentration of a specific sample component to total composition analysis, into an analytically useful signal’. Thus the sensor can be regarded as both a designed molecule and an analytical

1.1 General Definitions and Formulation of Key Problems

3

device that delivers real-time and online information on the presence of specific compounds in complex samples (Thevenot et al. 1999). In a narrow sense, the sensor is a molecule or assembled supra-molecular unit, such as nanoparticle or interface, that is able to selectively bind the target molecule (or supra-molecular structure) and provide information about this binding by the change of any parameter that can be measured. In a broader sense it should include control and processing electronics, interconnecting networks, software and other elements needed to make the signal not only recordable but understandable. The general problem in any sensor technology can be formulated as follows. We have the target molecule, particle or cell dispersed in a medium that may contain many similar molecules, particles or cells. We have to provide a sensor that has to be incorporated into this medium or exposed to contact with it. The presence of target should be revealed by its selective binding to the sensor. The binding should be detected and, if necessary, quantified in target concentration. This information on sensor-target binding requires some transduction mechanism that connects the binding (molecular event) and its detection by the instrument, on the scale of our vision and understanding.

1.1.1 Sensors or Assays? Assay is a broader term than sensor, since it may involve diverse manipulations with the tested system such as different chemical and biochemical reactions, addition, extraction or separation of components. Sensing is always a more direct procedure, which is based on an interaction between the target and a particular sensor unit designed for detecting this target and generating detectable response to this interaction. The latter can be a molecule, a particle or a solid surface, in which additional reactions are not needed or can be used only for amplification of primary effect of such interaction. The distinction between ‘sensors’ and ‘assays’ is still not very strict. Moreover, the same molecules with selective target-binding function can serve in both assay and sensor techniques. Therefore, critical here is the issue, how the reporter signal is provided. An example is the antibody, which is the most frequently used molecule with the function of biological recognition (Fig. 1.2). For participating in bioassays, this molecule can be labeled at the periphery, at any site outside its target-recognition unit. This is enough for providing response in heterogeneous format to immobilized target. In contrast, for its participation in sensing in homogeneous format, stringent additional requirements should be satisfied. The label should provide the detectable reporting signal directly or indirectly on the interaction with the target. One of solutions to achieve that could be to locate the label at the target-binding site to be a part of recognition unit (Enander et al. 2008). Details of application of antibodies as recognition units in immunoassays and immunosensors can be found in Volume 2, Chap. 4).

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1 Principles and Techniques in Chemical and Biological Sensing

a

b

Fig. 1.2 The labeling of antibodies with fluorescence dyes for use in bioassays and biosensors. IgG antibody is shown. It is a protein molecule composed of two light (L) and two heavy (H) chains forming domains cross-linked by disulfide (–S–S–) bonds. The VL and VH domains form the target antigen (Ag) binding site. The site of labeling determines the range of applications of antibodies. a The fluorescent dye is bound at the periphery of antibody molecule and serves only for the purpose of labeling. Its response is insensitive to antigen binding. b The reporter dye is located close to antigen binding site so that the interaction with antigen changes its fluorescence parameters providing the reporting signal

It has to be noted that different analytical methods use fluorescent reagents that irreversibly bind to targets in analyzed materials and change on binding their fluorescence. Such reaction-based probes (also referred to as activity-based probes) (Bezner et al. 2020) provide their functional groups to react with an analyte of interest. Here for analyte detection the selectivity versus other molecules is determined by molecular reactivity rather than by molecular recognition (Bruemmer et al. 2019). Such procedures consuming the reagents cannot be considered as sensing. Likewise, the determination of enzyme activity by monitoring the concentration of a reactant or product that is involved in the enzymatic reaction is not the sensing. Many of these assays use fluorescent substrates or products in direct or coupled reactions. These cases will not be discussed here, and the reader is addressed to a vast chemical and biochemical literature.

1.1.2 Homogeneous and Heterogeneous Formats Both assays and sensors can operate in heterogeneous or homogeneous formats. Experimental formats without any separation or washing steps (only “mix and measure”) are called homogeneous, whereas the formats for assays and sensors that involve multistep separation setups are referred to heterogeneous ones. The latter formats are those that require separation of the sensor-target complex for subsequent detection by any analytical method, including fluorescence. Most convenient way to realize that is to use a heterogeneous system, in which the sensor elements are immobilized on a solid support. In this case, after incubation in the tested medium

1.1 General Definitions and Formulation of Key Problems

5

the unreacted species that are present in the system can be removed prior to analysis, simply by washing. If necessary, additional reagents can be added for visualization or generation of response signal (Fig. 1.3). The reader may note that in the described above case, after incubation in the tested medium, only the reacted species generate analytical signal, so that remaining unreacted species should be removed. The role of fluorescence or any other (lightabsorbing, radioactive) reporters in this case is passive; they are used as the “labels”, just to indicate the binding. Sensitivity of these assays can be dramatically increased

a

Expose

R

R

R

b

R

R

R

R

Wash

R

R

c

R Detect I

R

R

R

R

R

Fig. 1.3 An example of heterogeneous assay that commonly consist of three steps. a The plate with immobilized receptor (R) molecules is exposed to the probed sample. The targets (T) become strongly and specifically bound, whereas the contaminating compounds (depicted as orange balls) remain in solution. b The plate is washed with removal of contaminating species. After that it contains only bound targets, and all unbound components of the mixture are removed. c The plate is exposed to solution of molecules or particles that are able to recognize the target and bind to it at a different site. Such indicators (I) contain fluorescent label. Alternatively, the bound target can be removed from the plate and analyzed with the methods of analytical chemistry. Here and in other illustrations below, geometrical fitting indicates specific binding (molecular recognition)

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1 Principles and Techniques in Chemical and Biological Sensing

by application of fluorescence reporters with increased brightness. It could be the nanoparticles or appended enzymes generating chromogenic or fluorescent product after incubation with proper substrate. In solution, this type of assay is also possible, but it always requires separation of target-sensor complexes by chromatography, electrophoresis, etc. As we will see below, employment of this assay principle in sensor technologies allows us to achieve the broadest dynamic range of quantitative determination of the target. Because of the implied washing step, the result is less sensitive to interference of non-specifically bound components of the tested system. However, the assay is limited to high-affinity binding, since only in this case the target-sensor complexes remain intact during manipulations with the sample. All additional operations, such as separation and washing, are time-consuming, which does not allow one to obtain immediate results. In contrast, homogeneous assays are those that provide the necessary signal upon target binding in the test medium without any separation or washing. Figure 1.4 illustrates different possibilities for realizing the sensor response in this format. In

a R

R

b R R

Fig. 1.4 Two examples of homogeneous assay. a Sensor molecules are composed of receptor (R) containing reporter groups that change their fluorescence on target (T) binding. b Two different sensor molecules possessing different reporters, one with EET donor site (marked black) and the other able for bright emission bind the target at two different binding sites. On interaction with target (T) of both sensors their fluorophore reporters become at close distance that allows recording of EET signal. In both cases, the assay may occur without the sensor immobilization and without the separation of the complex from unreacted components. Numerous possible interfering species (depicted as orange balls) should not influence the analysis

1.1 General Definitions and Formulation of Key Problems

7

the first case the target binding perturbs the emission of appended fluorophore. The other case requires participation of two fluorophores. One is the excitation energy transfer (EET) donor and the other—the acceptor. The sensor signal appears when they are simultaneously bound to the same target and located at a short distance. Such tests can be provided in homogeneous test media (solutions) and conform to the definition of ‘direct assays’ (Altschuh et al. 2006). Since no manipulations with the sample are needed, the sensors with this function are applicable for detecting the target distribution in the media of complex composition, including living cells. They should provide the broadest range of response by detectable changes of one or more parameters of its emission. A strong advantage of homogeneous assays formats is the possibility of quantitative determination of targets in the case of their relatively low affinities, so that a dynamic concentration-dependent equilibrium should be always established between free and bound target. However, in this case, the dynamic range of the assay (the target concentration range, in which the variation of reporter signal is detected) is much narrower, usually within two orders of magnitude. Such limitation is dictated by mass action law (see Chap. 2 of Volume 2). It is a matter of terminology, but some authors prefer to call heterogeneous assays as the ‘assays’ while homogeneous assays as the ‘sensors’. This is because in homogeneous assays it is much easier to develop sensors that will not need any manipulation in the course of measurement or after it and that are applicable for continuous monitoring of target concentration. According to the International Union of Pure and Applied Chemistry (IUPAC) nomenclature recommendations, a biosensor (that can be extended to any type of sensor) is defined as a self-contained analytical device, which is capable of providing quantitative or semi-quantitative analytical information using a biological recognition element either integrated within or intimately associated with a physicochemical transducer (Thevenot et al. 2001). It is clearly affirmed that “a biosensor should be clearly distinguished from a bioanalytical system which requires additional processing steps, such as the addition of reagents”. Meantime, this definition is not supported by many researchers, and different other definitions exist (Kellner et al. 2004). For instance, some authors suggest considering as the sensors also the devices, in which the response is produced in a chemical reaction with the target.

1.2 Elements of a Typical Sensor The term ‘sensor’ is often used in a very broad sense. In a narrow sense, however, the sensor should be a unit able to perform two important functions: (1) providing the target (analyte) binding; (2) reporting on this binding by generating an informative signal. Coupling of these functions requires a functional linker or linking mechanism that is called transduction (Fig. 1.5). Every type of sensors can be classified

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1 Principles and Techniques in Chemical and Biological Sensing

Receptor

Transducer

EmiƩer Detector

Target

ReporƟng RecogniƟon Fig. 1.5 Basic functional elements of fluorescent chemical sensor and biosensor. The primary event of target binding by receptor (recognition element of the sensor) should generate the response signal, which is provided by a reporter element. These functions are different but they must be strongly coupled via transducer

according to three basic elements of their operation that correspond to performing these functions. Receptor is the unit responsible for target recognition, reporting element (in figure depicted as fluorescence emitter) is the necessary element of any sensor device and their coupling element is the transducer. Different physical principles can be applied in sensor technologies to generate a measurable signal in response to sensor-target interaction.

1.2.1 The Recognition Element The recognition element (receptor) can be as small as a group of atoms chelating a metal cation and as big as a large protein molecule, DNA, membrane and even the living cell. In order to provide information on the presence, concentration or location of the target, the sensor-target interaction should proceed in a highly selective way, recognizing it from other objects of similar structure and properties that can be present in the probed system. The structure responsible for that is called the recognition unit or receptor. Addressing the demand of target detection, it can also be the object of any size and complexity. The target-binding properties of sensor receptors are specific for every target. Each of them should exhibit high affinity to a target and high level of discrimination against the species of similar structure. This can be achieved in many ways: by using complementarity in DNA and RNA sequences, by applying monoclonal antibodies and their recombinant fragments, by introduction of natural protein receptors and their analogs, by creation of combinatorial peptides and polynucleotide libraries and compounds forming inclusion complexes, by pore formation in imprinted polymers, etc. This issue is well elaborated in the literature and it will be extensively discussed in Volume 2 of this book. The target-receptor affinity interactions involve reversible multiple binding through noncovalent bonds, such as ionic bonds, hydrogen bonds, and van der Waals

1.2 Elements of a Typical Sensor

+

9

Hydrophobic surfaces

-

-

+

-

Target

+

Charge complementarity

Sensor

+

Signal production

-

Fig. 1.6 Molecular recognition between sensor and its target provided by steric complementarity and multipoint binding that is realized by hydrophobic interactions and interactions between complementary charges

interactions, π-π stacking, etc. (Bishop et al. 2009). The product of such affinity interaction is a specific molecular association complex. In order for such a complex to form, the involved species should be complementary with respect to shape and provide maximal amount of attractive interactions (Fig. 1.6). Optimized realization of such steric and energetic factors is essentially the molecular recognition. For providing stable intermolecular complex formation, the cumulative effect is achieved by assembling the interaction forces demonstrating structural complementarity. Each of them may not be strong enough to compete with the solutesolvent interactions, to withstand thermal fluctuations or to compensate entropy loss on binding, but the collective effect is much stronger than that expected from summation of individual interactions. Thus, structural and energetic complementarity drives molecular recognition. The multivalency, is a key principle in realizing molecular recognition for achieving strong, specific, yet reversible interactions between two or more binding sites. Being applicable to both biological (Mammen et al. 1998, Meyer and Knapp 2014) and synthetic (Badjic et al. 2005) systems, it states that multi-point binding between interacting structures can be realized only on formation of multiple, interconnected molecular binding modules. Multivalency allows providing sufficient stability of formed intermolecular assemblies and achieving specificity in these interactions. The positive cooperativity can be realized in multivalent binding, as the target binding at one site will restrict the mutual location at other sites, facilitating further binding events. In this way the multivalent binding affinity (often called avidity) depends not only on the monovalent binding affinity but also on the ‘valence’, the number of sensor-target binding pairs. Such increase of efficiency can be very strong, so that the dissociation constant in the case of multivalency is commonly described by exponential law. The power index of this function is an indicator of degree of cooperativity (Mammen et al. 1998). The term specificity is also frequently used. It can be defined as the ability to discriminate between molecular recognition from other binding of lower affinity. More in detail, these issues will be discussed in Volume 2, Chap. 2.

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1 Principles and Techniques in Chemical and Biological Sensing

1.2.2 The Reporter Element The receptor-target interactions are ‘blind’ until they are detected. Therefore, the other function of the sensor is to ‘visualize’ them, to report about the interaction events by providing the signal that can be analyzed and counted. The structure that is responsible for generation of this signal is called a reporter. A variety of reporters and the mechanisms in the background of their operation will be discussed in this book with a focus on fluorescence reporting. Optical (light absorption, reflection, fluorescence), electrochemical (amperometric, conductometric), mass, heat and acoustic effects can be also used for the purpose of designing the reporter elements (Banica 2012, Barsan et al. 2000) (see Sect. 1.3). Sensor as an analytical tool also needs a detector. It involves instrumentation, which is important as an interface between the micro-world of molecules and macroworld in which we live. In the micro-world the elementary events in sensing occur, and in the macro-world we have to analyze the results and make our decisions. Definitely, the role and construction of detectors depends on the selected analytical tool. In most of sensor technologies the response signal (optical, heat, mass, etc.) is transformed into an electrical signal that can be quantitatively measured. Sensor performance depends strongly on reporter unit, and to characterize it, different quantitative measures were introduced (Gauglitz 2018). One of them is the limit of detection (LOD), the limiting amount of analyte that can be analyzed with reasonable precision. The sensor sensitivity can be defined as the slope of the calibration curve plotted as a dependence of signal units vs. target concentration.

1.2.3 The Transducer The transformation of the signal about the binding event into the response of a reporter is called transduction, and if additional element of structure or functional unit is needed for that, it got the name transducer. In a broader sense, the transducer is also the physical mechanism that couples the receptor and reporter functionalities, such as conformational change or electronic charge transfer in the recognition unit modulating the analytical signal. Thus, the mechanisms of coupling are diverse and strongly connected with reporting element. They can be electronic, conformational, through the medium (e.g. due to pH change), or by coupled chemical or electrochemical reaction. Chapter 4 describes in detail several examples of such transducers that can be realized in fluorescence sensing. They could be electron and proton transfer and conformastional isomerization in the sensor unit. Translated to the sensor response it could be also the aggregation-disaggregation or binding-release of additional factors, such as protons and ions.

1.3 Diversity of Sensing Techniques and Their Operation Principles

11

1.3 Diversity of Sensing Techniques and Their Operation Principles The sensors and biosensors used in successfully realized and potential applications (Cooper 2003), are based on different physical principles (Fig. 1.7). They develop in parallel, competing and enriching each other. A number of excellent books and reviews have been published in the field of chemical sensors, biosensors and nanosensors (Banica 2012; Barsan et al. 2000; Fraden 2010; Perumal and Hashim 2014). By addressing them together with a number of related publications, the reader can make his own comparative analysis of different sensing strategies: electrochemical (Palecek et al. 2002; Warsinke et al. 2000); microcantilever (Carrascosa et al. 2006); optical (Baird et al. 2002; Baird and Myszka 2001) including surface plasmon resonance (SPR) (Baird et al. 2002; Homola 2003) and microrefractometricmicroreflectometric techniques (Gauglitz 2005). Below we provide only a brief analysis of their strong and weak points, in comparison to fluorescence that is the major subject of this book.

a

b

ANGLE

HEAT

d

c MASS

ELECTRICAL SIGNAL

Fig. 1.7 Schematics illustrating the operation principles of sensing based on several alternative technologies. a Calorimetr. The heat effects are recorded due to interaction between the target and receptor when they are mixed in a calorimeter. b Surface plasmon resonance (SPR). The layer of receptors is formed on the surface of a thin layer of gold or silver. The target binding causes the change of refractive index close to this surface that modulates the angular dependence of the reflected light beam. c Micro-cantilever technique. The target binding results in the mechanical signal (the change of vibration frequency) that is detected as the change of mass. d Electrochemistry. The target binding usually results in electron transfer between the electroactive compound and the transducer (electrode) or in a change of the existing electrochemical signal

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1 Principles and Techniques in Chemical and Biological Sensing

1.3.1 The Label-Free Techniques Label-free techniques that are developed to the level of research and practical application involve those with optical detection, such as surface plasmon resonance (SPR), and also acoustic, cantilever and calorimetric biosensors. Calorimetry (Fig. 1.7a) is an example of a general label-free technique that allows measurements in solutions without the necessity of immobilization of sensor molecules or of their labeling (Ladbury and Chowdhry 1996). This method is based on the principle of generation of heat effects that appear in the course of sensor-target binding. Since the detection is based on a general property of reaching the thermodynamic balance in the system after the formation of new target-receptor bonds, this method seems to be broadly applicable. The process of binding can be detected as a heat effect in solution; therefore the sensor immobilization is not needed. It should be noted, however, that this method does not possess any structural resolution. In complex mixtures, a strong binding by specific targets existing in small concentrations is not distinguishable from that of nonspecific binding by species present at high concentrations. Thus, the advantage of measurement in label-free conditions in solutions is compromised by very restricted range of applications. However, such applications exist, for instance, in detection of explosives and other highly energetic compounds (Liu et al. 2005). Surface plasmon resonance (SPR) is a typical example of immobilization-based but label-free technique (Fig. 1.7b). Description of an SPR principles and a review of its applications can be found in many articles (Homola 2003; Homola et al. 2005; Rich and Myszka 2006). The method is based on observing the change in refractive index close to the surface of a thin (~50 nm) metal (usually gold) film, illumination of which creates the so-called evanescent wave that generates optical signal. On this surface the receptor molecules or particles are immobilized. The target, when bound from a contacting solution phase, produces a detectable local increase in refractive index. This signal is observed as the change of the angle at which a strong decrease of intensity of reflected light occurs. Evanescent wave decreases exponentially as a function of distance from the surface of the sensor film on a scale up to 200 nm, which is convenient for the detection of large molecules. Unbound molecules and particles remain in the sample solution phase outside the detection layer and do not interfere with the measurement. This method has many attractive features. Because the necessity of pre-treatments or after-treatments to visualize the result is absent, it records the target binding in real time. Due to this important feature, it has become a valuable technique not only for determining the receptor-target affinities but also for sorptiondesorption kinetics of the target. Its application for simultaneous determination of multiple targets is difficult, but this problem can be overcome (Homola et al. 2005) and developed into SPR imaging sensor technology towards high-throughput multianalyte screening (Wong and Olivo 2014). Meantime, as every label-free method, SPR cannot distinguish specific and non-specific binding and therefore is not secured from false positive results.

1.3 Diversity of Sensing Techniques and Their Operation Principles

13

It is interesting to note that a hybrid between fluorescence and SPR can be created. The method is called evanescent wave fluorescence and will be discussed in more detail in Chap. 13. Like SPR it can measure surface-specific binding events in real time. In this method, the excitation is by evanescent wave and response is provided by the detection of fluorescence. Thus, only the species bound to the surface will be excited and they will produce fluorescence signal. Very interesting are the developments of sensors based on the change of mass on target binding (Fig. 1.7c). Two techniques have been developed using this principle. One is the acoustic sensing. It is based on a highly sensitive detection of mass changes measured via surface acoustic wave (Lange et al. 2008). The sensor needs to be coupled to the surface of piezoelectric material, such as quartz. Its applicability for accurate detection of protein targets has been demonstrated. The second one uses a microcantilever (vibrating microbalance). The spring constant of this vibrating nanoscale mechanical device is directly related to the increase of mass on target binding (Battiston et al. 2001). The latter technology needs a sensor immobilization to a specially prepared surface with special properties. The targets, interacting with the sensor, become captured on the surface and are detected. This technology has developed into piezoelectric microelectromechanical systems that offers labelfree detection but requiring the sensor immobilization for responding to binding of different targets (Pang et al. 2012). Electrochemical sensors (Fig. 1.7d) are very popular because they use simple instrumentation and allow direct recording of target binding to immobilized receptors in the form of an electrical signal. Electrical signal can be obtained in oxidationreduction reaction at the electrode. This can be either direct reaction with the participation of target or a coupled reaction that may involve a catalyst (Palecek 2005; Palecek and Jelen 2005). Electrochemical sensors are attractive by their simplicity, and because of this feature they are presently the best established and most used analytical sensors oriented for detection of single analyte. Different output parameters can be recorded, and according to them a classification is made into potentiometric (voltage), amperometric (current), and impedimetric (impedance) based sensors, of which the voltammetric and conductometric sensors can be considered as particular cases. In all these versions of technology performed in solutions, the sensor immobilization is always needed, and in most cases the protocol involving the coupled electrochemical reaction to target binding needs to be developed. In biosensing they are mostly based on enzymatic activity generating a detectable product (Bollella and Gorton 2018; Nguyen et al. 2019), and therefore are restricted to monitoring of substrate(s) or effector(s) of a particular enzyme. Thus, these methods possess important obviously positive feature: only the bound target molecules produce the response. Therefore, if the sample contains a complex mixture of different compounds, only the target will produce the signal and no separation or washing steps will be needed. That is why these methods may allow direct kinetic measurements.

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1 Principles and Techniques in Chemical and Biological Sensing

There are many possibilities to apply the label-free approach in solutions with the separation of high-affinity target-sensor complexes and their subsequent chromatographic separation. These complexes have to be analyzed by different analytical techniques, such as mass spectrometry, spectroscopy, enzyme inhibition or antibody binding. However, such procedures are always difficult and time-consuming. They are limited to the cases of very strong sensor-target interactions that do not have to be disrupted by separation procedures. Finally, it should be noted that high cost of instrumentation should be accounted in using the general label-free and immobilization-free approaches.

1.3.2 The Label-Based Approach The techniques exploring the label-based principle are those that use for analyte detection the electron-dense, NMR-contrasting, radioactive, light-absorbing or fluorescent labeling. Applicable, in principle, to any molecular interaction, this approach can be used to label at least one of the interacting partners, but such attachment of label should not change the binding affinity. The label-based techniques can be used in conjunction with different analytical devices, such as fiber-optic or gravimetric sensors (Luppa et al. 2001). In many of these cases the unbound labeling species have to be removed to allow recording only the labeled target-sensor complexes, as it is shown in Fig. 1.3. This is a simple and efficient way to achieve high contrast against the background. It is extensively used in different methods of practical importance, including Enzyme-Linked Immunosorbent Assay (ELISA) and its fluorescencebased analogs that allow important miniaturization of analytical tools (Eteshola and Balberg 2004). However, the most important advantage of these techniques is their use for constructing the sensors, in which the recognition element is labeled in such a way that a measurable physico-chemical property of the construct changes upon the complex formation in solution (Fig. 1.4). This allows providing homogeneous assays that avoid any separation steps (see Chap. 2). In principle, such technology is of general applicability; it is easy-to-implement and inexpensive. Particularly, in fluorescence sensing applications the sensor operation can be made ‘direct’, without additional manipulations or reagent additions (Altschuh et al. 2006; Gauglitz 2005). Here the informative signal can be based on a variation in several recorded parameters that change upon complex formation (see Chap. 3). When comparing this approach with the label-free methods based on detection of changes of refractive index, charge or mass at the sensor surface or heat effects, one has to note the following important point. Every analysis often involves side effects produced by contaminants in the test media. These contaminants also have mass and charge; they change refractive index and produce heat effects. This results in a

1.3 Diversity of Sensing Techniques and Their Operation Principles

15

background signal that may induce a serious complication, especially when these techniques are used for the testing of realistically complex samples, such as blood serum. In contrast, introduction of labels in sensing technologies always makes the response more specific leading to reduction of false positive complications in results.

1.4 Distinguishing Features of Fluorescence Technology Why fluorescence is so important and so widespread in molecular sensing and imaging? Here we will try to respond in short to this question. For acquiring more details the reader is encouraged to address other chapters of this book.

1.4.1 Fluorescence as the Technique of Choice Fluorescence sensing is essentially a label-based approach, in which the lightemitting molecules, nanoparticles, thin films or other reporting tools are introduced into the probed system with the purpose of obtaining analytical information. (Intrinsic fluorophores in biological systems, such as tryptophan in proteins and NADH in cells, can be considered as naturally imposed labels). Reporting techniques that are actively used in sensor technologies will be described in Chap. 3. A series of recently published books discussing different structural and mechanistic aspects of fluorescence reporters (Demchenko 2010a, 2010b, 2011; Jameson 2014; Lakowicz 2007; Valeur and Berberan-Santos 2012) may be also useful for the interested reader. Thus, what distinguishes fluorescence from all other methods suggested for reporting about sensor-target interaction? Figure 1.8 illustrates these features. Primarily it is the ultra-high sensitivity. With proper dye selection and proper experimental conditions, the absolute sensitivity may reach the absolute limit of detecting single molecules. This feature is especially needed if the analyte exists in trace amounts and it may allow avoiding the costly and time-consuming enrichment steps. The single molecular sensors are now a reality (Gooding and Gaus 2016). Meanwhile, one has to distinguish the absolute sensitivity, which is the sensitivity of detecting the fluorescent dye (or particle) from that required for recording the response to target binding. The latter is needed for providing the necessary dynamic range of variation of the recorded fluorescence parameters in detecting the sensor-target interaction. This is a much harder problem, and different possibilities for its solution will be discussed in detail in Chap. 4. The second distinguishing feature of fluorescence is the high speed of response to the change in molecular interactions. This response can be as fast as 10−8 to 10−10 s and is limited by fluorescence lifetime and the speed of photophysical or photochemical event that provides the response. Usually such high speed is not needed but sometimes it is essential. For instance, probing the rate of action potential propagation in excitable cells needs the sub-millisecond time resolution. However,

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1 Principles and Techniques in Chemical and Biological Sensing

Ultra-high sensitivity

Very high spatial resolution

Recording at a distance

Ultra-high time resolution

Fluorescence

Non-invasive Non-destructive

Detection of light polarization

Multiple output channels

Fig. 1.8 The distinguishing features of fluorescence detection in sensor technologies

in most of applications the speed of sensor response is not limited by fluorescence reporting. It is limited by other factors, such as the rate of target-sensor mutual diffusion and of establishment the dynamic equilibrium between bound and unbound target. Important is a very high spatial resolution that can be achieved with fluorescence. This allows recording detailed cellular images and operating with dense multianalyte sensor arrays. In microfluidic devices (Sect. 15.6) the detection volume can be reduced to nanoliters and in photon correlation spectroscopy (Sect. 16.3) even to femtoliters. The resolution in common microscopy is limited to 200–500 nm (in visible light). The limit is due to the effect of light diffraction when the dimensions become shorter than the wavelength. Even this limit can be overcome with the application of new methods of imaging and data analysis providing the possibility to increase the resolution to several nanometers. Thus, we observe transformation of microscopy into ‘nanoscopy’. Chapter 15 of Volume 2 will address this issue with demonstration of bright visual examples. Human eye is insensitive to visible light phase and polarization, but optical instrumentation allows recording their changes. That is why the optical anisotropy (Sect. 3.3) has become an important parameter that is actively used in fluorescence sensing. The non-destructive and non-invasive character of fluorescence sensing is beneficial primarily for many biological and medical applications. In fluorescence sensing, the reporter dye and the detecting instrument are located at a certain distance and are connected only via propagating light. That is why the fluorescence detection is equally well suited for remote control of chemical reactions in industrial reactors and for the sensing different targets within the living cells. The greatest advantage of fluorescence reporting, therefore, is its versatility. It comes from the basic events of the fluorescence response, which are the absorption

1.4 Distinguishing Features of Fluorescence Technology

17

and emission of light. Those are essentially the photophysical events coupled to molecular events of sensor-target interaction. That is why the sensing can be achieved in any environment: in solid, liquid and gas media and at all kinds of interfaces between them. The basic mechanism of response, remaining always the same, does not impose any limit on the formation of any supramolecular structures, incorporation of reporter into any nano-composite, attachment to solid support, etc. This allows not only creating smart molecular sensing devices but also their attachment to the surfaces in heterogeneous assays or integration into nanoparticles, endowing new functional possibilities. Due to these facts, homogeneous assays in liquid media develop into nanosensor technologies, in which, in addition to fluorescence, different self-assembling configurations and combining with the carriers of magnetic, NMRcontrasting, electron-dense, photoacoustic and other properties (see Chap. 12) can be explored. Heterogeneous assays develop into multi-analyte microarrays (sensor chips), which allow simultaneous detection of several hundreds and thousands of analytes (Chap. 15). The two-photon and confocal microscopies serve for obtaining the 3-dimensional images, which allows localizing target compounds in space.

1.4.2 Large-Scale Manipulation with Emission Colors and Lifetimes Optical signals do not mix and do not interfere. This important property of optical transfer of information is seen as the absence of perturbation of signals produced by photons of different colors (Fig. 1.9). This is the reason why optical fiber wires can transmit much larger amount of information than the electrical wires. Visible light communication technologies also use this principle (Lee et al. 2015). And since the transmission of multicolor signal proceeds without information losses, the possibility of generation, transmission and recording of informative multicolor signal can be used in sensors detecting simultaneously many analytes. The technologies of multiplex sensor developments include multiple response elements in optical fibers

Fig. 1.9 Illustration of the fact that propagating photons of different energies and representing different colors being differently reflected from the walls of optical fibers propagate independently. This allows constructing sensors detecting many analytes simultaneously, particularly with the aid of optical fibers

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1 Principles and Techniques in Chemical and Biological Sensing

(Sect. 15.3), nanobeads in homogeneous assay (Sect. 15.5) and spotted nanosensors (Sect. 15.4). In fluorescence sensing and imaging the information capacity of response signal can be very great. If 5–6 different colors distributed over visible spectrum generated by fluorescent organic dyes or semiconductor nanocrystals is used with their natural spectral overlap, together with recognizable variation of their relative intensities, up to one million color versions can be generated and recognized. This property of light opens practically unlimited possibilities for simultaneous detection of different targets in homogeneous assays based on the principle of optical barcoding (Han et al. 2001), see Sect. 15.5. The principle here is to recognize the microscopic object in the same way as checking the parallel line bars of typical macroscopic barcoding. The fluorescence sensing technologies based on the application of differently optically coded fluorescent microbeads were developed (Jun et al. 2012), and they proved to be efficient in recognizing different disease biomarkers (Giri et al. 2011). Different optical materials (organic dyes, quantum dots, carbon dots, lanthanide nanocrystals) can be used for formation of such color patterns, and their selection allowed to decrease the size of recognizable emitters to nanometer level (Shikha et al. 2017). This technology has potential for the development of wireless barcode-based multiplexed diagnosis of infected patients with smartphones as detection devices (Ming et al. 2015), see Sect. 15.2. Optical encoding based on resolution of fluorescence lifetimes offers much smaller possibilities due to quite narrow variation of lifetimes of organic dyes and semiconductor quantum dots that occupy the range rarely exceeding 10 ns. This may be not so attractive in view of the problems of recovery of individual components in complex decay curves. There is a bright possibility, however, to explore the long-lifetime emission of rare earth metal ions (such as of terbium and europium) with their long luminescence lifetimes ranging from micro to milliseconds. Remarkably, they can transfer their long lifetime emission properties to other emitters serving as excitation energy acceptors (see Sect. 3.4). Realizing these possibilities, different technologies of multiplex detection were developed. Attractive is a suggestion for manipulating the luminescence emission color and decay lifetimes simultaneously (Zhou et al. 2018). With a special design of upconverting nanoparticles (Sect. 7) the encoding capacity can be increased by orders of magnitude in comparison with conventional color/intensity way. In fluorescence imaging, recognition of barcoded objects can be coupled with their localization in space (Shikha et al. 2017). There are many suggestions to introduce barcodes into multiple imaging in the living cells (Andreiuk et al. 2017; Maetzig et al. 2018). The near-IR range, in addition to visible spectral range, is actively used in view of relative transparency of human tissues in this range, and barcoding based on detection and analysis of multiple emission colors and their intensities as well as lifetime barcoding have started to be in active use (Ortgies et al. 2018).

1.4 Distinguishing Features of Fluorescence Technology

19

1.4.3 Broad Diversity of Molecular and Nanoscale Emitters Fluorescence (and in more general terms luminescence) response can be generated in different ways. Optical excitation is the method the most popular and easiest to produce. The light absorption of UV, visible or near-IR light is only needed for that. Light absorption can be provided in single-photon and multiphoton mode (see Sect. 16.1). In later cases the excitation energy can become double or even triple by adding the energies of absorbed photons. Regarding luminescent materials, their phosphorescence can proceed with dramatic increase of emission lifetimes. Electronic excitation to light emissive states can be also achieved by providing chemical and biochemical reactions and in redox processes at electrode generated by electric fields (Chap. 14). All these features offer a broad choice for selecting possibilities in designing the reporter units. Remarkable is the broad choice of photoluminescent materials that allow realizing different sensing technologies. They will be overviewed in short below. Organic fluorescent dyes were and remain at the forefront of fluorescence sensing. Their small size, chemical reactivity, high absorbance and commonly high fluorescence quantum yield make them often the first choice for labeling molecules and nanostructures and for their incorporation into different types of materials, including living cells and tissues. Their properties are discussed in Chap. 5, where their weak points will be also marked. They often demonstrate undesired reactivity in different conditions of their molecular environment, which also involves photobleaching (irreversible loss of optical properties). The strong advantage is the possibility of their participation in different photophysical transformations, such as electronic charge and proton transfers (see Chap. 4). In this function, they may serve as valuable reporters by changing strongly their emission color. When organic dyes are assembled into nanoscale aggregates, their properties may change dramatically (Chap. 8). These aggregates can be formed by dyes themselves or they can be included into different polymeric structures. These structures can be both amorphous and highly ordered. They can demonstrate both aggregationinduced enhancement and aggregation-induced quenching. They can also interact between themselves demonstrating excitonic effects that dramatically modify their light absorption and emission. Their ‘superradiance’ and ‘superquenching’ can be actively used in sensing technologies. Migration of excitation within dye aggregates allows achieving focusing the excitation energy on certain fluorophores (antenna effect) and displaying a number of excitation-dependent Red-Edge effects. The nanostructures formed of carbon atoms (carbon dots) and also graphene and graphene oxide dots open a new story in the study and application of fluorescence emitters (Chap. 9). Lanthanide atoms being chelated by organic molecules likewise the clusters of several atoms composed of gold, silver and copper can be luminescent and they already occupy strong positions in sensing technologies (Chap. 6). Lanthanides europium and terbium demonstrate the long-lasting luminescence, whereas the emission of noble atom clusters resembles the fluorescence of organic dyes.

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Of special value are the upconverting nanoparticles that are made of lanthanide ions. Being excited in the near-IR region by two photons, they demonstrate series of sharp emission bands in the visible range of spectrum. Such conditions of their excitation and emission make them unique in the family of luminescent materials (Sect. 7.5). When applied for sensing and imaging, they allow avoiding totally the background. The reader can imagine an innumerable number of possibilities for assembling these molecular and nanoscale emitters for achieving their favorable outcome. Their assembly into nanocomposites allows achieving novel highly desirable properties (Chap. 10). Moreover, they can become multimodal when they incorporate the units possessing magnetic, electron-dense or NMR-contrasting properties (Chap. 12). Such composite structures can perform their work both in liquid and solid environments. Even more, in biological systems fluorescent nanocomposites can be loaded with different drugs. In this role they can serve not only as the drug carriers but also the indicators of drug distribution in living bodies. The realization of these fantastic possibilities will be discussed in Volume 2. Thus, measuring only several parameters of fluorescence (luminescence) emission, such as fluorescence excitation and emission spectra, and also relative intensities, anisotropies (or polarizations) and lifetimes at particular wavelengths (see Chap. 3) but possessing extremely broad assortment of light emitting materials the researcher has to make optimal choice. However, in order to benefit from this range of possibilities and to make efficient sensors, a proper mechanism of transduction (Chap. 4) coupling with the target recognition unit should be also selected.

1.4.4 Possibilities for Enhancement of Reporting Signal The sensitivity and limit of detection of sensor systems as well as contrast in microscopic images depends strongly on the brightness of fluorescence reporting units. Operating with fluorescent dyes and nanoparticles, what are the means to increase it? Such possibilities exist (Fig. 1.9) and those of them that are of general importance are discussed below (Fig. 1.10). Primarily, the dye response in intensity depends on medium conditions that determine the fluorescence (luminescence) quantum yield characterizing the distribution of probability of relaxation to the ground state between emissive and nonemissive pathways. In a recommended classical review (De Silva et al. 1997), the reader may find a lot of information for organic dyes showing that their behavior is not so simple and predictable. The solvent may influence the n-π energy level inversion, intersystem crossing to phosphorescent state, ionization potentials. The effects of intermolecular and intramolecular hydrogen bonding may be very important. Water may play a special role in fluorescence quenching being a strong electron donor and acceptor. It may also quench dramatically the emission of lanthanide clusters and semiconductor nanocrystals. The ways of protection from solvent effects include

1.4 Distinguishing Features of Fluorescence Technology

Engineering medium condiƟons

21

Assembly into nanoparƟcles

Fluorescence enhancement Plasmonic enhancement

CatalyƟc amplificaƟon

Fig. 1.10 The possibilities for enhancing the sensitivity of fluorescence reporters

incorporation of organic dyes into inclusion complexes with cyclodetrins and cucurbiturils (Sayed and Pal 2016). Protecting shells were also devised for different types of nanoparticles. In general, aggregation of organic dyes and their inclusion into nanoparticles increase the probability of their emission, but only on a condition that the excitation energy transfer to nonfluorescent aggregated forms is excluded. The principles of formation of fluorescent aggregates will be discussed in Chap. 8. A special role here can be played by the so-called H- and J-aggregate forming regular structures and possessing excitonic properties. Operating with emission intensities in nanocomposites allows generating antenna effects and superquenching. A strong enhancement of fluorescence emission can be achieved near metallic surfaces that can be made either in the form of thin films generating propagating plasmons or nanoparticles with the properties of localized plasmons. Generating strong local electromagnetic fields, they enhance the radiative decay rates (Chap. 13). The strongest effect of plasmon-enhanced detection is observed for weakly emitting and even completely nonfluorescent species and it facilitates ultrasensitive singlemolecular target detection (Taylor and Zijlstra 2017).

1.4.5 Catalytic Enhancement in Sensors and Biosensors A catalytic sensing and biosensing deserves broader overview since it is a special fluorescence-based technology aiming at increasing the response sensitivity. Catalytic are the sensors, the mechanism of operation of which involves the catalytic event with the generation (or consumption) of fluorescent species (Goggins and Frost 2016; Scrimin and Prins 2011). Therefore, in a strict sense they cannot be considered as ‘sensors’. The generation of fluorophores, however, plays a secondary role

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1 Principles and Techniques in Chemical and Biological Sensing

Receptor site

Product

Catalytic site

Substrate NO REACTION

REACTION

Fig. 1.11 The principle of allosteric enzyme sensors. Specific binding of target at the allosteric binding site influences enzyme catalytic activity with the production of a chromogenic or fluorescent reaction product. Fluorescence of this product can serve as a measure of target binding

highlighting only the target recognition. Catalysis can be used for amplification of reporting signal produced by other sensors. Such fluorogenic substrate transformation being specific for particular catalyst (enzyme, catalytic antibody, ribozyme) can be due to the allosteric modulation of enzyme activity. The activity of many enzymes can be modulated by binding specified molecules to the sites that are different from a catalytic site. Such enzymes are called allosteric enzymes, the sites of binding are called allosteric sites, and the molecules producing modulation of activity are the allosteric effectors. These allosteric effectors can be determined as the targets. In this case, the rate of catalytic transformation of substrate with the appearance of fluorescent products can be used as a quantitative measure of target concentration. The binding of the effector modulates catalytic activity by coupling with a conformational change transmitted to the catalytic site (Villaverde 2003). Generating fluorescent product responds to a target binding (Fig. 1.11). This principle of catalytic amplification is not limited to enzyme reactions. It can be applied to organometallic and supramolecular catalysis (Zhu and Anslyn 2006).

1.4.6 Single-Analyte Sensors and Multi-analyte Arrays When developing the sensors, the researchers commonly try to achieve the highest receptor-target binding affinity. The sensor technologies that involve washing or separation steps (such as depicted in Fig. 1.3) require keeping the target in the bound form during these procedures. This imposes great difficulties in constructing the sensors

1.4 Distinguishing Features of Fluorescence Technology

ExcitaƟon

23

Emission

Scanning Fig. 1.12 Suspension array for multiplexed sensing. Array can be scanned in two dimensions to collect from every spot the fluorescence signal. With a broad range of selectivities and binding affinities it can be efficiently used for sensing single analyte in broad range of its concentrations and also of many analytes as a single recognition pattern

limiting them to targets of high affinity. Therefore the most successful applications of high-affinity single analyte sensing were demonstrated in the cases of serial targets that combined high affinity and selectivity. Such is the recognition of DNA sequences by hybridization of complementary chains and of proteins by monoclonal antibodies. The multi-analyte sensing technologies can be successfully applied to these cases. They are the spotted arrays (with the sensors immobilized on plate surface, Fig. 1.12) and suspension arrays (with the sensors being in solutions). These two versions of sensor technology will be discussed in detail in Sects. 15.4 and 15.5, correspondingly. The range of applications of sensor arrays can be also quite variable. The arrays can be composed of series of sensors, each of them may interact with the target weakly but together they provide unique characteristic pattern of binding affinities and this pattern has a recognition value (Lavigne and Anslyn 2001). Alternatively, the researcher (or the user) may not be interested in detecting a single analyte but wants to analyze complex mixtures, in which there are many components with subtle structural differences (discussed in Volume 2). Thus, characterizing the aroma of perfume or dangerous air pollution requires analysis instead of single analyte, some pattern of analytes (Li et al. 2018). The sensor arrays addressing these requirements are called the optoelectronic noses (Askim et al. 2016; Geng et al. 2019). The compositions of many compounds in liquids forming characteristic taste of vine, juice or milk product can be also characterized with sensor arrays simulating our perception (Han et al. 2017). They are called optoelectronic tongs (Chen et al. 2019). They allow extracting more versatile information about the studied systems without separation and characterization of components. It should be accounted that in any sensor design the molecular recognition cannot be ideal, even in the case of monoclonal antibodies targeting particular protein. Even more difficult is the determination of small molecular weight compounds in the presence of structurally similar analytes, since the cross-reactivity can be great. When applying the designed array, each receptor may bind to multiple analytes, but

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1 Principles and Techniques in Chemical and Biological Sensing

with variable affinity. The resulting matrix of data provides characteristic patterns for the individual analyte but also can characterize complex mixtures comprised of multiple analytes (Parastar and Kirsanov 2020). The sensor arrays with fluorescence detection are useful in many areas, from environment monitoring and control of industrial processes to clinical diagnostics. Different examples of sensor construction and of various applications can be found in different chapters of this book.

1.5 Trends for Future Development Fluorescence sensing and imaging is a subject of not only academic interest. The sensor and biosensor industry has grown exponentially over the past few decades, and it is now projected that in the next decade this sector will reach a multibillion economic value. Comparing the cheap paper strips that offer rapid answer to particular analyte, the fluorescence sensors is expected to provide higher information per cost efficiency (Wilson et al. 2019). The application areas of these sensors cover a broad spectrum of analytes including clinical diagnosis (laboratory based and pointof-care), environmental testing (soil, water, and air), detection of warfare agents, and industrial process monitoring. This is a great challenge for research and industry to offer valuable product to users. What will be the most essential tendencies in these developments? Users will need convenience and speed of operation together with friendly design. Requested are also facile and rapid information processing, transmission and storage. Smartphone-based technologies (see Sect. 12.2) will continue its evolution for mediating and translation of sensor response. The color vision together with image formation, the ability of processing the information and its wireless transmission conform to requests of fluorescence sensing. It is much easier to adapt the technology that is accepted by billions of users rather than start its development from the beginning. In the next Chapter the reader will become acquainted with several frequently explored assay formats. They include the targets labeling, the competitor displacement assays, the sandwich assays and, finally, the direct reagent-independent sensing. Here we make prognosis on the strong move from passive application of fluorescence emitters realized in a plain manner in assays, staining, and labeling that require manipulation with reagents and irreversible steps. The sensors will become active, compact and multi-use devices composed of direct fluorescence signaling molecular/supramolecular sensors that will be able for fully reversible target binding. Reagent addition and washing steps will not be required and the real-time tracking of signal changes will be always possible. Thus, the tendency is seen in transition for fluorescent molecules and nanoparticles from being just detectors of sensing events to true nano-size sensor devices. For that, every elementary sensor unit should combine the properties of receptor and emitting reporter. Parallel analyte detection in complex mixtures (multiplexed assays) (such as shown in Fig. 1.12) and processing the images results in high information content

1.5 Trends for Future Development

25

that will be analyzed within short time and at low cost. The further development of micro- and nanoarrays is required for (1) gradual change of affinity to capture broader range of analyte concentrations, (2) to capture a greater number of targets simultaneously and (3) to provide recognition pattern and quantitative assay for important low-affinity target. All that will be done by forming a single recognition pattern. Will the fluorescence sensing substitute human senses? They have started to do that, and in some areas they already demonstrate better performance (Wong and Khor 2019). Regarding the imaging technologies, one of the most efficient applications in the studies of living cells is expected in drug discovery and in establishing the mechanisms of their action. The elementary acts of distribution of administered drug in the body, its entry into the cell and targeting particular organelles, the binding particular receptors will be visualized in most details. The organelle and whole-body tomography critically depends on the novel reporting dyes absorbing and emitting light in the near-IR range 700–1000 nm that is invisible to human eye but possessing relatively high transparency in tissues of living body. This is the requirement for the development of fluorescence tomography. The problem here is not in light sources or detectors but in smart sensing fluorescent dyes (see Sect. 5.4). It has all prospects to be resolved. One cannot predict in details the long-run future developments of sensor technologies. But what is sure, they are rapidly becoming a part of everyday life.

1.6 Sensing and Thinking. High Time for New Endeavor The reader must be very much surprised by the fact that in such important field as chemical sensing and biosensing there is no single leading idea and dominant technology. A variety of technologies develop in parallel, and every one of them has its advocates and critics. In this respect, fluorescence sensing develops in a very stimulating and productive environment. Its positions are very strong but not dominant, and in many application areas, the competition with other technologies is very tough. Answering the questions listed below will help the reader to move from passive reading to thinking, analysis and creativity. 1. 2.

3.

Explain the principle of molecular recognition and how it is realized in sensing technologies. Provide your choice between analyte detection using the probe based on chemical reactivity and the sensor based on molecular recognition in the cases: (a) the analyte is a small compound containing SH-group and (b) it is a protein with a number of such groups exposed on the surface. List all possible application areas of molecular sensors that you know. Explain what advantages appear on sensor immobilization to the surface compared to an assay in solution and how this expands or limits their application areas.

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4.

Explain why the potentially most general approach based on label-free and immobilization-free principles has a rather narrow range of applications in practice. In what sense and in which conditions the techniques based on radioactive labeling (isotope tracing) and fluorescent labeling are analogous? What are the requirements for the fluorescence reporters in such similar applications? What imaging technologies, except fluorescence, do you know? Are they able to sense particular targets in addition to providing the images? Compare fluorescence with other methods that you know of structural and dynamic analysis of materials in terms of sensitivity, space and time resolution, other important features. Evaluate comparatively the label-based methods using fluorescence detection. What is better to label, the receptor, the target together with the whole pool of potential targets, the capture antibody or the competitor? What range of applications is expected on application of any of these possibilities? What are the application areas of indirect and direct sensors? Which are broader? In the cases if both techniques are applicable, what is preferable based on such criteria as complexity, time consumption and cost? Fluorescence detection occurs through transparent space but it may use wires (optical fibers). What is their advantage over electrical wires? Why the sensing could be coupled with enzyme reactions? What is the difference in application between sensing based on an enzyme reaction and a ligandbinding (inhibitor-binding) allosteric sensing with the same or similar enzyme? How sensor arrays operate? Evaluate the range of their applications. The absence of high sensor selectivity, is that always bad? How to use this property?

5.

6. 7.

8.

9.

10. 11.

12. 13.

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Battiston FM, Ramseyer JP, Lang HP, Baller MK, Gerber C et al (2001) A chemical sensor based on a microfabricated cantilever array with simultaneous resonance-frequency and bending readout. Sensors Actuators B-Chem 77:122–131 Bezner BJ, Ryan LS, Lippert AR (2020) Reaction-based luminescent probes for reactive sulfur, oxygen, and nitrogen species: analytical techniques and recent progress. Anal Chem 92:309–326 Bishop KJ, Wilmer CE, Soh S, Grzybowski BA (2009) Nanoscale forces and their uses in selfassembly. Small 5:1600–1630 Bollella P, Gorton L (2018) Enzyme based amperometric biosensors. Curr Opin Electrochem 10:157–173 Bruemmer KJ, Crossley SW, Chang CJ (2019) Activity-based sensing: a synthetic methods approach for selective molecular imaging and beyond. Angewandte Chemie Int Edn 59:13734–13762 Carrascosa LG, Moreno M, Alvarez M, Lechuga LM (2006) Nanomechanical biosensors: a new sensing tool. Trac-Trends Anal Chem 25:196–206 Chen Z-H, Fan Q-X, Han X-Y, Shi G, Zhang M (2019) Design of smart chemical ‘tongue’ sensor arrays for pattern-recognition-based biochemical sensing applications. TrAC Trends Anal Chem 115794 Cooper MA (2003) Label-free screening of bio-molecular interactions. Anal Bioanal Chem 377:834–842 De Silva AP, Gunaratne HQN, Gunnaugsson T, Huxley AJM, McRoy CP et al (1997) Signaling recognition events with fluorescent sensors and switches. Chem Rev 97:1515–1566 Demchenko AP (ed) (2010a) Advanced fluorescence reporters in chemistry and biology I: Fundamentals and molecular design, vol 8, Springer series on fluorescence, Springer, Heidelberg Demchenko AP (ed) (2010b) Advanced fluorescence reporters in chemistry and biology II: Molecular constructions, polymers and nanoparticles. vol 9, Springer Series on Fluorescence, Springer, Heidelberg Demchenko AP (ed) (2011) Advanced fluorescence reporters in chemistry and biology III: Applications in sensing and imaging. Springer Series on Fluorescence, Springer, Heidelberg Enander K, Choulier L, Olsson AL, Yushchenko DA, Kanmert D et al (2008) A peptide-based, ratiometric biosensor construct for direct fluorescence detection of a protein analyte. Bioconjug Chem 19:1864–1870 Eteshola E, Balberg M (2004) Microfluidic ELISA: on-chip fluorescence imaging. Biomed Microdevice 6:7–9 Fraden J (2010) Handbook of modern sensors. Springer Gauglitz G (2005) Direct optical sensors: principles and selected applications. Anal Bioanal Chem 381:141–155 Gauglitz G (2018) Analytical evaluation of sensor measurements. Anal Bioanal Chem 410:5–13 Geng Y, Peveler WJ, Rotello VM (2019) Array-based “chemical nose” sensing in diagnostics and drug discovery. Angew Chem Int Ed 58:5190–5200 Giri S, Sykes EA, Jennings TL, Chan WC (2011) Rapid screening of genetic biomarkers of infectious agents using quantum dot barcodes. ACS Nano 5:1580–1587 Goggins S, Frost CG (2016) Approaches towards molecular amplification for sensing. Analyst 141:3157–3218 Gooding JJ, Gaus K (2016) Single-molecule sensors: challenges and opportunities for quantitative analysis. Angew Chem Int Ed 55:11354–11366 Han M, Gao X, Su JZ, Nie S (2001) Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules. Nat Biotechnol 19:631–635 Han J, Ma C, Wang B, Bender M, Bojanowski M et al (2017) A hypothesis-free sensor array discriminates whiskies for brand, age, and taste. Chem 2:817–824 Homola J (2003) Present and future of surface plasmon resonance biosensors. Anal Bioanal Chem 377:528–539 Homola J, Vaisocherova H, Dostalek J, Piliarik M (2005) Multi-analyte surface plasmon resonance biosensing. Methods 37:26–36 Jameson DM (2014) Introduction to fluorescence. CRC Press

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Jun B-H, Kang H, Lee Y-S, Jeong DH (2012) Fluorescence-based multiplex protein detection using optically encoded microbeads. Molecules 17:2474–2490 Kellner R, Mermet J-M, Otto M, Valcarcei M, Widmer HM (2004) Analytical chemistry. WileyVCH, New York, p 1181 Ladbury JE, Chowdhry BZ (1996) Sensing the heat: the application of isothermal titration calorimetry to thermodynamic studies of biomolecular interactions. Chem Biol 3:791–801 Lakowicz JR (2007) Principles of fluorescence spectroscopy. Springer, New York Lange K, Rapp BE, Rapp M (2008) Surface acoustic wave biosensors: a review. Anal Bioanal Chem 391:1509–1519 Lavigne JJ, Anslyn EV (2001) Sensing a paradigm shift in the field of molecular recognition: from selective to differential receptors. Angew Chem Int Ed 40:3118–3130 Lee SH, Jung S-Y, Kwon JK (2015) Modulation and coding for dimmable visible light communication. IEEE Commun Mag 53:136–143 Li Z, Askim JR, Suslick KS (2018) The optoelectronic nose: colorimetric and fluorometric sensor arrays. Chem Rev 119:231–292 Liu YS, Ugaz VM, Rogers WJ, Mannan MS, Saraf SR (2005) Development of an advanced nanocalorimetry system for material characterization. J Loss Prev Process Ind 18:139–144 Luppa PB, Sokoll LJ, Chan DW (2001) Immunosensors—principles and applications to clinical chemistry. Clin Chim Acta 314:1–26 Maetzig T, Morgan M, Schambach A (2018) Fluorescent genetic barcoding for cellular multiplex analyses. Exp Hematol 67:10–17 Mammen M, Choi S-K, Whitesides GM (1998) Polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors. Angew Chem Int Ed 37:2754–2794 Meyer T, Knapp EW (2014) Database of protein complexes with multivalent binding ability: Bivalbind. Proteins Struct Funct Bioinf 82:744–751 Ming K, Kim J, Biondi MJ, Syed A, Chen K et al (2015) Integrated quantum dot barcode smartphone optical device for wireless multiplexed diagnosis of infected patients. ACS Nano 9:3060–3074 Nguyen HC, Park H, Shin H-J, Nguyen NM, Nguyen ATV et al (2019) Fluorescent immunosorbent assay for Chikungunya virus detection. Intervirology 62:145–155 Ortgies DH, Tan M, Ximendes EC, Del Rosal B, Hu J et al (2018) Lifetime-encoded infraredemitting nanoparticles for in vivo multiplexed imaging. ACS Nano 12:4362–4368 Palecek E, Fojta M, Jelen F (2002) New approaches in the development of DNA sensors: hybridization and electrochemical detection of DNA and RNA at two different surfaces. Bioelectrochemistry 56:85–90 Palecek E (2005) Electroactivity of proteins and its possibilities in biomedicine and proteomics. (Ch. 19). In Palecek E, Scheller F, Wang, J (eds), Electrochemistry of nucleic acids and proteins. Towards electrochemical sensors for genomics and proteomics (pp 690–750). Amsterdam: Elsevier Palecek E, Jelen F (2005) Electrochemistry of nucleic acids In Electrochemistry of nucleic acids and proteins. In: Palecek E, Scheller F, Wang J (eds), Towards electrochemical sensors for genomics and proteomics (pp 74–174). Amsterdam: Elsevier Pang W, Zhao H, Kim ES, Zhang H, Yu H, Hu X (2012) Piezoelectric microelectromechanical resonant sensors for chemical and biological detection. Lab Chip 12:29–44 Parastar H, Kirsanov D (2020) Analytical figures of merit for multisensor arrays. ACS Sensors 5:580–587 Perumal V, Hashim U (2014) Advances in biosensors: Principle, architecture and applications. J Appl Biomed 12:1–15 Rich RL, Myszka DG (2006) Survey of the year 2005 commercial optical biosensor literature. J Mol Recognit 19:478–534 Sayed M, Pal H (2016) Supramolecularly assisted modulations in chromophoric properties and their possible applications: an overview. J Mater Chem C 4:2685–2706 Scrimin P, Prins LJ (2011) Sensing through signal amplification. Chem Soc Rev 40:4488–4505

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Chapter 2

Overview of Strategies in Fluorescence Sensing

The first step to any successful fluorescence sensing technology is the proper choice of optimal strategy. In this Chapter we focus on different strategies for obtaining the quantitative information on sensor-target interaction. There are different possibilities for that, and the technologies that are currently in use are critically analyzed. They include (a) the labeling of all pool of potential targets as it is commonly used in DNA hybridization assays, (b) competitor displacement assays that can be applied to a broader range of sensor-target affinities, (c) sandwich assays, the most developed of which are the immunoassays and (d) direct reagent-independent sensing. The latter is the most advanced strategy that still is difficult to realize. Finally, the reader will find the Section “Sensing and thinking” with the list of questions and problems addressed to the readers. The specific interaction between the sensor and its target can proceed in solutions and also when one of the partners is immobilized on solid support. The problem is to visualize this interaction in the most convenient and efficient way. In previous Chapter we introduced the basic concepts of sensing, the background of which is the molecular recognition and which can be realized in heterogeneous and homogeneous formats. A general sensor design was presented, the functional elements of which are the receptor, transducer and emitter. Among different technologies of molecular sensing that use different physical principles of detection we outlined the fluorescence technology and highlighted the broad range of its possibilities. Here we focus on different strategies for extracting the information on sensor-target interaction and on the possibilities for its transformation into quantitative target assay.

2.1 Labeling Targets in Fluorescence Assays Historically, fluorescence sensing came into play to substitute the radioactive isotope detection technique and to a large extent borrowed its methodology. Radioisotopes are insensitive to intermolecular interactions and all the methods based on their © Springer Nature Switzerland AG 2020 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-030-60155-3_2

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usage allow only the quantitative detection of their concentration in the desired locations. Their substitution by fluorescent dyes made the analysis cheaper, safer and much more sensitive. Many fluorescence technologies were developed following this line. Here the fluorescence emission of the dyes indicates the presence of a given target compound in the analyzed system and provides quantitative measure of this compound. Such dyes are in fact a part of the target system but not of the sensor system. In this technology, the whole pool of potential targets has to be labeled (Fig. 2.1). Then, after reacting with the receptor, the receptor-target complexes should be isolated from the pool. If the receptor is immobilized on the surface, this can be easily done by the procedure of washing-out the unreacted target analogs. However, in the case when the reaction occurs in solutions, the procedure should be different. There should be a chromatographic or electrophoretic separation of a receptor-target complex (Fig. 2.2), and only after that the analysis has to be made. Several examples of application of this concept are given below.

Fig. 2.1 Illustration of the principle behind the initial labeling the whole pool of potential targets present in the sample. The sensors are represented by only the unlabeled receptors (R). They bind the targets (T) and immobilize them on the surface leaving all labeled interfering species in solution (depicted as orange balls). The latter are removed on subsequent steps. This allows the quantitative analysis of bound labeled targets

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Fig. 2.2 Schematic illustration of procedures involved in sensing requiring the labeling of the whole pool of potential targets. In the case of immobilized receptors, the numerous unbound labeled interfering species are removed by washing, and in the case of assay in solution, a separation of sensor-target complexes and unbound components is required

2.1.1 Arrays for DNA Hybridization Presently the most popular methods of detecting specific DNA sequences are based on labeling the whole pool of these molecules in a studied system and on ‘hookingout’ of target sequence by hybridizing them with unlabeled sensor sequence. Nucleic acid hybridization is a particular kind of affinity interaction resulting in formation of a double-strand association complex connected by bonding within base pairs (Freeman et al. 2000), as described in more detail in Volume 2, Chap. 11. The target and sensor sequences should be complementary and form specific hydrogen bonds between two distinct base pairs, which are G-C and A-T in DNAs (or G-C and A-U in RNAs). Commonly, the solid-state hybridization is used, in which the sensing nucleic acid strands tethered to a solid support (‘probe’ strands) bind the strands of single-chain DNA or RNA molecules (‘target’ strands). For identifying specific recognition between complementary sequences, the whole pool of poly- or oligonucleotides that may contain the ‘target’ strands is labeled with a fluorescent dye before application to the sensor-containing plate. On the next step, the supportive plate is washed to remove unbound material and is applied to a fluorescence reader. If comparative studies are needed, the two nucleic acid pools can be labeled using different fluorescent dyes (usually a red-emitting cyanine dye Cy5 and a green-emitting dye Cy3), and applied to the same plate after mixing. DNA or RNA microarrays (sometimes called microchips) are the results of miniaturization of this technology (Shoemaker and Linsley 2002; Venkatasubbarao 2004). Using hybridization on microarrays, thousands of solid-phase hybridization reactions can be performed simultaneously to determine gene expression patterns or to identify genotypes. This technique is the basis of a variety of important applications,

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Fig. 2.3 DNA microarrays using target labeling (Khodakov et al. 2016). The RNA or cDNA to be profiled is the fluorophore-labeled using a terminal transferase enzyme. The labeled targets are then hybridized to the microarray that is surface functionalized with different probe sequences at different positions. Fluorescent intensity at a particular position indicates the concentration of its corresponding labeled target

from finding peculiarities of gene expression in disease, such as cancer, to analysis of its changes in ontogenesis and aging, detecting harmful microbes, etc. A version of DNA microarray technology for profiling RNA expression is schematically presented in Fig. 2.3 (Khodakov et al. 2016).

2.1.2 Labeling in Protein-Protein and Protein-Nucleic Acid Interactions Detection of protein targets based on microarray or any other high-throughput technology is highly needed for research and various applications. Very important is the development of ‘protein function’ arrays, which should consist of thousands of native proteins immobilized in a defined pattern and used for massive parallel testing of protein functions (Kodadek 2001, 2002). Such techniques are highly needed in proteomics research and also to satisfy the practical needs of pharmacology. The other type of arrays developed as ‘protein detection’ devices with arrayed protein-binding agents that are strongly needed for proteomics. Examples of their application could include cytokines, growth factors, apoptosis-related and other biological systemsrelated arrays. They must allow detecting from dozens up to several thousands of analytes. It has to be recognized that compared to labeling the whole pool of DNA or RNA molecules, the fluorescence labeling of a protein pool is much more difficult and much less efficient. One may claim, that covalent labeling at amino groups (N-terminal and that belonging to Lys residues) is a very common procedure in protein chemistry. But the fact is that every protein in the pool will bind the dye at different sites and in different quantities, and this property is determined by its three-dimensional structure. Since the charged Lys residues are commonly exposed to the surface of protein molecules, their modification may hamper the binding to the receptor. This does not allow proper calibration for quantitative measurements.

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Similar approach to that described above is used in antibody micro-array techniques (micro-array immunosensors). In a number of currently used assays, immobilized unlabeled antibodies assembled into micro-arrays are used for testing protein antigens. In order to record selective binding, all potential antigens in the sample have to be preliminarily labeled with fluorescent dye (MacBeath and Schreiber 2000). Thus, this approach shows the same disadvantages as the DNA and RNA hybridization assays with possibly greater errors. They can arise from nonspecific binding of analyte antigens and non-analytes present in the sample. Thus, labeling of protein pools has to be avoided; other methods described in this book that allow omitting this procedure could be more appropriate for protein assays.

2.1.3 Advantages and Limitations of the Approach Based on Pool Labeling Pool labeling is a simple procedure that can usually be performed in one step. Extracting and analyzing the labeled target is based on well-elaborated procedures. Nevertheless, this approach has a number of disadvantages and limitations. Some of them are technical and some derive from the fact that in such systems it is hard to achieve a true thermodynamic equilibrium between bound and unbound species that is required for quantitative analysis. This is because of the following. 1. The correct quantitative measurement of target concentration is possible only when all target molecules (or particles) present in the system are bound to the sensor. Therefore, the measurements cannot be made in the studied system directly but only in aliquots taken from it. In order to achieve complete target binding, the concentration of receptors should be higher than that of the targets. 2. The procedure requires a step of labeling with the dye, an incubation step for target binding and the washing step to remove unbound target analogs that contain the fluorescent label. These steps are time-consuming. Therefore, this method is not applicable for continuous monitoring of the target concentration. 3. The method can be applied only to strong binders, i.e. to the cases in which the sensor-target complex can remain intact under the washing procedure. Therefore, the targets with relatively low affinities (dissociation constant K d 10−5 to 10−7 M), which are of the order of magnitude to that in enzyme-substrate and enzymeinhibitor complexes, cannot be analyzed reliably using this approach. 4. The label itself may influence the target recognition. There should be an independent proof that there is no modification of target affinity by the attached fluorescent label. It is not surprising therefore, that this approach is attractive only when the tested compounds are of the same chemical nature and their labeling can be achieved quantitatively using the same general procedure. Fortunately, this is the case of many DNA and RNA hybridization assays.

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The major advantages of this approach are its simplicity and the absence of limitation imposed on reporter dyes. This approach is also not limited to sensing molecules. It can be extended to sensing large particles and even living cells. The label can be introduced to their pool, and specific targets can be analyzed after a separation step using electrophoresis, chromatography or flow cytometry.

2.2 Sandwich Assays The sandwich as a component of everyday food is known to everyone. It has three layers: bread at the bottom, then ham, cheese or butter in the middle and, again, bread on the top. The same is the composition of sandwich-type fluorescence assay unit, in which three layers are formed on target binding (Fig. 2.4). In a sandwich configuration the receptor molecules are at the bottom. Being attached to solid support, they form a layer. In immunosensor techniques the receptors are antibodies that recognize large-size antigens. They can be also the antigens that recognize antibodies. The target molecules, when bound to receptor molecules are in the middle of such sandwich. Both sensors and targets are not labeled, so their complex formation does not produce any response signal. Therefore, a third component is needed for detecting the sensor-target interaction, which we will call the indicator. Its function is to visualize the formed complex with the aid of attached

Fig. 2.4 Illustration of basic idea behind sandwich assays with fluorescence detection. After the target (T) binding to immobilized receptor (R) and forming their complex on the surface, an indicator (I) that interacts with the target is introduced. The indicator is labeled, which provides labeling of the immobilized complex. Numerous contaminating species (depicted as orange balls) do not interact with the labeled indicator

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label, so it should be applied on the top. In immunoassays it is the antibody that is specially prepared for recognizing the immobilized target at a different binding site than the receptor. In the case if the receptor-target complex is formed, the indicator has to label this complex and if not—to be removed by washing.

2.2.1 Sensing the Antigens and Antibodies This type of analysis is important primarily in clinical practice. Viruses and bacteria are the antigens, as well as different foreign materials that can get inside the human body or to which it can contact. Antibodies are the molecules forming the response to antigens. Their detection is also very important. Clinical diagnostics, response of individual to treatment, prognosis of disease and other issues can be resolved with this analysis. In both tasks of sensing the antigens or antibodies the sandwich approaches are commonly used (Gopinath et al. 2014). This is because of strong binding between antigens and antibodies (with K d on the level of picomoles or nanomoles) there is often the possibility to substitute the real antigen (e.g. microbe) by a molecule of a rather small size (e.g. peptide or glycolipide) that retains the antibody-binding properties. The structural elements of even smaller size, called antigenic determinants (epitops), are recognized by antibodies. The assay configurations for detection of antigens and antibodies are schematically presented in Fig. 2.5. The secondary antibody serving as the indicator (the capture antibody) can be modified in different ways for providing the most sensitive response. Because of that, the mechanism of indicator operation is rather general, in line with that depicted in Fig. 2.4. In immunoassays detecting the antibodies, it is simply a different antibody reacting with the target antibody. This prevents many problems. The function of indicator is universal, since it can display broad range of site-sensitivities. There is also no need in reporting function of the label. The label serves only for measuring the amount of bound indicator (Marquette and Blum 2006).

2.2.2 Labeling with Catalytic Amplification High sensitivity of detection system is a very desirable factor that in many cases determines the strategy of sensor development. Coupling the response function with catalytic or biocatalytic transformation of reporter signal is one of the earliest ideas in the field of sensing, and it is still very actively explored. In this way, a single analyte binding event can be coupled with the generation of multiple reporting molecules resulting in substantial signal amplification (Makhlynets and Korendovych 2014). Most of these applications are related to sandwich (e.g. ELISA) techniques, where a second binder (an indicator) should carry the label, and it is the brightness of this label that determines the overall sensitivity (Fig. 2.6). Here the idea of connecting the sensing and catalysis is simple. An indicator is chemically coupled to an enzyme

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a

Indicator Target Receptor

b Indicator

Target

Receptor

Fig. 2.5 Schematic representation of assay formats for sensing the antigens and the antibodies. a Detection of antigens. Antibodies serving as receptors (R) are immobilized on a solid support. Each antibody (usually, bivalent) captures its respective target (T) (called antigen) from a complex mixture. Detection occurs by recognizing the bound antigen by a second antibody serving as indicator (I) that contains optical label or enzyme producing optically detectable product. b Detection of antibodies. Antigen is the receptor (R) that is immobilized on a solid support. Antibodies specific to this antigen are the targets (T) captured from a complex mixture (e.g. blood serum). Detection occurs by recognizing the bound antibodies by other antibodies serving as indicators (I). They react with the target antibodies as with their own antigens. The latter antibodies can be labelled for providing an optical signal as in the case (a). The present interfering species are not immobilized. They are removed before the analysis

that, upon addition of substrate, generates colored (measured by light absorption) or fluorescent product at the site of binding. Then, if an excess of reaction substrate is provided, the sensitivity will depend on incubation time needed to generate the detectable product. Schematically this situation is depicted in Fig. 2.6. A popular version of this assay that uses the generation of colored product in enzyme reaction is called the enzyme-linked immunosorbent assay (ELISA). Here the indicator antibody can be fused to an enzyme, which, on addition of substrate,

2.2 Sandwich Assays

R

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I

E

Fig. 2.6 The scheme illustrating operation of an assay using the labeled indicator with catalytic amplification. Primarily the sensor exposing the recognition unit (R) captures the target (T) that on the next step is recognized by an indicator (I). The indicator is fused to the enzyme (E). When the ternary R–T–I complex is formed, then it can be detected by the transformation of nonfluorescent substrate (S) into fluorescent product (P) catalyzed by the enzyme. Numerous interfering species in the reaction media cannot produce this effect

produces the colored (light-absorbing) reaction product during the final incubation step (Mendoza et al. 1999). Upon incubation, each such molecular complex can generate many colored molecules. Intensity of their color can be analyzed as a function of concentration of the bound target. Application of fluorogenic (producing fluorescence) substrates allows increasing substantially the precision and sensitivity (Meng et al. 2005; Nguyen et al. 2019). Some enzyme-labeled fluorescence kits use phosphatase substrate (Zhao et al. 2019) that has weak blue fluorescence in solution but upon enzymatic cleavage forms a bright and highly photostable yellow-green fluorescent precipitate. The sites of binding of antibody-phosphatase complex can be detected by fluorescent precipitate deposited at the site of enzymatic activity. A very high sensitivity of these assays can be achieved, which ranges from picomoles to nanomoles in target concentrations. This is quite sufficient to satisfy many demands in clinical diagnostics. In many cases, however, the labeling without catalytic amplification can be quite sufficient for achieving this level of sensitivity, especially when the ultra-bright particles such as dye-doped nanoparticles (Sects. 8.3–8.4) or quantum dots (Sect. 7.2) are used for labeling. The effect of amplification appears due to multiple substrate transformations at a single site. In contrast, in common affinity-based sensors the sensitivity is limited by the 1:1 binding stoichiometry between the target and the sensor, which allows generation of response signal only in one reporter molecule for binding one target molecule. Therefore, ELISA and its fluorescence analog as the techniques that use catalytic amplification are superior in sensitivity to most biosensors with single emitters, whatever is the transduction principle. Catalyst and substrate are selected here for providing the most efficient response. Meantime, it is essential to note that the response relies on the correct assembly of the whole complex (R) − (T) − (I) = (E), which limits this methodology to heterogeneous sensor format.

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Fig. 2.7 Schematic representation of a sandwich DNA assay based on indicator DNA conjugated with dyes or fluorescent nanoparticles. The immobilized receptor DNA and labeled indicator DNA use for hybridization different segments of target DNA. Numerous interfering DNA species do not bind neither with receptor nor with indicator DNA

2.2.3 Ultrasensitive DNA Hybridization Assays The sandwich assay format can be applied for the detection of all types of analytes that contain two distinct molecular recognition sites, one for binding to the receptor and the other for attaching the indicator. The DNA hybridization assays are remarkable in this respect (Fig. 2.7). The recognition and detection of the single-chain DNA molecule can be provided in such a way that one part of it interacts with the receptor DNA forming a double helix. The remaining free part can interact with the indicator DNA that can carry fluorescent label. Here the most specific interaction with the formation of double helix can be realized too. The brightest (in all aspects) application of this principle can be found in the work with brightly fluorescent dye-doped silica nanoparticles. Their application allows detecting the DNA target molecules down to subpicomolar levels (Zhao et al. 2003).

2.2.4 Advantages and Limitations of the Approach The successful application of sandwich assays depends mostly on the possibility of recognizing in the target of two independent binding sites (Shen et al. 2014). This is because in this technology the target has to bind independently the receptor and the indicator. This limits the assay to rather large molecules (e.g. proteins) or to supramolecular structures and cells, in which the binding to two different sites can be independent. Many small target molecules do not possess multiple structurally separate sites for binding and therefore cannot be detected by sandwich assays. Since the indicator can be coupled with enzyme generating a light-absorbing or fluorescent product, the signal amplification can be achieved leading to a very high

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sensitivity of the assay. However, in all the cases the response is not immediate and not direct, because an assay in the presence of unbound indicator is not possible and its separation is always needed. The sandwich assay technique is well adapted to antibodies serving either as the receptors or as the targets. Regarding applications on a more general scale, their range is very limited. When used for the detection of single-strand DNA, the two sites of hybridization have to be used. It can be noted that in a variety of bioassays, particularly in ELISA with fluorescence detection, the amplified output signal is achieved by linking (Meng et al. 2005) or by activation of an enzyme (Villaverde 2003) or ribozyme (Navani and Li 2006) producing chromogenic or fluorescent product. Therefore, being dependent on reagent addition and lengthy incubation times, these methods are limited in applications. New fluorophores with dramatically increased brightness have strong potential of substituting the enzyme amplification methods in detection techniques. In addition, this costly and time-consuming technique is subjective to non-specific absorption effects that increase with the number of used components, especially of proteins that can denature and provide nonspecific binding.

2.3 Competitor Displacement Assays Many scientists are trying to develop technologies that could avoid the introduction of fluorescence label into analyzed samples, and the competitor displacement assay is one of these possibilities (Nguyen and Anslyn 2006; Wu et al. 2013). In this technology, neither receptor nor target may contain a fluorescence label. The presence of indicator is also not needed. Instead, an additional player in the target recognition mechanism is introduced, the fluorescent competitor (Fig. 2.8). Usually a competitor is the labeled target analog that binds to the same site on the receptor as the target. After this binding, a mixture of different compounds that contains the target is applied. Due to its specific interaction (molecular recognition) with the receptor, the target displaces the competitor from the binding site. The target can be analyzed quantitatively because it is the only species that can displace the competitor. The competitor reports about its displacement by changing the parameters of its fluorescence emission. These changes can be calibrated as a function of target concentration. There can be other configurations in competitor displacement assays. For instance, the competitor can be unlabeled and immobilized on the surface. It binds the sensor molecules added to solution that contains the fluorescence reporting units. When the sample that contains the target to be analyzed is applied, the target interacts with the sensor inducing its dissociation from the surface thus disrupting its interaction with competitor. A reporter signal is generated when the sensor changes from immobilized to target-bound form in solution.

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c R

R

R

R

Fig. 2.8 Illustration of the principle behind the competitor displacement assay. The response to target (T) binding is obtained when the labeled competitor (C), competing for the binding site on the receptor (R), is displaced from this binding site to the solution demonstrating a change in parameters of its fluorescence. Contaminating interfering species that may be present are depicted as orange balls

2.3.1 Unlabeled Sensor and Labeled Competitor in Homogeneous Assay Although the competitor displacement assays can be performed with immobilized receptors, this method allows homogeneous assays in solutions. In this case, there is no need to separate the receptor-target complexes from the target-free receptor molecules. It is essential that in this configuration the competitor does not interact directly with the target. It interacts with the receptor only via the reversible noncovalent binding. Upon release from the complex, the competitor exhibits a change of its interactions, so that its complex with the receptor dissociates in favor to interactions with solvent molecules (Wiskur et al. 2001). The fluorescence reporter signal in this case can be obtained as the change of different fluorescence parameters, such as intensity, polarization and lifetime (see Chap. 3). Figure 3.9 illustrates the application of anisotropy detection to this assay. In these cases the target is not involved in the transduction mechanism; it just substitutes the reporting competitor. The competition assay is the most convenient technique when the target is of relatively low molecular mass and if this compound or its analog can be coupled with a fluorescent dye to produce a labeled competitor. Then the competition for binding to a limited number of receptors can be exploited between unlabeled target molecules and their labeled analogs. In the presence of fixed amount of labeled competitor, the mole fraction of target bound to the receptor varies inversely with the total target concentration. In this competitive format, it is necessary that some

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fluorescence signal should change between the two states of the competitor, its free and bound forms. As a general feature, this method requires keeping the competitor concentration on a strictly defined level; it lacks adaptability to continuous monitoring of target concentration (Piatek et al. 2004). The details of concentration dependences of fluorescence response in the case of competition and other formats can be found in Volume 2, Chap. 2. This method has gained high popularity in detecting small molecules that can be recognized in complexes with cucurbit[7]urils, cyclodextrins, calixarenes and other macrocyclic hosts (Nguyen and Anslyn 2006; Zhou et al. 2018). Being displaced from the complex to polar environment, the fluorescent competitor commonly demonstrates the quenching of its emission. Dissociating from these complexes, the competitor may also enhance its emission due to disruption of the bond with the sensor causing the electron transfer quenching. Many proteins can bind fluorescent dyes at their ligand-binding sites. When the ligand replaces the dye, large changes in the dye fluorescence may be observed. Based on these observations, different rather simple procedures for the determination of particular ligands can be developed. It is known, for example, that avidin and biotin forming very strong and highly specific complex can be easily quantified using the fluorescence probe 2,6-ANS (2-anilinonaphthalene-6-sulfonic acid) (Mock et al. 1988). Binding of 2,6-ANS produces dramatic increase in avidin fluorescence, and biotin, when present in the system, completely displaces it with an easily recordable fluorescence quenching. Serum albumin has a function of binding and transporting many ligands in the blood, including different drugs. It can also bind some fluorescent dyes with high affinity at the same sites where it binds the drugs (Ercelen et al. 2005). Therefore, displacement of fluorescent ligands by the target ligands may provide a simple and efficient estimate of their concentrations in unknown media. Moreover, the tests for screening different ligands for their albumin-binding affinities can be established. Such tests, being used for the determination of lifetimes of toxic compounds and drug in the blood circulation, may be used in toxicology and drug discovery (Poór et al. 2013; Zhang et al. 2015). A displacement assay using fluorescent DNA intercalator dyes may serve as another example (Tse and Boger 2004). It can be applied for different compounds for establishing the DNA binding affinity and sequence selectivity. Intercalator dyes are the dyes such as ethidium bromide or thiasole orange (see Sect. 5.2) that can insert spontaneously between nucleic acid bases. These dyes are almost non-fluorescent in their free form in aqueous buffer but attain strong fluorescence only in the DNA bound form. Their binding occurs only when the DNA has a double-stranded helical structure. Intercalation between nucleic acid bases screens them from the quenching in contacts with water resulting in high-intensity emission. Application of target DNA binding compounds that destroy the double helix results in intercalator release and quenching of its fluorescence. Such possibility of generating the reporter signal indicates the fact that there is no absolute necessity for a competitor to be replaced by the target from the same binding site. The only requirement is that the sensor-target

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interaction should destroy the biding site for the competitor, which can be done in different ways.

2.3.2 Labeling of Both Receptor and Competitor There is an alternative possibility for implementing the competitor displacement assays, which finds a broad range of applications. The receptor can be additionally labeled for providing long-range interaction with the competitor that will allow excitation energy transfer (EET) measurements. In this case (see Sect. 4.3), the EET effect can be observed only in a competitor complex with a labeled receptor. When the unlabeled target replaces the competitor, the latter diffuses into the medium and the optical effect generated by the energy transfer is lost. This event can report on the act of target binding. An interesting realization of this idea is a fluorescent immunoassay, in which the competitor is designed by conjugation of a target compound to a fluorescently labeled protein. Bovine serum albumin can be used for that, and the whole complex can serve as a competitor in the determination of target binding to an antibody (Schobel et al. 1999). If the antibody is labeled with EET donor and albumin by EET acceptor, an enhancement of fluorescence emission of the donor can be observed at competitor displacement. This response can be analyzed in terms of target concentration. In such assays, the labeled albumin can be successfully substituted by quantum dots (see Sect. 7.2) or by other brightly fluorescent nanoparticles. This approach can be illustrated by the operation of competitive glucose biosensor, the design of which was reported by many authors. Glucose-binding proteins concanavalin A (Ballerstadt et al. 2004) or apo-glucose oxidase (Chinnayelka and McShane 2004) form stable complexes with labeled dextran, so that EET occurs between the two labels. Glucose displaces dextran in the complex, and this effect dependent on glucose concentration can be used for its determination. In DNA hybridization assays, the following procedure can be applied. The sensor sequence labeled with a fluorescent dye is first hybridized with a competitor sequence labeled with an EET acceptor (quencher). Hybridization with target DNA displaces the quencher and induces enhancement of fluorescence (Shoemaker and Linsley 2002). Another example comes from the sensing using the antibodies. In the sensor targeted at determining the concentration of the explosive 2,4,6-trinitrotoluene (TNT) in aqueous environments, an antibody fragment was bound to a quantum dot, which served as a EET donor. Trinitrotoluene analog was labeled with a dye that served as an acceptor (Goldman et al. 2003). In this case, the binding of the target TNT displaces this analog and induces enhancement of its fluorescence emission. Interesting examples of application of competitor displacement assays can be found with aptamer sensors that are the binders selected from large oligonucleotide libraries (Volume 2, Chap. 5). Fluorescently labeled DNA aptamer that detects oligonucleotide sequences can form a complex with short oligonucleotide

2.3 Competitor Displacement Assays

45

that contains a quencher. Dissociation of this complex that occurs on substitution by target on its binding brings the fluorescence enhancement (Nutiu and Li 2004). Similar strategy is used to study the ligand-RNA interactions (Zhang et al. 2010).

2.3.3 Advantages and Limitations of the Approach In principle, the approach based on competitor displacement is advantageous over techniques requiring labeling of a whole pool containing the required target. However, in practice it is difficult to implement it in many particular cases and even more difficult to make it generic. The affinity of receptor to target should be high enough to replace the competitor, but its affinity to competitor should be also high in order to withstand chromatographic separation (if the assay is performed in solution) or washing the plates (if the sensor is immobilized). Therefore, it is often not reasonable to use the attached dye only as a label, and in line with anisotropy detection this methodology develops in a direction requiring the application of responsive dyes (see Sect. 5.3). Some authors distinguish between the competition and displacement assays. In displacement assays the analyte should possess a high affinity to the receptor and be totally bound substituting the labeled competitor that may be of lower affinity but present in saturating concentrations. This condition allows operating with multianalyte sensor arrays and reading fluorescence after washing steps. In contrast, in competition assay both the target analyte and competitor may be of lower affinity, but an equilibrium condition should be established in their binding.

2.4 Direct Reagent-Independent Sensing It is known that different parameters of fluorescence emission can be modulated in broad ranges by weak intermolecular interactions of the dyes. It must be a good idea to use this property in a straightforward and most efficient way by making the sensor with direct response to sensor-target interactions (Altschuh et al. 2006; Arugula and Simonian 2014; Banala et al. 2013). In the case of success, we may get simple and universal means to provide fluorescence sensing without any limitations on the assay format.

2.4.1 The Principle of Direct ‘Mix-and-Read’ Sensing On the pathway to realization of the idea of direct sensing, the special requirements should be imposed on the properties of fluorescent dyes. Instead of being simple labels or tags they have to attain all the features of molecular instruments reporting

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on particular changes in van der Waals interactions, hydrogen bonds and/or electrostatic forces. These changes should generate the reporting signal. In many cases, sophisticated transduction mechanisms have to be developed to obtain the signal on elementary act of sensor-target binding and transform it into the signal from fluorescence reporter (see Chap. 4). Simple and very efficient may be the application of dyes responding to intermolecular interactions by wavelength shifting or switching the intensity between two bands. Let us specify, what we wish to achieve. First, these new instruments should be molecular sensors. This means that their operation (both sensing and response) has to take place at a molecular level. If necessary, they can be integrated into supramolecular and macroscopic devices, although their basic operation should not require that. But it should be accounted that the transduction between recognition and reporting is easier if the corresponding elements are located closer on molecular scale. Possessing such functions, they can be used for assays either in homogeneous phase (in solutions) or on their attachment to solid support. Secondly, they should be the direct sensors. This means that they should avoid any intermediate signal-transduction steps or additional manipulations in developing the sensing response. In addition, they should be reagent-independent (sometimes called reagentless) sensors, so that their operation should not require the supply and consumption of additional reagents. Incorporation into sensor molecules of a fluorescent dye that changes its emission properties during the recognition event is the most promising approach for detecting the sensor-target binding (molecular recognition). Also, there are many possibilities for operation with different kind of nanoparticles that may serve as nano-sized supports, as reporters or as modulators and amplifiers of the output signal. The illustration of the basic concept of direct sensing when applied to heterogeneous format is presented in Fig. 2.9. The fact that no additional factors are required for the operation of direct sensors is very important. The device made of such sensors can operate in a broad range of external conditions and in any heterogeneous environment that could be a waveguide surface, a microplate or the interior of a living cell. Moreover, since this technique does not require any separation or washing steps and allows distinction between bound and unbound analyte in thermodynamic equilibrium, it may serve in a very broad range of target affinities.

2.4.2 Contact and Remote Sensors In the case of direct sensing, the fluorescent reporter is an integral part of the sensor (Fig. 2.10), and its function is to provide the change of fluorescence parameters on analyte binding. This function can be introduced by covalent labeling of the recognition unit by the dye to form a molecular sensing device. Meantime, as is seen in Sect. 1.1.1, this straightforward and simple construction with efficient response function is often difficult to achieve, and there is no general methodology for that.

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Fig. 2.9 The principle of operation of direct sensor. Here the fluorophore labeling the receptor (R) is the integral part of the sensor. Its response is coupled directly with the recognition of target (T) in the medium that may contain a magnitude of non-target species

In order to provide the transduction of the sensing event directly into fluorescence response, one has to choose between several possibilities. Some of them are depicted in Fig. 2.10. (a) Contact sensing. Here the fluorescent reporter is involved in direct contact with the target and is a part of the recognition process. As a result of reporter— target interactions, one or more of its fluorescence parameters change. They can be scaled in target concentration. Since the reporter has to be present in sensor-target contact area, there is always a danger that it may influence the recognition of the target by producing steric and energetic barriers. Therefore, the compromise should be found between providing its strong involvement in target binding with the production of strong fluorescence response (that may also hamper the recognition) and a weaker involvement of the dye in binding that could be sometimes insufficient for providing detectable response in fluorescence. The direct contact between the target and fluorescence reporter can also be beneficial when it allows modulating the excited-state reaction occurring in the dye: isomerizations and electron, charge or energy transfers (see Chap. 4). The principle of contact sensing has found optimal realization in the design of small chemical sensors detecting the ions (de Silva et al. 1997; Valeur 2002). It has also found application in sensing with peptides and proteins. Particularly, the antibody sensors were designed, in which the environment-sensitive dye is attached close to antigen binding site (Enander et al. 2008; Gopinath et al. 2014; Renard et al. 2002); the knowledge of three-dimensional structure of antibody

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a

b

c

Fig. 2.10 Different possibilities for direct sensing of target (T) by the sensor (S) labeled with fluorescence reporter. a In contact sensing the reporter is involved in direct contact with the bound target. b In remote (allosteric) sensing the reporter responds to conformational change in the sensor molecule occurring on target binding, and its direct contact with the target is not needed. c Anisotropy sensing, in which the rotational mobility of the sensor decreases on the target binding (due to increased mass of a complex), and this change is reflected as an increase of fluorescence anisotropy

allowed suggesting this site (see Fig. 1.2). This is an example of structure-based ‘rational design’ approach. Meantime, the approaches using peptide or nucleic acid libraries, in which the dye is included into their covalent structures, may appear extremely efficient due to possibility of selection based on optimization of both sensing and reporting functions. (b) ‘Remote’ sensing. In the case of remote (e.g. allosteric) sensing, the reporting dye is located distantly from the site of analyte recognition. To provide the response, the sensor molecule should exhibit some conformational change reflected by reporter dye located at the periphery, away from the binding site. The possibilities of realizing this principle are limited to those sensor molecules, in which relatively strong global conformational changes accompany the recognition event. Nevertheless, if these changes do occur, they allow many possibilities for selecting the dye and optimizing its positioning in the sensor structure. The changes of protein conformation on binding various ligands, including protein-protein and protein—nucleic acid interactions, are very common (Gerstein et al. 1994), up to the complete unfolding-folding (Demchenko 2001). This allows realizing many possibilities in sensor design. Recent applications of

2.4 Direct Reagent-Independent Sensing

49

this principle include rearrangement of domains in sensing proteins and disruption of beacons, as in oligonucleotide sensors. Sensing with ligand-binding proteins (Dwyer and Hellinga 2004; Sapsford et al. 2006) often follows the principle of remote fluorescence detection. The sensing with molecular beacons and other polymers and biomolecules exhibiting conformational change deserves discussion in more detail (Sect. 4.4). (c) A general property of the sensor should change on target binding, such as rotational mobility that can be observed as the change of emission anisotropy (Sect. 3.3). It allows providing target determination in homogeneous formats (Sect. 1.1) without separation steps. Based on this concept, the immunoassays (Shapiro 2016) and detections of small molecules using sensing aptamers (Perrier et al. 2018) have become very popular in research and clinics (Hall et al. 2016; Minagawa et al. 2019; Zhang et al. 2019). The mechanisms coupling the binding event with signal transduction for the generation of response in emission will be discussed in different sections of this book. We will observe that the level of structural integration between the receptor and the reporter functional elements can be very different. This produces the basis for the development of many simple direct ‘mix-and-read’ methods.

2.4.3 Advantages and Limitations of the Approach A very attractive feature of direct sensors is the possibility of using nearly all possible biopolymer and mimetic systems, in which molecular recognition can be realized or simulated. This makes the background for the development of a wide range of molecular biosensor technologies, which include the use of antibodies and their fragments, ligand-binding proteins (natural and raised by selection using several scaffolds), enzymes and their chemically modified products, synthetic peptides and natural or synthetic polynucleotides. A wide variety of targets can be also analyzed, from small ions to supramolecular structures and living cells. Still the sensitivity of direct sensors is not as high as of indirect sensors and immunosensors that operate on the basis of multi-step coupled reactions and that use signal amplification enhancing the level of targets (such as PCR in detection of specific nucleic acids), or the response on sensor-target binding (such as ELISA in detection of antigens and antibodies). Different methods for the dramatic increase of sensitivity in reporter response have been suggested recently (see Sect. 1.4), and now we observe that the concept of a direct reagent-free sensing offers new fascinating possibilities for the development of sensing systems. The strong advantage of direct sensors is the fact that they explore potentially simple one-step binding reaction with the response on a time scale of measurement. This allows continuous monitoring the analyte concentration (Galbán et al. 2012). Since this reaction allows immobilization and integration of sensor molecules into arrays, all the range of molecular, nanoscale, microscopic and macroscopic devices

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can be adapted to this concept. Multiple advantages make direct sensors progressive and prospective for future applications. Another major advantage of direct sensors must be their versatility, which is out of competition. Direct sensors do not need the plates or any other solid supports but can use them to achieve spatial resolution for providing simultaneous response to many targets. In direct sensing, the reaction of target binding can be performed with the sensor molecules already in dilute solutions, and they can also be incorporated into nanoparticles, membrane vesicles, living cells, etc. No less important is the advantage of high speed of analysis that in this case is limited only by target diffusion in the medium (Brune et al. 1994) and by the rate of conformational changes in the sensor (or target) molecule if they occur on target binding to provide fluorescence response. This allows monitoring the process of interest on the fastest possible time scale. With direct sensors, the range of quantitatively determined target concentrations can be extremely broad. This is the result of rapid establishment of dynamic equilibrium between free and bound targets in accordance to mass action law (see Chap. 2 of Volume 2). Only the bound analyte generates the fluorescence response, which allows easy determination of its total concentration in the volume of any size. Such sensor can be adapted for particular range of target concentrations by variation of the binding constant. Moreover, since direct sensors allow measuring the target binding at equilibrium, they, in principle, can be used many times without regeneration and may allow continuous monitoring. Finally, let us together with an interested reader provide critical comparison between two strategies in sensing that develop in parallel—competitor displacement and direct sensing. We find a common feature: either the competitor or reporter should possess the response function. In the first case this function has to be realized by recording the difference between reporting signals coming from receptor-bound form of (often) target-like competitor and its free form. In contrast, in direct sensing the reporting signal can be similar but it should be generated by the receptor. Because of simplicity, an idea to use the direct sensor format is preferable. However, it is not easily realizable in practice. Possibilities for its realization will be discussed in Chap. 4.

2.5 Sensing and Thinking. The Cost of Brightness and Amplification in Sensor Response Different sensing formats are in development and realization in practice and the reader has to evaluate their advantages and disadvantages when applied to particular targets and particular conditions with the account of different situations of sensor use. Answering the questions listed below will help the reader to move from passive reading to thinking, analysis and creativity.

2.5 Sensing and Thinking. The Cost of Brightness and Amplification …

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1.

Compare the background mechanisms of operation of sensors described above. Which of them allows (a) continuous monitoring of industrial process, (b) miniaturization down to single-molecular level, (c) development into high-throughput micro(nano)array format? 2. How does the DNA hybridization occur? How to use this process for detecting particular DNA sequence? What is the role of fluorescent labels in these assays and how in this case the fluorescence response can be technically realized? 3. Describe the problems that appear in labeling of the protein targets. Why it is better to avoid the labeling of a protein pool? Why this approach is not applicable to low-affinity targets? 4. Why in sandwich assays the third partner in target recognition, the indicator (e.g. capture antibody), is needed? What is gained and what is lost in its application? 5. Explain in detail the principle of operation of ELISA. How the reporting signal is generated and how the effect of amplification is achieved? Why this assay is so good with antibodies but is less practical with other binders? 6. What is the role of fluorescence reporter in competitor displacement assay? Suggest examples for proper applications of these assays. Can the sensor operate with immobilized competitor? How such sensors operate in the case of DNA intercalating dyes? What can be additionally achieved by labeling both sensor and competitor? 7. Think about an application of competitor displacement assay in complex situations, such as the case of two or more types of binding sites with different affinities. 8. The dyes can respond to a target binding directly, by the change of parameters of their fluorescence. Where they should be located in this case? What properties of the sensor should correspond to different locations of the reporting dye? 9. Find the common features in the mechanisms of operation between competitor displacement and direct sensing. Indicate the differences in their realization in view of presence of third component, the competitor. 10. Select one protein target (let it be insulin) and explain, how you will apply all sensor strategies described above. Provide their comparative analysis in terms of speed, complexity (the number of needed manipulations), skills needed by the performers, etc.

References Altschuh D, Oncul S, Demchenko AP (2006) Fluorescence sensing of intermolecular interactions and development of direct molecular biosensors. J Mol Recognit 19:459–477 Arugula MA, Simonian A (2014) Novel trends in affinity biosensors: current challenges and perspectives. Meas Sci Technol 25:032001 Ballerstadt R, Polak A, Beuhler A, Frye J (2004) In vitro long-term performance study of a nearinfrared fluorescence affinity sensor for glucose monitoring. Biosens Bioelectron 19:905–914 Banala S, Arts R, Aper SJ, Merkx M (2013) No washing, less waiting: engineering biomolecular reporters for single-step antibody detection in solution. Org Biomol Chem 11:7642–7649

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Brune M, Hunter JL, Corrie JET, Webb MR (1994) Direct, real-time measurement of rapid inorganic phosphate release using a novel fluorescent probe and its application to actomyosin subfragment 1 ATPase. Biochemistry 33:8262–8271 Chinnayelka S, McShane MJ (2004) Resonance energy transfer nanobiosensors based on affinity binding between apo-enzyme and its substrate. Biomacromol 5:1657–1661 de Silva AP, Gunaratne HQN, Gunnaugsson T, Huxley AJM, McRoy CP et al (1997) Signaling recognition events with fluorescent sensors and switches. Chem Rev 97:1515–1566 Demchenko AP (2001) Recognition between flexible protein molecules: induced and assisted folding. J Mol Recognit 14:42–61 Dwyer MA, Hellinga HW (2004) Periplasmic binding proteins: a versatile superfamily for protein engineering. Curr Opin Struct Biol 14:495–504 Enander K, Choulier L, Olsson AL, Yushchenko DA, Kanmert D et al (2008) A peptide-based, ratiometric biosensor construct for direct fluorescence detection of a protein analyte. Bioconjug Chem 19:1864–1870 Ercelen S, Klymchenko AS, Mely Y, Demchenko AP (2005) The binding of novel two-color fluorescence probe FA to serum albumins of different species. Int J Biol Macromol 35:231–242 Freeman WM, Robertson DJ, Vrana KE (2000) Fundamentals of DNA hybridization arrays for gene expression analysis. Biotechniques 29:1042–1055 Galbán J, Sanz-Vicente I, Ortega E, del Barrio M, de Marcos S (2012) Reagentless fluorescent biosensors based on proteins for continuous monitoring systems. Anal Bioanal Chem 402:3039– 3054 Gerstein M, Lesk AM, Chothia C (1994) Structural mechanisms for domain movements in proteins. Biochemistry 33:6739–6749 Goldman ER, Anderson GP, Lebedev N, Lingerfelt BM, Winter PT et al (2003) Analysis of aqueous 2,4,6-trinitrotoluene (TNT) using a fluorescent displacement immunoassay. Anal Bioanal Chem 375:471–475 Gopinath SC, Tang T-H, Citartan M, Chen Y, Lakshmipriya T (2014) Current aspects in immunosensors. Biosens Bioelectron 57:292–302 Hall MD, Yasgar A, Peryea T, Braisted JC, Jadhav A et al (2016) Fluorescence polarization assays in high-throughput screening and drug discovery: a review. Methods and applications in fluorescence 4:022001 Khodakov D, Wang C, Zhang DY (2016) Diagnostics based on nucleic acid sequence variant profiling: PCR, hybridization, and NGS approaches. Adv Drug Deliv Rev 105:3–19 Kodadek T (2001) Protein microarrays: prospects and problems. Chem Biol 8:105–115 Kodadek T (2002) Development of protein-detecting microarrays and related devices. Trends Biochem Sci 27:295–300 MacBeath G, Schreiber SL (2000) Printing proteins as microarrays for high-throughput function determination. Science 289:1760–1763 Makhlynets OV, Korendovych IV (2014) Design of catalytically amplified sensors for small molecules. Biomolecules 4:402–418 Marquette CA, Blum LJ (2006) State of the art and recent advances in immunoanalytical systems. Biosens Bioelectron 21:1424–1433 Mendoza LG, McQuary P, Mongan A, Gangadharan R, Brignac S, Eggers M (1999) Highthroughput microarray-based enzyme-linked immunosorbent assay (ELISA). Biotechniques 27:778–788 Meng Y, High K, Antonello J, Washabaugh MW, Zhao QJ (2005) Enhanced sensitivity and precision in an enzyme-linked immunosorbent assay with fluorogenic substrates compared with commonly used chromogenic substrates. Anal Biochem 345:227–236 Minagawa H, Shimizu A, Kataoka Y, Kuwahara M, Kato S et al (2019) Fluorescence polarizationbased rapid detection system for salivary biomarkers using modified DNA aptamers containing base-appended bases. Anal Chem 92:1780–1787

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Mock DM, Lankford G, Horowitz P (1988) A study of the interaction of avidin with 2Anilinonaphthalene-6-sulfonic acid (2,6 Ans) as a probe of the biotin binding-site. Clin Res 36:A895–A95 Navani NK, Li YF (2006) Nucleic acid aptamers and enzymes as sensors. Curr Opin Chem Biol 10:272–281 Nguyen BT, Anslyn EV (2006) Indicator–displacement assays. Coord Chem Rev 250:3118–3127 Nguyen HC, Park H, Shin H-J, Nguyen NM, Nguyen ATV et al (2019) Fluorescent immunosorbent assay for Chikungunya virus detection. Intervirology 62:145–155 Nutiu R, Li YF (2004) Structure-switching signaling aptamers: transducing molecular recognition into fluorescence signaling. Chem-a Eur J 10:1868–1876 Perrier S, Guieu Vr, Chovelon B, Ravelet C, Peyrin E (2018) Panoply of fluorescence polarization/anisotropy signaling mechanisms for functional nucleic acid-based sensing platforms. Anal Chem 90:4236–4248 Piatek AM, Bomble YJ, Wiskur SL, Anslyn EV (2004) Threshold detection using indicatordisplacement assays: an application in the analysis of malate in Pinot Noir grapes. J Am Chem Soc 126:6072–6077 Poór M, Kunsági-Máté S, Czibulya Z, Li Y, Peles-Lemli B et al (2013) Fluorescence spectroscopic investigation of competitive interactions between ochratoxin A and 13 drug molecules for binding to human serum albumin. Luminescence 28:726–733 Renard M, Belkadi L, Hugo N, England P, Altschuh D, Bedouelle H (2002) Knowledge-based design of reagentless fluorescent biosensors from recombinant antibodies. J Mol Biol 318:429–442 Sapsford KE, Pons T, Medintz IL, Mattoussi H (2006) Biosensing with luminescent semiconductor quantum dots. Sensors 6:925–953 Schobel U, Egelhaaf HJ, Brecht A, Oelkrug D, Gauglitz G (1999) New-donor-acceptor pair for fluorescent immunoassays by energy transfer. Bioconjug Chem 10:1107–1114 Shapiro AB (2016) Investigation of β-lactam antibacterial drugs, β-lactamases, and penicillinbinding proteins with fluorescence polarization and anisotropy: a review. Methods Appl Fluoresc 4:024002 Shen J, Li Y, Gu H, Xia F, Zuo X (2014) Recent development of sandwich assay based on the nanobiotechnologies for proteins, nucleic acids, small molecules, and ions. Chem Rev 114:7631– 7677 Shoemaker DD, Linsley PS (2002) Recent developments in DNA microarrays. Curr Opin Microbiol 5:334–337 Tse WC, Boger DL (2004) A fluorescent intercalator displacement assay for establishing DNA binding selectivity and affinity. Acc Chem Res 37:61–69 Valeur B (2002) Molecular fluorescence. Wiley VCH, Weinheim Venkatasubbarao S (2004) Microarrays—status and prospects. Trends Biotechnol 22:630–637 Villaverde A (2003) Allosteric enzymes as biosensors for molecular diagnosis. FEBS Lett 554:169– 172 Wiskur SL, Ait-Haddou H, Lavigne JJ, Anslyn EV (2001) Teaching old indicators new tricks. Acc Chem Res 34:963–972 Wu P, Xu C, Hou X (2013) Exploration of displacement reaction/sorption strategies in spectrometric analysis. Appl Spectrosc Rev 48:629–653 Zhang H, Wu Q, Berezin MY (2015) Fluorescence anisotropy (polarization): from drug screening to precision medicine. Expert Opin Drug Discov 10:1145–1161 Zhang H, Yang S, De Ruyck K, Beloglazova N, Eremin SA et al (2019) Fluorescence polarization assays for chemical contaminants in food and environmental analyses. TrAC Trends Anal Chem 114:293–313 Zhang J, Umemoto S, Nakatani K (2010) Fluorescent indicator displacement assay for ligand—RNA interactions. J Am Chem Soc 132:3660–3661 Zhao D, Li J, Peng C, Zhu S, Sun J, Yang X (2019) Fluorescence immunoassay based on the alkaline phosphatase triggered in situ fluorogenic reaction of o-phenylenediamine and ascorbic acid. Anal Chem 91:2978–2984

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Zhao X, Tapec-Dytioco R, Tan W (2003) Ultrasensitive DNA detection using highly fluorescent bioconjugated nanoparticles. J Am Chem Soc 125:11474–11475 Zhou Y, Gao L, Tong X, Li Q, Fei Y et al (2018) Supramolecularly multicolor DNA decoding using an indicator competition assay. Anal Chem 90:13183–13187

Chapter 3

Fluorescence Detection in Sensor Technologies

In this Chapter we concentrate on the methods of fluorescence observation. Starting from introducing the definitions and explaining fundamentals in fluorescence spectroscopy, the discussion proceeds to different methods of fluorescence that are valuable for sensing technologies. We discuss intensity-based sensing with and without intrinsic reference and the methods that do not need such reference and require the measurements of anisotropy and lifetime. The methods based on excimer and exciplex formation and Excitation Energy Transfer (EET) involve interactions in the system of fluorescence reporters. In contrast, the sensing response based on wavelength-shifting and two-band wavelength-ratiometry relies on modulation of weak intermolecular interactions of a single fluorophore with its environment. The final section “Sensing and thinking” discusses the optimal choice of fluorescence detection techniques with the list of questions and problems addressed to the reader. Fluorescence is one of very interesting and important phenomena that are observed on interactions of light and matter. Different photophysical processes can be involved in these interactions, and fluorescence emission is one of them (Fig. 3.1). Light can be reflected from the sample surface; it can pass through it exhibiting refraction and also be absorbed within the sample demonstrating decreased transmission due to specific absorption or light scattering. These processes changing the light intensity do not change the energies of transmitted light quanta and therefore there is no change in their recording on wavelength scale. In contrast, luminescence, and fluorescence in particular, is observed with the change in wavelength. The other its specific features are the propagation in all directions, the delay between light absorption and emission and characteristic behavior in polarization of emission. They appear because on absorption of light quantum the molecule appears in the excited state and stays there during definite lifetime. Light emission always competes with non-emissive processes resulting in quenching that influences all emission parameters. During the lifetime of the excited state, different processes can proceed, such as molecular rotations and electron, proton and energy transfer reactions. These processes are the sources of valuable information used in fluorescence sensor technologies. © Springer Nature Switzerland AG 2020 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-030-60155-3_3

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3 Fluorescence Detection in Sensor Technologies ReflecƟon

Sample

RefracƟon

Transmission ScaƩering

Detector

AbsorpƟon

Light source Monochromator

Emission

Detector Fig. 3.1 Different phenomena observed on interaction of light and matter

3.1 Fluorescence Fundamentals Fluorescence is the phenomenon of emission of light quanta by a molecule or supramolecular structure (fluorophore) after initial electronic excitation in a lightabsorption process. After excitation by absorbing the light quanta, a molecule resides for some time in the so-called excited state and its fluorescence emission can be observed usually with a lower energy (longer wavelength) than the excitation. Such change in fluorescence emission color with respect to the color of incident light was first observed by Irish scientist George Stokes and is called the Stokes shift. The time scale of fluorescence emission depends on both the fluorophore and its interactions with local environment. Thus, for organic dyes the fluorescence lifetime is in picosecond (ps) to nanosecond (ns) time range, typically 10−8 –10−11 s. Fluorescence is a part of a more general phenomenon, luminescence. The latter includes the emission of species excited in the course of chemical reactions (chemiluminescence), biochemical reactions (bioluminescence) or upon oxidation/reduction at electrode (electrochemiluminescence). Important for sensing is also the emission with long lifetime from triplet state (phosphorescence). Duration of these types of luminescence can be much longer than the fluorescence. For semiconductor nanocrystals it can be tens of nanoseconds (ns); for organometallic compounds and lanthanide complexes—hundreds of nanoseconds, up to milliseconds (ms).

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3.1.1 The Light Emission Phenomenon The absorption and emission of light quanta (photons) involves electronic transitions between quantized energy levels. This means that only the quanta of wellspecified energies can be absorbed and emitted. The distribution of such electronic transitions probabilities on their energies forms the light absorption and emission spectra. Longer wavelengths correspond to lower energies. Our interest in fluorescence sensing and imaging technologies is in the usage of visible range of spectrum, from 400 nm (violet-blue color) to 760 nm (red- near-IR edge) extending to longer wavelengths that are not visible by human eye. The absorption of light quanta involves the transition from the ground singlet state to the excited states. This process occurs so rapidly (in femtoseconds) that electronic sub-system changes its energy without the change in atom nuclei configuration. Then different processes of relaxation in the excited states start to proceed, including vibrational relaxation (transition to the lowest in energy vibrational state) and internal conversion (transition to the lowest excited singlet state with the loss of energy). Then the molecule or nanoparticle has a chance to return to the ground state emitting photon in the form of fluorescence or without emission. A transition from singlet to triplet state (in which the energy is split into triplet seen in paramagnetic resonance studies) is also possible. This process is called intersystem crossing, and from this state the phosphorescence emission is observed. Since the latter process involves low probable spin inversion, it proceeds on much slower time scale than the fluorescence. The proper selection of excitation and emission wavelengths is important for fluorescence sensing. Since the fluorescence spectrum is shifted with respect to excitation spectrum to longer wavelengths (the Stokes shift) (Fig. 3.2), the highest fluorescence intensity will be observed if the excitation is provided and emission recorded at the wavelengths of the correspondent band maxima. The complicating factor is the elastic light scattering that occurs at the excitation wavelength and the spectral profile of which corresponds to the profile of incident light. The light-scattering complications are especially strong if the studied sample contains macromolecules or particles, the size of which is larger than the wavelength of light. Therefore, excitation and emission wavelengths should always be separated and the dyes with large Stokes shift are preferred. The Raman scattering bands present in any solvent are shifted with respect to the main scattering band and overlap the fluorescence spectrum. They may produce complications in fluorescence measurements if the emission intensity is very low. In practical sense, the excitation and emission wavelengths should be selected to optimally match the radiation wavelengths provided by light source (e.g. if it is a laser) and the optimal sensitivity range of the detector. The Kasha’s rule states that the excess energy got in absorption and resulting in excitation to higher electronic and vibrational excited states is dissipated on a very fast scale before the emission (internal conversion), so that the emission occurs from the lowest excited state (Fig. 3.3). Practically this means that we cannot achieve the generation of new fluorescence bands at shorter wavelengths if we shift the

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Excita on

Fig. 3.2 Typical absorption and fluorescence emission spectra. Fluorescence spectrum is located at longer wavelengths with respect to the absorption spectrum. The area between absorption and emission spectrum is shadowed. The distance between their band maxima is called the Stokes shift that is usually expressed as a change in energy on a wavenumber scale ν, and ν (cm−1 ) = 107 /λ (nm). Fluorescence excitation spectrum usually matches the absorption spectrum, and excitation wavelength is usually selected close to this band maximum. The major light-scattering band is observed at the wavelength of excitation. The Raman scattering band is shifted by a value of energy of vibrations, it is seen only if the fluorescence intensity is low

Wavelength

S2 Energy

Fig. 3.3 Illustration of the Kasha’s rule. The position of fluorescence emission spectrum remains unchanged whether excitation is provided at the absorption band maximum, its high-energy slope or separate band corresponding to the singlet S2 state located at higher energies. Due to ultrafast internal conversion the fluorescence proceeds from the lowest in energy S1 excited state to S0 ground state

S1 S0

Internal conversion Fluorescence emission

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excitation to the second short-wavelength absorption band. The same rule operates also for phosphorescence, which is the emission from triplet state. For in-depth understanding of light absorption and emission phenomena and of the laws that govern these processes the reader may address the excellent textbooks on photochemistry (Birks 1970; Turro et al. 2009) and the books with particular focus on fluorescence (Lakowicz 2006; Valeur and Berberan-Santos 2012). Here we stress characteristic features of luminescence (and, particularly, of fluorescence) that are explored actively in fluorescence probing, sensing and imaging. – It is the wavelength-shifted emission, which allows separating optically the absorbed (and scattered) from emitted light quanta. – It is the emission delayed in time. This allows excluding or substantially reducing not only the light scattering but also the short-living background emission. – Many fluorophores can be excited selectively by polarized light and, depending on the conditions, emit light of variable polarization.

3.1.2 Fluorescence Parameters Used in Sensing Several parameters of fluorescence emission can be recorded and all of them can be used in sensing (Fig. 3.4). Intensity F can be measured at given wavelengths of excitation and emission (usually, band maxima). Its dependence on emission wavelength, F (λem ), gives the fluorescence emission spectrum. If this intensity is measured over the range of excitation wavelengths, one can get the fluorescence excitation spectrum F (λex ). Emission anisotropy, r (or the similar parameter, polarization, P) is the function of fluorescence intensities obtained at two different polarizations, vertical and horizontal. Finally, emission can be characterized by the fluorescence lifetime τF , which is the inverse function of the rate constant of the excited state emissive depopulation, k r . All of these parameters can be determined as a function of excitation and emission wavelengths. They can be used for reporting on sensor-target interactions, so that a variety of possibilities exist for their employment in sensor constructs. A combination of these parameters in recording fluorescence is possible. The intensity, polarization and lifetime are usually measured with resolution in wavelength. Time-resolved (Suhling et al. 2005) and polarization-selective (Jameson and Croney 2003) measurements allow increasing efficiency in analytical applications by suppressing the background signal and increasing selectivity of response. Anisotropy with time resolution can characterize the fast rotational motions of fluorophores (Clayton et al. 2002). Meantime, as we will see in other chapters of this book, the assembly of molecules and nanoparticles into nanocomposites that allows their interaction and collective photophysical events must put some corrections in interpretation of “classical” results. For instance, drop in anisotropy can occur not only because of suppression of rotations, but also due to homo-EET that may occur without detectable spectroscopic changes. Only the observation of Red-Edge effects (Demchenko 2002) allows differentiating between these two phenomena.

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Fig. 3.4 Fluorescence parameters used in sensing. The simplest is the fluorescence intensity F measured at particular excitation (λex ) and emission (λem ) wavelengths. Dependence F (λex ) gives the excitation spectrum and F (λem ) the emission spectrum (wavelength domain). The rate of emission decay gives its inverse function—the fluorescence lifetime (time domain). Recording fluorescence emission at two orthogonal (perpendicular) polarizations gives emission anisotropy (anisotropy domain). Different combinations of these parameters can be realized in fluorescence techniques

Specific fluorophore design allows observation of multiple emission bands that can be due to designed combination of non-interacting fluorophore but also due to the excited-state reactions within single fluorophores or between them. This allows applying λ-ratiometry, i.e. collecting the ratio of intensities at two or more wavelengths (Demchenko 2010). This approach improves dramatically the quantitative analysis of fluorescence signal (Demchenko 2005c) and can be combined with other fluorescence detection methods. Thus, the output signal is always the number of emitted light quanta that can be integrated and presented as fluorescence intensity. When the intensity is recorded as a function of time, its decay can be characterized by the fluorescence lifetime and gives rise to lifetime-based sensing. Excitation with linearly polarized light also results in polarized emission (optical anisotropy). Any rotational motion or transfer of energy to another fluorophore decreases the polarization. When the light intensity is recorded with spectral resolution, the resulting fluorescence emission spectra carry the information on the fluorophore interactions and reactivity in the excited state (that may lead to the generation of new bands), which, in turn, opens a new channel of information in sensing. In the following sections of this Chapter we will discuss different possibilities in using these parameters of fluorescence emission and the corresponding detection methods for design of operational fluorescence sensors.

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3.1.3 Parameters Characterizing the Properties of Fluorescence Emitters In view of tremendous diversity of structures, chemical reactivities and spectroscopic properties, one needs certain practically useful criteria for the dye selection for the purpose of sensing and imaging. They can be formulated in the form of spectroscopic parameters that have to be optimized. 1. High molar absorbance, ε. It is the parameter describing the ability of molecule to absorb light at a particular wavelength. The absorbance E measured at any wavelength on spectrophotometer is proportional to the dye concentration c (in mol/l) and the light path length in a sample l (in cm). In this relation, ε plays the role of coefficient of proportionality, so that E = εcl. The value of molar absorbance is obtained by purifying and drying the dye, dissolving it at low concentration and measuring E on a spectrophotometer. The broadly used term extinction includes absorbance and light scattering. Absorbance is a unique characteristic of a molecule or nanoparticle under certain environmental conditions. In general, the bigger is the fluorophore size, the greater the probability that the photon will be absorbed. The light-absorbing power of the dye is commonly characterized by a value εmax at the maximum of absorption band. The absorbance is related to optical crosssection, and for organic dyes, εmax varies from ca. 103 l mol−1 cm−1 for small dyes, such as coumarins to ca. 105 l mol−1 cm−1 for larger fluorophores such as cyanines and phthalocyanines (Lavis and Raines 2008). It should be selected as high as possible, but the limiting factor is the fluorophore size. Using the dyes with lower εmax values is not reasonable, and much higher values are hardly achievable. 2. High fluorescence quantum yield,  . Quantum yield of any process is the ratio of a number of quanta participating in this process to a total number of absorbed quanta. For fluorescence, it is the ratio of the number of light quanta emitted as fluorescence to the total number of quanta that were absorbed. The term ‘brightness’ that determines the absolute sensitivity of fluorescence detection (Wetzl et al. 2003) is commonly defined as the product of molar absorbance and quantum yield, . The quantum yield is an intrinsic fluorophore property that can be modulated in a most dramatic way by interactions of the dye with its environment. The  values may vary from very low numbers (in relative units) to almost 100%, see Sect. 3.1.4. The sensing technique, in which the change of fluorescence intensity is recorded as an effect of quenching (Sect. 3.2), is based on variations of quantum yield. 3. Optimal excitation wavelength. In order to achieve the highest brightness, one has to excite fluorescence at wavelengths close to the absorption band maximum. Since the dye brightness is estimated as the product of molar absorbance ε(λex ) (at the applied excitation wavelength) and fluorescence quantum yield , it is important that the excitation wavelength, λex , should be chosen optimally with this account.

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Other factors are important for selecting the dyes with excitation band at particular wavelength, such as the availability of light sources, necessity to avoid autofluorescence background in living cell studies, etc. To avoid the cells autofluorescence, the excitation at wavelengths longer than 400–460 nm is preferable. For the diagnostic sensors implemented under the human skin, one has to account that the skin has a very low transparency throughout the whole visible range due to absorption by porphyrins and skin pigments. Such absorption is dramatically reduced in the near-IR, until the vibrational overtones of water appear, so the range 650–1450 nm is optimal for this application (Pansare et al. 2012). 4. Optimal emission wavelength. The range of operational wavelengths should be selected with the account of wavelength dependence of the sensitivity of detector. If the detector is a human eye, then the emission should be in the visible range of 400–700 nm. Many spectrofluorimeters available on the market are equipped with photodetectors with the cutting edge of 750–800 nm, which is not sufficient for recording of spectra of many dyes. However, the special “red-sensitive” photomultipliers can be supplied at a special request. Different red-infrared detectors using semiconductor elements have become available in recent time. 5. Large Stokes shift—the shift between absorption and emission band maxima on the energy (wavenumber) scale. The Stokes shift reflects the loss of excitation energy in different relaxation processes before the emission and is commonly observed in the direction of low energies and long wavelengths. The strongest Stokes shifts are observed for dyes with asymmetric structure and stronger charge distribution within the fluorophore. It should be accounted that the most popular dyes as fluorescent tags and labels, fluorescein, rhodamines, cyanines and some BODIPY derivatives, possess a rather small Stokes shift. Light scattering can be a difficult problem with such dyes enabling to provide recording of excitation and emission aside from the band maxima. Meantime, stronger separation between absorption and emission bands allows reducing the light-scattering effects and provides possibilities for more efficient collection of emitted light using broadband filters or larger monochromator slits. Larger Stokes shift allows reducing homo-EET (excitation energy transfer between the same dye molecules) thus allowing to suppress the concentrationdependent depolarization and quenching (see Sect. 10.2). The high-density attachment of these dyes to macromolecules and their incorporation into nanocomposite structures is possible. In some sensing technologies the dyes possessing strong Stokes shifts behave within these high-density units as independent emitters. 6. Optimal fluorescence lifetime, τF . This parameter depends on the dye and cannot be longer than the radiative lifetime, which is the fundamental property of a particular dye. The lifetime also depends on the temperature and the dye immediate environment. So depending on the application, it can be selected long or short by choosing the dye and its environment. Long lifetime is easy to detect and analyze. It is usually associated with a high quantum yield (the absence or small involvement of quenching effects). However, the long lifetimes (longer than 10−7 s, such as that of pyrene) are quenched

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by oxygen diffusing in the medium, so it is sometimes difficult to control this parameter. In anisotropy sensing (polarization assays), to provide the strongest effect the lifetime should fit the time scale of molecular rotations (see Sect. 3.3). Its longer values are needed to detect rotations of larger molecular units. Meantime for some applications, short τF is preferable. So it is in flow cytometry, in which a small number of dyes are under intensive excitation and the response may be limited by the duration of excitation-emission cycle. It must also be noted that due to a fundamental limitation, long τF cannot be achieved with high εmax . 7. High photostability. Each fluorescent dye can be degraded (photobleached) after some number of excitation-emission cycles (Eggeling et al. 1998). Photobleaching is a consequence of higher dye chemical reactivity in the excited states. Degradation occurs due to some photochemical reaction that often involves molecular oxygen and is coupled with the production of singlet oxygen. Fluorescein molecules can survive between 104 and 105 excitation-emission cycles before decomposing. The dyes differ strongly in their photostability, so coumarins are among the least stable and perylene, rhodamins and cyanines are much more photostable. Commonly the dyes are distinguished as being more photostable or less photostable than fluorescein (see Sect. 5.1). In sensing technologies that do not require exposure to intensive light, photostability is not a big problem. It may not be important in spectroscopic studies, in which the narrow excitation slits and fast recording of spectra are used. In flow cytometry and microplate reading, the photo-bleaching is generally not a problem because the sample remains in the laser beam for a very short period of time. Otherwise, oxygen can be removed from the system, which requires oxygen-free atmosphere or consumption of oxygen in any coupled reaction. Such possibility may not exist in fluorescence microscopy of living cells, and this often limits obtaining cellular images to a short time scale. Photostability is an important issue in single molecular studies and in super-resolution microscopy (Volume 2, Chap. 15), where the dye molecules are subjected to high light intensities (Ha and Tinnefeld 2012). 8. Optimal solubility—penetration—reactivity in the studied system. Each dye has particular physical and chemical properties (polarity, charge distribution, reactivity to form covalent bonds, ability to participate in π–π stacking, hydrogen bonding and other noncovalent interactions). These properties determine the dye distribution in a heterogeneous system (e.g. on a liquid-solid interface, in cell cytoplasm or biomembrane) and determines the extent of labeling of particular macromolecules or organelles. In addition, the dye has to be chemically stable and its fluorescence should be insensitive or low sensitive to the factors like pH or temperature if this sensitivity is not wanted. The quantum yields, lifetimes and photostabilities of organic dyes are variable in broad ranges and they can be substantially improved by encapsulating them into macrocyclic host molecules (Dsouza et al. 2011) such as cyclodextrins and cucurbiturils (Biedermann et al. 2012). Such macrocycle-protected dyes can be used in nanocomposite constructions being incorporated into the particles made of inorganic

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(Liang et al. 2010) or organic (Borisov et al. 2010) polymers (see Chap. 8). The most attractive are the dye-doped composites based on dendrimers, which allow maintaining the definite size and molecular order of composed structures (Bergamini et al. 2010). With this fundamental knowledge we will address the parameters of fluorescence emission, the changes of which can be used as reporting signals. This will be done in the sections that follow.

3.1.4 Fluorophore Emissive Power: The Brightness and Quantum Yield The quantum yield  is one of the major parameters characterizing fluorescent materials (Crosby and Demas 1971; Fery-Forgues and Lavabre 1999; Rurack 2008). Being used to quantify the efficiency of the emission process, it determines the suitability of such materials for applications in chemical sensing, bioanalysis and fluorescence imaging as well as for the active components in optical devices. Fluorescence quantum yield, , is defined as the ratio of the number of emitted quanta to the whole number of the quanta absorbed. In more general terms, it is the probability to emit one photon in the form of fluorescence after absorbing one photon. Fluorescence intensity at any wavelength, F (λ), is proportional to the fluorescence quantum yield . The excited state population decays as a function of time, which can be expressed by correspondent rate constants (first derivatives of concentration of excited-state species as a function of time). The quantum yield is determined by the ratio of radiative (k r ) and the sum of all non-radiative (k nr ) rate constants of excited-state decay: =

1 kr  =   kr + knr 1 + knr kr

(3.1)

In strongly fluorescent dye, the emission determined by k r should occur much faster than all the ‘dark’ processes occurring with the rates k nr . Primarily,  depends on the dye structure, and it is not easy to predict it on the basis of calculations. We can only estimate the efficiency of non-emissive channels of excited-state decay, such as internal vibrations. In addition to fluorescence, the dyes can participate in different dark non-emissive reactions. So,  is used as a measure of efficiency of fluorescence emission compared to other processes occurring during the excited-state lifetime, such as static and collisional quenching, transition to triplet state or photochemical transformations. For accurate  determination, different methods were developed. One is the “absolute” method that is based on collecting the whole spatially distributed fluorescence emission or reconstructing this spatial profile. The other is a comparative method

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that relies on the previous knowledge the quantum yield of some standard material, for which  is well determined (Würth et al. 2011). The later method requires only simple light absorption and fluorescence measurements in the same experimental conditions with conventional instrumentation. The tabulated  values for the references covering the whole spectral range are available (Brouwer 2011; Lakowicz 2006). This method is based on one important condition, which is the perfect matching of the sample excitation band with the absorption band (Vavilov’s law). This law derives from the fact that every absorbed light quantum over the absorption band has equal probability of emission, and this makes  independent of excitation wavelength λex . This is common for organic dyes studied in solutions, but may not be observed in some rare specific cases. In the following sections the most commonly used sensing technologies are presented together with their critical analysis.

3.2 Intensity-Based Sensing The change from light to dark is easily observed. This change can be efficiently reproduced on molecular level with recording of response from fluorescent dyes that switch from an emissive to a dark non-emissive state. Thus, everybody knows fluorescein for its bright fluorescence emission. Fluorescence of this dye can be almost completely quenched on its binding to anti-fluorescein antibody. This provides the reader an estimate of dynamic range of intensity sensing of intermolecular interactions: from extremely bright to almost totally dark. If fluorescein is attached through a flexible linkage to another dye, tetramethylrhodamine, the opposite effect on the binding to anti-fluorescein antibody can be shown. Two dyes make non-fluorescent stacking dimer, and the binding to antibody results in disrupting this dimer with dramatic enhancement of fluorescence by a rhodamine dye (Kapanidis and Weiss 2002). Thus, for various sensing applications, modulation of fluorescence intensity can be observed in both directions, the quenching and the enhancement, and in the broadest possible ranges.

3.2.1 Peculiarities of Fluorescence Intensity Measurements The measurement of fluorescence intensity changes at a single wavelength (usually at the emission band maximum) is the simplest and still very sensitive method to obtain information from fluorescence reporter. In many cases, it is relatively easy to provide the coupling of this enhancement/quenching response with the sensing event. The variation in intensity is the reflection of changes in the fundamental parameter of emission, the fluorescence quantum yield.

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Proper selection of excitation and emission wavelengths is important for intensity sensing. Commonly, the fluorescence spectrum is shifted with respect to excitation spectrum to longer wavelengths (the Stokes shift) (Fig. 3.2). As it was indicated above, the highest fluorescence intensity will be observed if the excitation and emission are provided at the wavelengths of the correspondent band maxima and if possible complications arising from light scattering are accounted. Many dyes that can be used for providing the response to target binding can change their fluorescence emission intensity on the variation of their weak noncovalent interaction with the local environment, and this change is mediated by non-radiative rate constants k nr . A change in fluorescence intensity from very high values (that correspond to quantum yield  ~ 100%) to zero or almost zero values between the target-free and the target-bound forms can be used in the background of operation of intensity-based sensors exploring the quenching effects.

3.2.2 How to Make Use of Quenching Effects? The switching between the emissive and dark states should involve some mechanism providing substantial quenching the fluorescence of reporter dye. This can be realized in different ways: (1) The quenching can be intrinsic to the reporter molecule, which means that the reporter dye molecule itself can switch between ‘light’ and ‘dark’ states. This is possible, but the dye molecule should be properly constructed. For instance, if the intrinsic mechanism of quenching is the intramolecular electron transfer (IET), then the dye should contain electron-donor and electron-acceptor fragments. There are many possibilities to make such constructions (see Sect. 4.1) and they are extensively explored, especially in ion sensing. (2) The quenching requires intermolecular interactions. Fluorescent dyes in their excited states may be electron-rich or electron-poor compared to the target molecules. So on their complex-formation an electron-transfer quenching can be observed. The quenching can also be observed on the dye interactions with heavy and transition metal ions. Fluorescence quenching can be connected with conformational flexibility that may exist in some dye molecules (Sect. 4.4). Should the rigidity in environment of these dyes increase, a dramatic enhancement of fluorescence can be achieved (McFarland and Finney 2001). Classical examples of this type of behavior are triarylmethane dyes, such as Crystal Violet and Malachite Green. They are nonfluorescent in liquid media but become strongly fluorescent on absorption to rigid surfaces and on binding to proteins. Several families of dyes demonstrating the effect of aggregation-induced emission have been synthesized and characterized as it will be discussed in Sect. 8.3. (3) The quenching involves the participation of solvent. Formation-disruption of hydrogen bonds with solvent molecules and different solvent-dependent

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changes of dye geometry can be observed in many organic dyes. Dramatic quenching in water (and to lesser extent in some alcohols) may occur due to formation by water molecules of the traps for solvated electrons. In addition, the solvent can influence on the dye energy states, particularly on inversion of n (non-fluorescent) and π (fluorescent) energy levels. The latter effect can be explained based on Kasha rule (see Fig. 3.3), according to which the fluorescence occurs from the lowest excited energy state. If the non-emissive state becomes the lowest, then fluorescence is quenched. These and other factors can also modulate strongly the fluorescence intensity (de Silva et al. 1997). Practically for sensing applications, this means that exposing the fluorescence reporter to the solvent or screening from it may influence dramatically the emission intensity and in this way to provide the necessary response (Fürstenberg 2017). From this short discussion, one can derive that there exist a variety of possibilities for exploring fluorescence quenching effects. Some of them may operate together. The researcher has a lot of choice for constructing a sensor with its response based on the principle of intensity sensing.

3.2.3 Quenching: Static and Dynamic Fluorescence quenching differs not only by its intrinsic mechanism but also by the way, in which it is applied. The quencher can form the long-lasting contact with the reporter dye, so that its emission is abolished but the emission of the same closely located dyes that do not interact with the quencher may not change. Such quenching is called static. It can totally decrease fluorescence intensity without changing the lifetime. This is because the dyes interacting with the quencher do not emit light, and those, which do not interact, emit normally. The other limiting case is the dynamic quenching. Here the effect of quenching competes on a time scale with the emission and is determined by the diffusion of quencher in the medium and by its collisions with the excited dye. In this case, the relative change of intensity, F 0 /F, is strictly proportional to the correspondent change of fluorescence lifetime, τ0 /τ, where F 0 and τ0 correspond to conditions without quencher. Fluorescence lifetime is the reverse function of the rate of the excited-state depopulation: τ0 = 1/(kr + knr )

(3.2)

The Stern-Volmer relationship establishes the correlation of these changes with the quencher concentration [Q] by introducing the rate constant proportional to this concentration: F0 /F = τ0 /τ = 1 + kq τ0 [Q] = KSV [Q]

(3.3)

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Here k q is the rate constant of quenching. In the case of 100% efficiency of quenching it is equal to quencher diffusion constant (being typically in the range of 109 -1010 M−1 s−1 ). K SV = k q τ0 is called the Stern-Volmer quenching constant. It can be determined from the slope of the graph displaying dependence of intensity or lifetime on [Q], expressed by Eq. (3.3). If the lifetime does not change in proportion to the change of intensity or if the quenching effect in intensity exceeds the diffusional limit, this indicates the contribution of static quenching. In the case if all the quenching is static, a dye bound to a quencher appears immediately (much faster than the decay) in a dark nonemissive state. The unbound fluorophores exhibit their natural lifetimes, and the slope of F 0 /F versus [Q] yields a binding constant. The static and dynamic quenching can be distinguished also by the temperature effects. If the quenching is static, it decreases with the increase of temperature because of disrupting molecular complexes that produce quenching. On the contrary, the dynamic quenching increases because of increasing the quencher diffusion rate. Both static and dynamic quenching can be used for providing the fluorescence response. In sensing technology, the target or the competitor can be used as dynamic quencher, and its concentration can be determined from Eq. (3.3). This condition could be better applied to detecting small molecules, the diffusion of which is fast. Meantime, in many cases the employment of such sensor constructs that use the static quenching produced by intramolecular or intermolecular complex formation is very efficient.

3.2.4 Non-linearity Effects When the fluorescence intensity is measured at low dye concentrations and low intensities of incident light and dye distribution in the studied system does not depend upon its concentration, this intensity should be strictly proportional to the dye concentration. Non-linear effects in quenching can appear due to several factors. They are: (a). Those depending on sample volume and geometry. In the case of strongly absorbing (low transparent) samples, the light passing through the sample gets weaker and excites fluorescence at a lower level (primary inner filter effect). Also, in the cases of high fluorophore concentration, the fluorescent light emitted from illuminated volume can be re-absorbed in a dark volume instead of passing to detection device (secondary inner filter effect). The involvement of these factors can be diminished or even eliminated by reducing the optical path or the sample volume, by special sample geometry or introduction of correction factors (Fonin et al. 2014). This effect does not create problems at absorbance levels 0.05-0.1 and lower.

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(b). Those depending on the properties of the dye. Some dyes form the nonfluorescent dimers and aggregates and their formation is concentrationdependent. In addition, at short distances between the dye molecules the excitation can migrate between the dyes so that the dye absorbing light can transfer its energy to neighboring dyes and these non-fluorescent species may serve as the light traps. This explains why one cannot load many dye molecules (fluorescein or rhodamine derivatives) on single protein molecule (e.g. antibody); such complexes become non-fluorescent. The mechanisms of these processes will be explained in Sect. 8.1. The energy migration to nonfluorescent associates can be reduced by using the dyes with large Stokes shifts. However, there is an efficient application of self -quenching effects. They may bring useful information in the cases when the observed phenomenon involves a strong change in the local dye concentration. For instance, the dyes trapped at high concentrations in inner volume of phospholipids vesicles (liposomes) can be non-fluorescent. But when the vesicle integrity is disrupted, the dye comes out from vesicle and being diluted increases dramatically its fluorescence. Hence, there appears the control for vesicle integrity (Han et al. 2005). (c). Those occurring at high intensities of excitation light. In this case, the nonlinearity is explained by the fact that a large portion of molecules appears in the excited state so that the ground state becomes depopulated. Since fluorescence intensity is proportional to the ground-state concentration, the so-called light quenching effect can be observed (Lakowicz et al. 1994). All these effects do not appear at common excitation and emission conditions and the commonly used dye concentrations but they have to be accounted for if the researcher starts working outside this range.

3.2.5 Internal Calibration in Intensity Sensing Calibration in fluorescence sensing means the operation, as a result of which at every sensing element (molecule, nanoparticle, etc.) or at every site of the image the fluorescence signal becomes independent of any other factor except the reported concentration of bound target (Vogt et al. 2008). The problem of calibration in intensity sensing is very important because commonly the fluorescence intensity F is measured in relative units that are proportional to the number of emitted quanta and have no absolute meaning if not compared with some standard measurement. When the fluorescence spectrum is recorded, the intensity at one wavelength is compared with intensities at other wavelengths; therefore, the spectrum has an informative value. However, one cannot compare the fluorescence intensities of two samples measured today and one year after because of the difference in experimental conditions. Likewise, one cannot compare the fluorescence intensities at two neighboring spots in the sensor array with the labeled target, in which the amount of sensor

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molecules is different. Similar problem exists in cellular imaging of target distribution: the image formed of light intensities depends not only on the target distribution but also on the distribution of sensors themselves. In the commonly used instrumentation, the fluorescence signal is obtained in relative units (the quanta counts or the analog signal obtained on their averaging by the detection system). In this case, the fluorescence response depends not only upon the sensor—target binding, but also upon such technical parameters as the intensity of incident light, geometry of the instrument, detector sensitivity, monochromator slit widths, etc. It is sensitive to every fluctuation in light source intensity or in sensitivity of the detector. Thus, the recorded changes of intensity always vary from instrument to instrument, and the proper reference even for compensating these instrumental effects is difficult to apply. In common spectrofluorimeter, the measured signal can be compared with the signal coming from the reference sample. This reference could be the sensor molecules in a given concentration calibrated with the addition of given concentrations of the target. By variation of target concentration a calibration function can be obtained. It is then used for determining the target concentration from the readout of fluorescence response from the sample. This approach is hard to realize on a microscopic level or in simultaneous analysis of different sensor-target compositions. We have to analyze why this difficulty appears with an aim to find proper solutions. (a) It is difficult to achieve a strictly determined concentration (or density) of sensor molecules in analyzed volume. It is common also that both the sensing properties and their reporting abilities degrade in time due to either inactivation or photobleaching. (b) It is difficult to provide a separate reference to every sensor unit in the case if we deal with large amount of them on arrays and each of them responds to binding of a different target. (c) Even if this is done, it is hard to equalize the concentrations of sensor molecules in a sensing reference element or to provide correction for their difference. Thus, if our aim is obtaining the quantitative information about concentrations of many different targets simultaneously, we have to develop a special methodology. We have to follow the requirement that each element of the array should become self -calibrating. This means that its response should contain not only the signal reporting on the target binding but also a different signal (or signals) that could allow providing the account or compensation of all the effects that influence the fluorescence parameter(s) besides the target binding. In the simplest case of reversible binding with stoichiometry 1:1, the target analyte concentration [A] can be obtained from the measured fluorescence intensity F as:  [A] = Kd

F − Fmin Fmax − F

 (3.4)

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Here F min is the fluorescence intensity without binding and F max is the intensity if the sensor molecules are totally occupied. K d is the dissociation constant. The differences in intensities in the numerator and denominator allow compensating for the background signal and the obtained ratio can be calibrated in target concentration. However, since F, F min and F max are expressed in relative units, they have to be determined in the same test and in exactly the same experimental conditions. This is difficult and often not possible. The most efficient and commonly used approach is the introduction of reference reporter. This additional dye can be introduced into a sensor molecule (or into support layer, the same nanoparticle, etc.), so that it can be excited together with the reporter dye and emit light at a different wavelength and it should not change its signal on target binding. This should provide an additional independent channel of information that could serve as a reference (Fig. 3.5). If the reference is properly selected, then one can observe two peaks in fluorescence—one from reporter with a maximum at λ1 and the other from the reference with a maximum at λ2 . Their intensity ratio can be calibrated in concentration of the bound target. Thus, if we divide both the numerator and denominator of Eq. (3.4) by F ref (λ2 ), the intensity of the reference measured in the same conditions, we can obtain target concentration from the following equation that contains only the intensity ratios R = F (λ1 )/F ref (λ2 ), Rmin = F min (λ1 )/F ref (λ2 ), and Rmax = F max (λ1 )/F ref (λ2 ):  [A] = Kd

R − Rmin Rmax − R

 (3.5)

This approach to quantitative target assay has found many applications. As an example, it was used in the construction of double-labeled molecular biosensors

TT +

T +

S Target T

S

R

Target T

Fig. 3.5 The scheme illustrating the advantage of self-calibrated signal on sensor (S)—target (T) interaction with the aid of reference dye (R). The sensor—target interaction produces quenching of fluorescence spectra in the case of intensity sensing (left) and intensity sensing with the reference (right). The two dyes, sensing and referencing, are excited and detected simultaneously. The reference dye allows recording the ratio of two intensities detected at wavelengths λ1 and λ2

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based on glutamine and glucose binding proteins (Ge et al. 2004; Tolosa et al. 2003). In other research, for the design of ratiometric Cl− sensor a molecular hybrid was synthesized that contained two covalently linked dyes (Jayaraman et al. 1999). They can be excited simultaneously but exhibit separate emission spectra. One of them is strongly quenched by Cl− ion, while the other does not interact with Cl− and serves as a reference for the sensor concentration. The sensor designs with the introduction of additional dyes as the references are also used in cation and pH sensing (Koronszi et al. 1998). Summarizing, we outline what is achieved and what is not achieved with the introduction of reference dye. The two dyes, responsive and non-responsive to target binding, can be excited and their fluorescence emission detected simultaneously. In principle, the results should be reproducible on the instruments with a different optical arrangement, light source intensity, slit widths, etc. In addition, if the two dyes are distributed very similarly in the illuminated volume, the two-band ratiometric signal can be calibrated in target concentration. This calibration, in some range of target concentrations, will be insensitive to the concentration of sensor (and reporter dye) molecules. It should be remembered however that the sensor molecules can exhibit degradation and the dye molecules can photobleach as a function of time. These effects may provide the time-dependent and target-independent changes of the measured intensity ratios. In addition, because the reporter and reference dyes emit independently and their sensitivity to quenching (by temperature, ions, etc.) may be different, every effect of fluorescence quenching unrelated to target binding will interfere with the measured result. This can make the sensor non-reproducible in terms of obtaining precise quantitative data even in serial measurements with the same instrument. In these cases, other detection methods that will be overviewed below are preferable.

3.2.6 Intensity Change as a Choice for Fluorescence Sensing A good story about intensity sensing is the great range of possibilities to choose the organic dyes, noble metal clusters, quantum dots and other types of fluorescence emitters that can be used. All of them can change the emission intensity, the simplest way of fluorescence response. Such property of these materials will be demonstrated in the following chapters. The weak point of this approach is the necessity to resolve the problem of calibrating the sensor element. The light intensity depends upon the concentration of sensor molecules, excitation wavelength, optical density at this wavelength and on the emission wavelength, complicating the calibration. It is difficult to obtain a linear response in fluorescence intensity operating with fiber optical waveguides. But what is really difficult or even impossible, it is to account or compensate the effects of light-absorption and light-scattering. This can be a serious problem, especially if the studied medium is heterogeneous, such as the colloid suspension, the solution of high-molecular-weight polymer or the living cells. If the target molecules are of large size and if they can aggregate during the

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measurement, this may create irresolvable problems. Finally, any time-dependent processes occurring with the fluorophore, especially photobleaching, will make the sensor non-reproducible even in serial measurements with the same instrument and when all above listed conditions are kept constant or properly corrected. Discussing the disadvantages of sensing based on the changes of fluorescence intensity we do not have to forget its important merits. One such merit is the simplicity in instrumentation, in the measurement and in adjustability of this instrumentation for the use in integrated sensing devices and microarrays. Another merit is the highest possible absolute sensitivity, in view that in other methods the application of higher resolution in spectra or resolution in time and anisotropy results in the loss of absolute intensity. If the interfering factors such as background fluorescence and light scattering are not very important, the integral intensity over the whole emission band can be collected. Thus, the sensors based on registration of F changes (sometimes called intensiometric sensors) can be recommended for the use in qualitative or semiquantitative detection technologies where a high sensitivity is very desirable but high precision of quantitative measurement is of secondary importance.

3.3 Anisotropy-Based Sensing and Polarization Assays Linearly polarized light is the light that propagates with its electric field vector keeping a unique direction in space. Such light wave can excite the dyes only if they are in definite orientation. If these dyes are immobile on a time scale of fluorescence decay, they emit also the polarized light. However, due to rotational diffusion they may randomly change their orientation, so that being initially anisotropic their orientation becomes isotropic with time. This leads to depolarization of emitted light. If the dye represents a fluorescent reporter and in the course of sensing event either the size of rotating unit or local viscosity increases, then the rotation will slow down and we will get an increase of fluorescence polarization and anisotropy. The time window for this rotation is also important. It is given by fluorescence lifetime τF . When τF increases, our sensor will then have more time to rotate and anisotropy (or polarization) will decrease. Thus, the rotation rate and the fluorescence emission rate determine the response in anisotropy. Recording of anisotropy requires the measurements of the ratio of fluorescence intensities obtained at two different polarizations of the emission light, vertical and horizontal (Guo et al. 1998; Jameson and Croney 2003). Primarily fluorescence polarization measurements were used to study rotational mobility of labeled macromolecules, the presence of their flexible fragments, association-dissociation of protein subunits and of intermolecular interactions influencing this mobility. Later on, different polarization assays were developed based on the same principle: the slowing down of rotational mobility of sensor molecule on target binding, which increases the size of rotating unit. Their development led to anisotropy-based sensor arrays, in which small rotating sensor units are immobilized on support and the target binding is detected as the decrease of its rate of rotation.

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3.3.1 Background of the Method The measurement of steady-state anisotropy, r, is simple and needs two polarizers, one in excitation and the other in emission beams of the instrument (Fig. 3.6). When the sample is excited by vertically polarized light (indexed as V ) and the emission intensity is measured at vertical (F VV ) and horizontal (F VH ) polarizations, then one can obtain r from the following relation: r=

1 − G × (FVH /FVV ) FVV − G × FVH = , FVV + G × 2FVH 1 + G × 2(FVH /FVV )

(3.6)

where G is an instrumental factor (Jameson and Ross 2010). Polarization P is a similar parameter that is defined as P = (F VV − F VH )/(F VV +F VH ). Its relation to r is simple: r = 2P/(3−)

(3.7)

Polarizer Light source

a

b Analyzer

Fig. 3.6 Geometry of fluorescence anisotropy experiment and response of polarized emission to fluorophore rotation. Fluorophores in the cuvette are excited with short pulses of vertically (z) polarized light causing photoselection. An analyzer in fluorescence channel can be rotated from the vertical F VV to the horizontal F VH position. In the insert: Connection between light depolarization and rotational mobility. Linearly polarized light is absorbed by molecules predominantly oriented in a particular way, for example, parallel to the electric field vector of the incident light. If the molecules emit linearly polarized light and their orientation during the lifetime of excited state τF is not changed (a), the total emission will be polarized. If the orientation of the molecules during lifetime τF becomes chaotic (b), the emission is depolarized

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It is also used for characterizing polarized emission. Though it is less convenient, it was historically the first parameter introduced, providing essentially the same information. Equation (3.6) shows that r in fact is a ratiometric parameter that does not depend upon the absolute intensity of light emission because the variations of intensity influence proportionally the F VV and F VH values. Therefore the anisotropy, similarly to the lifetime (Sect. 3.4), is low sensitive to variation of reporter dye concentration or to its change in the course of experiment (e.g. to photobleaching). Thus, we may consider that anisotropy and polarization of emission that are derived from the intensities collected at the same wavelength at vertical (F VV ) and horizontal (F VH ) polarizations are the intrinsic properties of the dye molecule in particular environment. In the simplest case of rotating dye, when both the rotation and the fluorescence decay can be represented by single-exponential functions, the range of anisotropy variation (r) is determined by the ratio of fluorescence lifetime (τF ) and rotational correlation time (τφ ) variation. The latter describes the rotation of dye molecule or the structure to which it is rigidly attached: r=

r0 1 + τF /ϕ

(3.8)

Here r 0 is the fundamental anisotropy. It can be defined as the limiting anisotropy obtained in the absence of rotational motion. It is an intrinsic parameter of a fluorophore that is determined by the angle between its absorption and emission dipoles.

3.3.2 Practical Considerations The dynamic range of anisotropy sensing is determined by the difference of this parameter between free sensor, which is the rapidly rotating unit and the sensortarget complex that exhibits a strongly decreased rate of rotation (Fig. 3.7). The reporter dye should be rigidly attached to the sensor for reflecting this change of mobility. The polarization experiment allows obtaining two-channel information on sensing event from a single reporter dye. The range of variation of r values in this event could extend from 0 to r 0 (which is commonly around 0.3–0.4) and it should not depend upon the instrumental factors or the dye concentration. As follows from Eq. (3.8) and the above discussion, the variation of anisotropy can be observed in two cases, on the variation of fluorophore rotational mobility (the change of τφ ) and on the variation of emission lifetime τF . At given τF the rate of molecular motions determines the change of r, so that in the limit of slow molecular motions (τφ τF ) the r value approaches r 0 , and in the limit of fast molecular motions (τφ  τF ) r is close to 0. This determines dynamic range of the assay.

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NO TARGET

Time

TARGET BOUND

T

T T

T

Time

T

T

T T

T

T

Fig. 3.7 Illustration of the basic principle of fluorescence anisotropy sensing (polarization assays). Dye molecules with their absorption transition vectors (arrows) aligned parallel to the electric vector of linearly polarized light are selectively excited. Due to rapid rotational diffusion, the distribution in their orientations becomes randomized prior to emission, resulting in low fluorescence anisotropy. The binding of a large, slowly rotating target molecule or particle prevents the rotation and results in highly polarized fluorescence. The difference between the two values of anisotropy provides a direct readout of the target binding

Since the change of anisotropy on target binding can be achieved by the change of two parameters, τφ and τF , it is essential that their ratio should exhibit the most significant difference between target-bound and target-free states of the sensor. This is possible, and it is highly preferable to realize the case when the change of τφ and τF occurs in opposite directions. Meanwhile, it is not uncommon that the target binding resulting in increasing τφ due to the increase of the size of rotating unit increases also τF due to dye immobilization or screening from the quenching effect of water. In order to provide high sensitivity of the assay these situations need to be avoided. Anisotropy sensing is well compatible with the methodology of direct reagentindependent sensing (Sect. 2.4). Attachment of the dye at the sensor-target contact areas in this case is not required, which increases the possibilities in application of this method. In competitor displacement assays (Sect. 2.3) the binding of small competitor molecule is easily detected as an increase in anisotropy. Meantime, the advantages of this method are often difficult to realize in heterogeneous assays, since for the sensor attached to the surface the rotational mobility can already be suppressed and on binding the target the anisotropy may not exhibit additional increase. Therefore, in such cases the fluorophore has to be attached to a flexible linker for maintaining its own degrees of rotational freedom relative to the sensor molecule, so that the loss of its mobility could report about the binding. Another problem is the resolution in the response of anisotropy sensing in the case of high molecular weight targets. In order to achieve a good resolution in anisotropy the rotational correlation time of the labeled target should correspond to

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the fluorescence lifetime. Using the well-known Stokes-Einstein relation for isotropic Brownian rotational diffusion, one can estimate the rotational correlation time of solute molecule or particle (assuming its spherical shape) on the basis of molecular mass (M): ϕ=

ηV ηM = (v + h) RT RT

(3.9)

Here η is the viscosity of the solution, and V is the molecular volume, R is the ideal gas constant, T is the absolute temperature, v is the partial specific volume in cm3 /g, and h is the hydration (typically for proteins, 0.2 g of H2 O/g). Assuming the typical v value for a protein, temperature 20 °C and the viscosity of water, one can use its simplified version: τφ (ns) = 3.05 × 10−4 × M

(3.10)

Anisotropy

It follows that for achieving the best sensing signal in assay for a protein of molecular mass 40,000 the fluorophore lifetimes should be approximately 12 ns. From this equation, using Eq. (3.8) one can evaluate the fluorescence lifetime that will be optimal to detect the variation of anisotropy in the necessary range. The calculations based on Eq. (3.9) correlate reasonably well with experimental data. Figure 3.8 presents the graphs built on this correlation (Guo et al. 1998).

Molecular mass, Daltons Fig. 3.8 Molecular-weight-dependent anisotropy for a protein-bound luminophore with luminescence lifetimes of 4, 40, 400, and 2700 ns. The curves are based on Eqs. 3.8 and 3.9 assuming an aqueous solution at 20 °C with a viscosity of 1 cP and (v + h) = 1.9 (Guo et al. 1998)

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From these estimates it can be immediately seen that common organic dyes with fluorescence lifetimes of the order of 1 ns are suitable for detection in anisotropy response of only the smallest and rapidly rotating sensors (or competitors) and do not properly fit the size of even small proteins. For labeling larger molecules, such as antibodies, the luminophores with longer lifetimes are needed. They should satisfy the best sensing conditions, so that τφ < τF before the binding and τφ > τF after binding the target.

3.3.3 Applications of Fluorescence Anisotropy in Sensing Like other methods of fluorescence detection, the anisotropy sensing is based on the existence of two states of the sensor, so that the switching between them depends on the concentration of bound target. In these states, the dye reporters exhibit different possibilities to emit light and to rotate. The major application of this technique in fluorescence sensing is to provide response to target binding by the change of rotational mobility of fluorescent reporter. Figure 3.9 illustrates the change of recorded anisotropy r between 0 and r 0 values as a function of target concentration in direct assay (the target binding increases anisotropy), see Fig. 3.9a. In competition assay the situation can be the opposite: the release of bound competitor increases its mobility and decreases the anisotropy. This figure also shows the target concentration effect in the case of different correlations between τφ and τF in the case of direct sensing. If the sensor size is small, anisotropy is low. With its increase, the τφ relative to τF value increases, which decreases the dynamic range for target determination, as illustrated by target binding curves (Fig. 3.9b). Thus, upon increase of τφ , or with the decrease of τF the response to target binding decreases and may vanish. Variation of sensor-target concentration ratio allows determining the dissociation constants K d , which are important for the analysis of protein-ligand interactions (Wang et al. 2013).

a

b T

T [T]

[T]

Fig. 3.9 Dependence of anisotropy sensor response on target concentration [T] in direct and competition assays (a) and this dependence for direct assay at different sizes of rotating units ϕ influencing the correlations between τφ and τF (b)

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This method has found its most extended application in sensing techniques based on antigen-antibody recognition. The so-called “polarization immunoassays” are based on the measurements of steady-state anisotropy in both heterogeneous and homogeneous assay formats. In a homogeneous fluorescence polarization immunoassay for antigens or antibodies detection (Nielsen et al. 2000) either the antigen or the antibody can be determined in solution with the labeled fluorescent partner. On the formation of their complex the steady-state anisotropy increases due to an increase of the rotating unit size. The fluorescence polarization assay can be also used for determining the antigen-antibody affinity (Smallshaw et al. 1998). Anisotropy measurements are also used in competitive immunoassays that have become common not only in research but also in routine clinical analysis. Primarily the complex of the sensor antibody with the target analog labeled with fluorescent dye is formed. Consequently, the target present in tested medium produces the change of fluorescence anisotropy by displacing the labeled competitor from the binding site. Since the free competitor is a smaller rotating unit than its complex with the sensor, a decrease of anisotropy is detected based on fluorescence labeling. This procedure can be performed in solution as a homogeneous immunoassay as it does not require any separation steps (Chen et al. 2019). Similarly to other competitive assays in solutions, it cannot be extended for measuring several targets simultaneously. An example of successful application of anisotropy detection technique in ion sensing can be the detection of Zn2+ picomolar to nanomolar concentrations by application of aminobenzoxadiazole dye covalently attached to genetically modified metalloprotein carbonic anhydrase (Thompson et al. 1998). The results demonstrate that using inexpensive instruments the free transition metal ions can be determined at trace levels in aqueous solution. In drug discovery, the fluorescence polarization (anisotropy) technology became a routine technique because of its convenience and low price (Hall et al. 2016; Owicki 2000). Originally, the polarization assays were developed for the singlesample analytical measurements, and the technology was rapidly converted to highthroughput screening. Fluorescence anisotropy imaging with two-photon excitation is actively used to perform this task (Vinegoni et al. 2019). There is no limit for generalization of anisotropy sensing technique to the largeformat proteomics arrays using probably the most advanced capture molecules, which are the nucleic acid aptamers (see Sect. 4.4). Aptamers can be immobilized on solid support at one point by a chain terminal and can rotate around this point of attachment with the observed decrease of anisotropy. The target binding decreases the rate of their rotation. Based on this principle, a biosensor in a chip configuration that is capable of quantifying several proteins with relevance to cancer was demonstrated (McCauley et al. 2003). A rapid, homogeneous aptamer-based bioanalysis was reported for the sensitive detection of immunoglobulin E (IgE) in the low-nanomolar range with high specificity (Gokulrangan et al. 2005). Summarizing, we outline three possibilities for using the fluorescence response in anisotropy (polarization) in sensing: (a) When anisotropy increases with the increase of molecular mass of rotating unit;

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(b) When anisotropy increases due to increase of local viscosity producing higher friction on rotating unit; (c) When anisotropy increases due to fluorescence lifetime decrease being coupled to any effect of dynamic quenching. All these effects are interrelated and observed within a rather narrow range of related parameters. Nevertheless, the possibilities of this technique application are very broad. There exist, however, the problems in application of nanosensors. They are twofold. First, many nanoscale emitters (e.g. quantum dots) do not exhibit detectable fundamental anisotropy. Second, the nanosecond lifetimes of fluorophores are too short to fit the microsecond rotational dynamics of nanoparticles. Therefore, the acceptable solution with anisotropy sensing is its use in competitor assays as it is shown in Fig. 3.9. For obtaining the sensing signal in anisotropy, either the dye should be small enough to provide the fast rotation rate, or the lifetime long enough to fit this rate scale (Chen et al. 2019).

3.3.4 Comparisons with Other Methods of Fluorescence Detection Anisotropy sensing allows direct response that is independent of sensor concentration and allows both direct and competitor substitution sensing. In the background of this independence is the independence of measured anisotropy (polarization) on absolute fluorescence intensity. In this respect, we have to stress the following. (a) Two information channels in this case are derived from a single dye molecule exhibiting a single electronic transition. Thus, the two parameters, (F VV and F VH ) are strongly coupled and proportional to the absolute intensity value. (b) If there appear additional perturbations, such as the variations of lifetime or the appearance of additional degrees of rotational freedom, the F VV and F VH values change in a different manner producing a change of anisotropy that overlaps on the response obtained on target binding. In particular, any factor that causes the decrease of fluorescence lifetime (produces quenching) increases the anisotropy. Dynamic range of the fluorescence response is determined by the “window” in anisotropy values between the readings obtained with the free sensor and the sensor with bound target (Jameson and Croney 2003); the possibility to increase this range is offered by long-lifetime luminophores. The scaling of bound target concentration is performed between these two limiting (low and high) values, and the position of the whole scale depends on fluorescence lifetime τF . In the case of presence of fluorescence quenchers, the whole scale will be shifted producing a systematic error. Due to this fact, the self-calibration that can be achieved in anisotropy sensing may not always be sufficient.

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It should be emphasized that the weak point of this detection technique is its great sensitivity to light-scattering effects, especially to those that may result in non-specific aggregation of the sample during the sensing procedure. This occurs because the scattered light is always 100% polarized, and its contribution cannot be removed in the steady-state measurements if there is a strong spectral overlap between scattered and fluorescent light. For avoiding the light-scattering artifacts, the dyes with a large Stokes shifts should be preferably used. This allows increasing the gap between excitation (and also light-scattering) and emission wavelengths.

3.4 Lifetime-Based Fluorescence/Luminescence Response Fluorescence is the process that develops in time after initial electronic excitation. Its intensity decays as a function of time in subnanosecond-nanosecond time range. It is hard to imagine such short periods of time; they are outside of our everyday experience. Nevertheless, the life of molecules in these time periods is very busy. Molecules move, rotate, collide, participate in different reactions. Fluorescence emission helps us to reveal mechanisms of all these events. Duration of fluorescence emission also depends on them. If we want to make a sensor, our target should also participate in these processes. Luminescence is the term broader than fluorescence and it includes the emission with with much longer lifetime, the phosphorescence.

3.4.1 Physical Background of Time-Resolved Detection Usually molecules emit light independently, so the decay function of their fluorescence emission is essentially concentration-independent. It is an intrinsic property of the fluorophore, of its interactions, dynamics and participation in reactions. Because the fluorescence decay is a random event, the emissions of individual quanta follow Poisson statistics. Therefore, in an ideal case of structurally identical and independent fluorescence emitters, the fluorescence decay is a single exponential function of time t: F(t) = F0 exp(−t/τF )

(3.11)

Here F 0 is the initial intensity, at time t = 0. The time constant τF is called the excited-state (fluorescence) lifetime, it is the reverse function of the fluorescence decay rate, k F . In many real cases, the decay is not exponential, but it is often possible to characterize it by averaged lifetime, . The necessary condition in lifetime sensing is the possibility of detecting the strong difference of between the target-bound and target-free forms of the sensor. Lifetime sensing is based on two different principles:

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(a) Modulation of lifetime by dynamic quencher. Molecular oxygen is a strong quencher of long-lifetime emission, operating by diffusional mechanism. The decrease of τF occurs gradually with oxygen concentration. This change obeys the Stern-Volmer relationship (Eq. 3.3), which allows determining the concentration of free oxygen. High mobility combined with potent quenching are the unique features of oxygen molecules. (b) Shifting of the sensor to a different discrete form with different lifetime. The presence of distinct difference in lifetime between two forms of the sensor, free (A) and bound (B) is necessary for lifetime sensing application of this type. Belonging to the same dye, these two forms can be excited at the same wavelength. When excited, they emit light independently and therefore the observed nonexponential decay can be decomposed into two different individual decays B with lifetime τA F and τF : F(t) = αA exp(−t/τFA ) + αB exp(−t/τFB )

(3.12)

Important, the two forms of the sensor (target-free and target-bound) can be excited at the same wavelength and their contributions must be obtained from deconvolution of two-component decays. These two principles explored in lifetime sensing are depicted in Fig. 3.10. In the first case, the decay does not change its initially monoexponential character but Target

a

T

Target

T

b T T

Life me

Life me

Fig. 3.10 Typical effect of target binding influence on fluorescence decay kinetics and derived lifetimes. Long fluorescence decay (large τF ) changes to short decay (smaller τF ) on target binding. a The fluorescence decays in logarithmic coordinates. Thin solid lines are the decays without the target, thick solid lines—with the saturation by target, the dashed line—on interaction with the target resulting in the change of lifetime between these values. b The lifetime values. In the case when the target (T) is the dynamic quencher (left), the decay becomes shorter gradually as a function of its concentration. In the case when the target binding changes the lifetime leading to it’s another constant value, the decay is non-exponential but can be resolved into two exponential components. The relative magnitude of these components changes as the function of target concentration, which allows its quantification

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the gradual decrease in τF can be observed. This happens in dynamic quenching, so that τF can be plotted as a function of quencher concentration. These cases are rare and generally are not interesting, since it is hard to couple dynamic quenching with molecular recognition. The transition between two sensor forms with different lifetimes on target binding is more typical. In relation to molecular sensing, the interest to lifetime detection techniques is due to the fact that fluorescence decay is an intrinsic characteristic of fluorophore and of its environment and does not depend upon fluorophore concentration. Moreover, the results are insensitive to optical parameters of the instrument, so that the attenuation of the signal in the optical path does not distort it. The light scattering produces also much lesser problems, since the scattered light decays on a very fast time scale and does not interfere with fluorescence decay observed at longer times. Most often, the decays do not depend on emission wavelength (with the exception of excited-state reactions occurring on the time scale of decay), which allows to collect them from the whole spectra and achieve relatively high sensitivity.

3.4.2 Technique Using the Time Resolution Fluorescence decay functions can be obtained with the aid of special instrumentation (Berezin and Achilefu 2010). It allows recording the decay directly using the single photon counting, stroboscopic techniques or observing the demodulation and phase shift of harmonically modulated excitation light. These methods are well described in the literature (Collier and McShane 2013; Lakowicz 2006; Valeur and BerberanSantos 2012). In brief, in the phase-modulation technique the excitation light is modulated with high frequency (~ 100–200 MHz), and the detector records the shift in phase of modulated signal (the phase angle) and the decrease in its modulation depth in fluorescence compared to that in excitation light. The slower is fluorescence decay (the longer lifetime), the larger phase shift and the smaller modulation. In time-correlated single-photon counting the excitation occurs by a light source producing short pulses, and single quanta are randomly selected from every pulse to form the experimental emission decay function. The emission decay function can be also derived by mathematical transformation of the primary data obtained by phase-modulation. Thus, the methods are equivalent though their technical realization is different. Lifetime measurements require a more complicated and expensive technique than that required for steady-state measurements, so the prospects for this method depend upon the development of simple and cheap ways for its realization. An important extension of this methodology is the development of assays that involve the long-lifetime luminescence of rare earth ions or explore the reactions with their participation.

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3.4.3 Time-Resolved Anisotropy The time-resolved detection can be combined with the measurements of anisotropy to give the time-resolved anisotropy r(t), see Fig. 3.3. Technically this can be achieved by incorporating the polarizer and analyzer into the time-resolved fluorimeter. The time resolution allows eliminating all light-scattering effects, to which the steadystate anisotropy is sensitive. If the two forms of a sensor, A and B, differ by anisotropy decay, then the observed decay will be composed by contributions provided by these individual forms:   r(t) = r0 FA exp(−t/φA ) + FB exp(−t/φB ) .

(3.13)

Similarly to the steady-state anisotropy, r(t) is an ‘intensity-weighted’ parameter in sensing, so that the intensity decays collected in two polarization channels depend on their initial intensities.

3.4.4 Applications of Time-Resolved Fluorescence Examples of application of lifetime sensing were presented for popular fluorescence probes, used for measuring cation concentration (such as Calcium Green) and pH (such as SNAFL-2 (Szmacinski and Lakowicz 1994). Remarkably, one of pH-sensitive dyes was incorporated into polymeric hydrogel, and it was shown that lifetime sensing provides reliable results in light-scattering media (Kuwana and Sevick-Muraca 2002). The other important application is the sensing of compounds that produce decrease of τF in molecular collisions. An example is the chloride sensing in living cells. The mechanism of response of many cellular chloride sensors is based on collisional fluorescence quenching by Cl− ion that decreases both the intensity and the lifetime. Using SPQ (6-methoxy-N-[3-sulfopropyl]quinolinium) chloride-sensitive probe, a comparative study of intensity-sensing and lifetime-sensing was performed and the strong advantages of lifetime sensing were demonstrated (Szmacinski and Lakowicz 1994). Recently, with the development of fluorescence lifetime imaging (Sarder et al. 2015), this method has started to be applied for mapping the microviscosity in living cells (Kuimova 2012; Lee et al. 2018). In this application, the dye molecules that change their fluorescence lifetimes due to their intramolecular rotations resulting in dynamic quenching are used. The photophysical transformations in these dyes, producing the TICT states, are described in Sect. 4.1. In general, it is not only the instrumentation that limits the lifetime-based sensing and imaging. The dyes that exist in two switchable emissive forms with different lifetimes are rare, and new efforts are needed for their synthesis and implementation. In this respect, squaraine dyes show promise for different applications including

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immunoassays with near-IR lifetime detection (Povrozin et al. 2009). The range of collisional quenchers as potential targets is also very limited. The dyes that gradually change their lifetimes may be of use as oxygen and viscosity sensors based on quenching effects. Variation of lifetimes of excitation energy donors is another possibility for implementation in sensing.

3.4.5 Phosphorescence and Long-Lasting Luminescence Phosphorescence exhibits much longer emission lifetimes than that of fluorescence. Due to high probability for its quenching in thermal diffusion, it is commonly observed only at cryogenic temperatures. But in special conditions (incorporation of dye into a rigid matrix and removal of mobile oxygen) it can be observed also at room temperatures with lifetimes covering a very broad range 10−6 —102 s. Rigidity of dye environment can be provided by the sol-gel or polymeric materials. The long duration of phosphorescence emission is because it involves a spin-forbidden transition from the triplet excited state to the ground state. Due to this fact, the quantum yield of phosphorescence is usually very low. Meantime there are the dyes that incorporate heavy atoms (e.g. eosin, erythrosine), so their bright phosphorescence can be observed at room temperatures (Sect. 5.4). Such enhancement of phosphorescence (the Kasha heavy atom effect) was known for many years. Optical sensors with phosphorescence response are expected to feature rapid advancement with the development of new dyes of pure organic origin (Koch et al. 2014; Xu et al. 2013), incorporating heavy and transition metal atoms and of nano-composites hosting them. Phosphorescence spectra are usually insensitive to environment perturbations, whereas the lifetimes may change by orders of magnitude. This makes phosphorescence very attractive for the development of sensor techniques based on decaytime measurements (Sanchez-Barragan et al. 2006). Present applications of phosphorescence lifetime sensors are addressing mostly the sensing of oxygen. Oxygen is a potent collisional quencher of phosphorescence and this effect is very strong due to long duration of this emission in comparison to oxygen diffusion rate. However, the current applications include sensing pesticides, antibiotics and CO2 (Sanchez-Barragan et al. 2006). Switching between fluorescent and phosphorescent emissions can be traced both in lifetime and λ-ratiometric (Sect. 3.6) domains. In addition to long emission lifetimes, phosphorescence is usually observed with strong Stokes shifts and strong temperature-dependent quenching. In the λ-ratiometric sensing technologies, only those dyes can be used that display strong room-temperature phosphorescence with the steady-state intensity comparable to that of fluorescence. They include platinum and palladium ions incorporated into porphyrins (Papkovsky and O’Riordan 2005) and chelating complexes of ruthenium (Castellano et al. 1998; O’Neal et al. 2004) that can be included into solid polymer matrices. Since phosphorescence band is strongly

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Excita on pulse Fluorescence Long-life me luminescence

Intensity

Fig. 3.11 The time-resolved measurements in the case of long-lifetime emitters. With time delay when collecting luminescence quanta the contribution of light scattering that appears with the short excitation pulse and the short-lifetime background fluorescence can be removed. In order to increase the intensity, the integrated signal can be collected

3 Fluorescence Detection in Sensor Technologies

Integrated signal

Time delay

Time, ms

shifted to longer wavelengths, a very precise, convenient and self-calibrating detection can be achieved in a steady-state λ-ratiometric recording (Hochreiner et al. 2005). Long lifetimes in the millisecond time range are characteristic also of luminescent lanthanide chelating complexes and phosphorescent metal-ligand complexes of ruthenium, rhenium and osmium ions (Zhang et al. 2018). Long-lifetime emission was also found in porphyrins incorporating platinum or palladium atoms. The properties of these luminophores will be discussed in Sects. 6.2 and 6.3. Being different in the mechanisms of their emission, they share common features regarding the benefits of emission decay detection on an extended time scale. Attractive is the simplicity of time-resolved detection technique compared to fluorescence. It becomes easy to discriminate the light-scattering background and contaminating fluorescence just by setting the time delay removing the ‘early’ channels in acquiring the data. Moreover, an integrated signal can be collected from light quanta emitted with specified time delay (Fig. 3.11). The latter advantage makes phosphorescence and long-living luminescence sensors attractive for intracellular studies and for sensing in different heterogeneous systems, in which the background emission creates the problems. In view that all listed above types of long lifetime emission demonstrate also the strong Stokes shifts and high spectral resolution together with high temporal resolution, they feature very good prospects in sensing and imaging fields.

3.4.6 Comparison with Other Fluorescence Detection Techniques Before starting the attempts to apply lifetime sensing for detecting particular target, one has to understand clearly that the cases when one of the forms of reporter fluorophore (with bound or unbound target) is quenched statically (becoming nonfluorescent) being very good for intensity measurement, cannot be analyzed with

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time-resolved technique. This is due to the fact that the static quenching changes the number but not the lifetimes of emitting species, so that the target binding will remain unnoticed. In contrast to static quenching, collisional quenchers produce dramatic change in lifetime. These features explain why the best results in application of lifetime sensing were obtained for determining the targets that are strong collisional quenchers of fluorescence (oxygen, chloride, sulfur dioxide, NO). Meantime, the effects of dynamic molecular collisions with the target are different from that of target binding: in these cases one can observe a gradual change of lifetime as a function of concentration of the quencher instead of intensity redistribution between two lifetimes belonging to forms with bound and unbound target (see Fig. 3.10). These facts indicate the necessity of synthesizing the “lifetime-sensing” dyes, for which the response in to environment changes could be significant. Very beneficial for lifetime sensing must be the cases when the sensor in the unbound form emits light with a short but well-resolved , and on the target binding the lifetime increases substantially. This condition can be realized with careful selection of the dye and the site of its binding (Guo et al. 1998; Kuwana and Sevick-Muraca 2002). Thus, the lifetime sensing can be recommended as a more advanced substitute of intensity sensing in the cases when the local sensor concentration varies and is unable to be measured or controlled, e.g. in microscopy. The significant expansion of assay methods combining highly time and spatially resolved fluorescence data by fluorescence lifetime imaging is expected (Meyer-Almes 2017), particularly for sensing inside the living cells (Sarder et al. 2015). Despite the dramatic loss of emission intensity, this method has the advantages over intensity and anisotropy measurements in all cases where the stray and scattered light is an important problem. It is reasonable to use it in the cases when there are no substantial spectral changes on target binding and the wavelength-ratiometric methods cannot be applied, also in high-throughput screening (Skilitsi et al. 2017).

3.5 Wavelength-Shifting Sensing Most of the information that human brain obtains from the surrounding world is acquired by visual perception. Colored vision and characterization of visual objects in real color is an important contribution to this process. Therefore it is not surprising that in spectroscopy and in its combination with microscopy the attempts to obtain information in real color in the visible range of spectrum are in the minds of many researchers. This situation was not changed with the application of electronic photometric devices, since the spectral shift information is often easier obtained and more unambiguously interpreted than the information obtained in the studies of anisotropy and lifetimes. Therefore, the fluorescent dyes that change the color of their emission are in great demand. In many respects the application of these dyes may substitute different sensing technologies based on excimer formation and resonance energy transfer (Sect. 3.6), since single labeling is always easier than the labeling with two dyes.

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According to the effects that are produced in original spectra, the λ-ratiometric dyes can be classified into two groups. One group combines the wavelength-shifting probes, in which the ratiometric effect is produced by the spectral shifts of a single band displayed in emission spectrum. Their properties and applications are overviewed in this Section. The other group of λ-ratiometric dyes, the two-band ratiometric, allows achieving interplay of two emission bands intensities. Such reporters will be discussed in the next Section.

3.5.1 The Physical Background Under the Wavelength Shifts The background of wavelength shifts in excitation or emission bands is the change of energy of correspondent electronic transitions under the influence of intermolecular interactions. Theory of these effects and the results of numerous experiments can be found in different textbooks (Lakowicz 2006; Suppan and Ghoneim 1997), and here we provide only their simplified description directed for practical use. It has to be recollected that any molecules that are able to absorb light possess discrete energy levels in the ground state (the electronic state in which it resides at equilibrium, without excitation) and in the excited state (the electronic state achieved on absorption of light quanta). Molecule can absorb and emit the light quanta only of the energy that corresponds to the energy gap between these states. This gap is inversely proportional to the wavelength position of the band maximum. In spectroscopy, the wavenumber scale ν (expressed in cm−1 units) is commonly used as an energy scale, so that ν (cm−1 ) = 107 /λ (nm). The spectrum recorded on a wavelength scale is in fact a reverse function of absorbed or emitted energy. The energies of both ground and excited states are influenced by intermolecular interactions: the stronger are the interactions—the lower is the correspondent level on an energy scale. This is true for the energies of both ground and excited states. But since not only the energy but often the spatial distribution of electrons changes dramatically on excitation, these states interact differently with surrounding molecules. If the interaction with the surrounding is stronger in the ground state, then, when this interaction increases, the difference in energy between the states increases. Then the molecule absorbs and emits light of higher energies and the spectra become shifted to higher energies, i.e. to shorter wavelengths. On the opposite, if the interaction energy is stronger in the excited state, then on increase of this interaction the spectra shift to longer wavelengths. For illustration, one may refer to classical Jablonski diagram that, for the purpose of our discussion, was modified and simplified (without showing vibrational levels, high-order electronic states and intramolecular relaxations). It is presented in Fig. 3.12. In highly fluorescent organic dyes the lowest ground-state and excited-state energy levels are formed by π-electrons. Commonly the π-electronic system with the inclusion of hetero-atoms becomes more polarized in the excited state, and becoming a stronger dipole it interacts stronger with its molecular environment. As a result,

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λ

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λ

λ

Fig. 3.12 A simplified Jablonski diagram of ground S0 and excited S1 energy levels and transitions between them. Vertical upward arrow shows the excitation and downward arrows show emissive (straight) and non-emissive transitions to the ground state. In condensed media, the energies of ground and excited states are decreased due to electronic interactions with the environment by arising from solute-solvent interactions solvation energies Wg and We correspondingly. In addition, in polar media there occurs an establishment of equilibrium in interactions of dye and surrounding dipoles (dipolar relaxation). As a result, the energy gap between S0 and S1 states decreases and the spectra shift to longer wavelengths. Typical absorption (blue) and fluorescence spectra (green, orange and red) are presented below

the energy gap between the ground and the excited states becomes smaller and the spectra shift to longer wavelengths. There are possibilities to increase this effect by introducing different chemical substitutions polarizing the π-electronic system. If we attach an electron-donor group (such as dialkylamino-) on one side of molecule and an electron acceptor (such as carbonyl) on its opposite side, the charge separation in the excited state becomes greater. Molecule will become a strong electric dipole, its interactions with surrounding dipoles will become stronger and the emission spectrum will exhibit stronger shift. Such structures are often called ‘push-pull structures’. The structures of the so-called ‘polarity-sensitive’ dyes follow this principle. The higher is polarity of the medium, the stronger is the shift of fluorescence spectra to longer wavelengths. Usually the dyes that exhibit the changes in emission spectra are called λ-ratiometric because they allow a convenient quantitative characterization of the changes in

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emission spectra by evaluation of intensity ratio at two selected wavelengths. The structures and properties of these dyes will be discussed in Sect. 5.3.

3.5.2 The Measurements of Wavelength Shifts in Excitation and Emission The shift of the whole fluorescence band results in increase of intensity at one of its edges and decrease at the other. As the information channels for collecting the fluorescence intensities one can use two selected wavelengths at the two edges, in which the effect is the strongest (Fig. 3.13). Commonly, these are the wavelengths of the maximal slopes of the spectrum (the wavelengths of maximum and minimum of its first derivative). Then, taking the intensity ratio at these points, one may benefit from two-channel λ-ratiometric measurements. Meantime, it should be kept in mind that the intensities at the slopes of the spectra are about one half lower than at the band maxima and the presence of impurities in the sample and light-scattering effects may influence differently the intensities at these short and long-wavelength wings. Wavelength shifts in excitation spectra are not frequently used in sensing. They are measured mostly for electrochromic dyes (e.g. aminostyryl derivatives) that are strongly sensitive to a variation of local electric fields (Sect. 5.3). Based on them, the binding of functional dyes to cell membranes and their phospholipids analogs (liposomes) allows the detection of variations in membrane potential (Gross et al. 1994). Also, the collective effects of charges that can be generated in phospholipid bilayers may be used in the development of nano-scale and whole-cell biosensors. Wavelength-ratiometric measurements of spectral shifts in emission are used more frequently. Some dyes exhibit very significant (up to 100 nm and more) shifts in wavelength positions of their fluorescence spectra in response to changes in their interaction with the molecular environment (Valeur and Berberan-Santos 2012). These

a

b

F2

F1

F1

F2

F2

F1

F1

F2 λ1

λ2

Wavelength

λ1

λ2

Wavelength

Fig. 3.13 Informative signal obtained on λ-ratiometric recording at two fixed wavelengths (Demchenko 2013a). a The sensor response is in the form of spectral shift (Nile Red is an example). b The response is in a decrease of intensity of one band λ1 and in an increase at a different band λ2 . Substituted 3-hydroxyflavones are the examples of such behavior. The quantitative measure in this λ-ratiometry is the change in the ratio F(λ1 )/F(λ2 )

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shifts are usually due to a combination of effects of polarity with the effects of intermolecular hydrogen bonding (Vazquez et al. 2005). This H-bonding being stronger in the excited state may produce strong additional shifts. Thus, if the dye is incorporated into the sensor molecule in such a way that its interaction with the environment changes as a result of a conformational change in the sensor or due to direct contact with the target, the event of sensing can be detected by the spectral shift (Sect. 5.3). This principle is used in the design of both chemical sensors and biosensors. It could be ideal to observe the environment-dependent spectral shifts without change or with insignificant change of integral intensity, since this will provide the highest signal-to-noise ratio and the broadest range of variation in intensity ratios. In reality, this condition is difficult to realize because the environment-dependent quenching is the common property of these dyes. The increase of their dipole moment on excitation produces an increase in interactions in the excited state with the environment (see Fig. 3.12) and results not only in the shifts of spectra to the red but also the activation of a number of factors quenching the fluorescence (Demchenko 2005b). This is the reason why the shift to longer wavelengths is often associated with decrease of intensity (Fig. 3.14). Sometimes, this decrease is dramatic. This makes the ratiometric measurements based on wavelength shifts imprecise and subjective to systematic errors. Fig. 3.14 Sensing with the wavelength-shifting fluorophores by recording the intensity ratios at two wavelengths, λ1 and λ2 . a Ideal case, when the target-free and target-bound form (located at shorter wavelengths) of the sensor exhibit similar fluorescence intensities. b Often observed reality when in unbound form, in addition to shift, the spectrum is quenched and broadened

a

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3.5.3 Wavelength-Ratiometric Measurements In principle, some of the important problems observed with the intensity measurements can be avoided if the sensing response is provided by observation of substantial shift of fluorescence band on the wavelength scale. This could allow instead of measuring one parameter, F(λ), obtaining variations of two intensity values at two different wavelengths, λ1 and λ2 , and taking the ratio of their intensities, F(λ1 )/F(λ2 ). In the dynamic range of sensing, this ratio may depend on the concentration of bound target but may not depend on the concentration of the sensor molecules. Photobleaching of the fluorophore that decreases the concentration of responsive sensors will then also be without influence on the result (if, of course, the photodegradation products are non-fluorescent). If the spectrum shifts (for instance, to the red), then the intensity at the short-wavelength slope decreases and at the long-wavelength slope increases (see Fig. 3.13), and this can allow convenient measurement of intensity ratios at two fixed wavelengths. The choice of λ1 and λ2 in the case of spectral shifts can be made in two different ways. One is to choose λ1 at the point of maximal increase of intensity and λ2 at the point of its maximal decrease. Then the dynamic range of change in F(λ1 )/F(λ2 ) will be the largest, and this change can be calibrated as a function of concentration of the bound target. The other possibility is to choose λ1 at the point at which the intensity exhibits maximal change and λ2 at the point, in which the two spectra cross and the intensity does not change at all (isoemissive point). Then λ1 can be chosen either at the blue (short-wavelength) or red (long-wavelength) slope, apart from λ2 and in the region of maximal spectral change. Since commonly the fluorescence spectra are not symmetrical but exhibit a shallower slope at longer wavelengths, then the changes at the blue side are usually stronger and the dynamic range of response broader. However in some cases if the Stokes shift is small, the readings at shorter wavelengths can be subjected to stronger light scattering perturbation. Formally, there is no advantage of wavelength-ratiometric sensing over twowavelength intensity sensing with molecular reference. In both cases the ratio of two intensities at two wavelengths, λ1 and λ2 , is obtained. The only difference is that instead of keeping a permanent value, the intensity at the second (reference) channel (at λ2 ) increases or decreases in a converse manner to its change in the first channel, at λ1 . If for recording the reference signal we select the wavelength at which all the calibrated spectra cross one another (isoemissive point), then we can determine the target concentration using Eq. (3.5). In a more general case, when λ2 is a different wavelength, (e.g. it is the maximum of the second band), then we have to include the factor that accounts for this intensity redistribution, which is the ratio of intensities of free and bound forms at λ2 :    R − Rmin FF (λ2 ) (3.14) [A] = Kd Rmax − R FB (λ2 )

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3.5.4 Application of λ-Ratiometry in Sensing In a number of studies, the fluorescence shifts were detected in response to the change in intermolecular interactions, and their λ-ratiometric display was used in molecular sensing. As an example, a strong red shift of fluorescence spectra of Ca2+ -binding proteins (parvalbumin, troponin C) labeled with acrylodan was demonstrated on interaction with calcium ions (Prendergast et al. 1983). This shift was accompanied by significant decrease of intensity. It was suggested that a conformational change occurs as a result of the ion binding, and the covalently bound dye located in low-polar environment becomes exposed to the protein surface. This idea was further developed for Ca2+ sensing by labeling of single-Cys mutants of troponin C (Putkey et al. 1997). It was also observed that a λ-ratiometeric dye attached to zinc finger responds strongly to zinc binding (Walkup and Imperiali 1996), since the binding induces formation of structured peptide conformation with the screening of fluorescence reporter from the solvent. Some progress was also achieved in fatty acid and saccharide sensing. A significant spectral shift (by 35 nm to the red) was observed for acrylodan selectively attached to Lys residue of fatty acid binding protein upon binding fatty acids (Richieri et al. 1992). This occurs due to a displacement of the dye from hydrophobic fatty acid binding pocket. The blue shifts were observed on binding the ligand to maltose binding protein (Gilardi et al. 1994). A wavelength-shift sensor for saccharides was made based on two asymmetric diphenylpolyenes, in which dimethylamino group served as an electron donor and boronic acid group functioned both as electron acceptor and as the sensor for saccharides (Di Cesare and Lakowicz 2001). These authors achieved a blue shift and an increase of intensity on target binding and recorded a λ-ratiometric response. They also observed the decrease in fluorescence lifetime on sugar binding and claimed that the lifetime sensing may be more convenient observation in this case. The prototype of direct molecular immunosensor based on wavelength shift principle has been described (Bright et al. 1990). For its construction the complex of human serum albumin (HSA) with anti-HSA antibody was formed and subjected to labeling by fluorescent dansyl derivative. This allowed labeling antibody outside the antigen-binding site at unspecified locations of amino groups. After removal of HSA and immobilization on solid support, the antibody becomes reactive to HSA binding demonstrating the blue shift of dansyl emission together with increase of its intensity. The outlined above are rather uncommon cases, in which the environmentsensitive dyes respond to target binding by fluorescence shift that is strong enough for ratiometric measurement. There are many examples, when these shifts were undetected, and therefore the measurements had to be reduced to single-wavelength records of intensity. This problem is clearly seen in the work (de Lorimier et al. 2002), in which a very detailed study of 11 bacterial periplasmic binding proteins modified with 8 environmentally sensitive fluorophores were conducted. The common

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spectroscopic effects on the binding of target ligands were only the changes in fluorescence intensities, whereas the spectral shifts were either insignificant or even totally absent. Only in two cases, of Glu/Asp and glucose binding proteins with the bound acrylodan dye the shifts (13 and 21 nm correspondingly) were sufficient for λ-ratiometric measurements. This insensitivity can only be understood if no significant change in polarity of fluorophore environment occurs on target binding and also if the fluorophores remain highly hydrated and retain hydrogen bonds with water molecules.

3.5.5 Comparison with Other Fluorescence Reporting Techniques Ideally, the wavelength-shift detection is optimal for realizing the concept of direct sensing. It would be ideal to obtain response to intermolecular interactions by spectral shifts only, without the change or with insignificant change of band intensity. In reality, this condition is difficult to realize because the same interactions may result in quenching (Sect. 3.2). Whereas in intensity sensing the strong change in intensity on target binding is a necessary requirement for generating the response, in wavelengthshift sensing it becomes a complicating factor. It is difficult to predict these quenching effects and to analyze them quantitatively. They can appear due to the decrease of energy separation between locally excited and intramolecular charge transfer (ICT) states (described in Sect. 4.1) up to the level inversion, quenching by electron transfer to solvent traps, quenching via newly formed intermolecular hydrogen bonds, or increased thermal quenching due to smaller gap between ground and excited-state energies (de Silva et al. 1997). Intramolecular flexibility in fluorophore may contribute strongly to the quenching effect. These quenching effects follow quite different regularities with respect to the spectral shifts, since the two effects of shift and quenching are often not correlated. The environment-sensitive dyes on the increase of polarity together with the longwavelength shift and decrease of intensity may commonly demonstrate an increase of the width of the spectrum. As a result, the combination of three factors, the shift, the change of intensity and the broadening, results in a shifted component that will be strongly quenched, so that in reality one will observe the spectrum depicted in Fig. 3.14,b. In these cases, the application of common environment-sensitive dyes is not justified, and the fluorophores specially designed for intensity measurements are to be preferred.

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3.6 Two-Band λ-Ratiometry with Single Fluorophore The previous section could give the reader a full impression on the advantages of operation of fluorescence reporter based on achieving the shift in fluorescence spectrum and on the difficulties associated with that. Therefore, the researchers try to develop the fluorescence reporters, in which the sensitivity to the change of intermolecular interactions could be greatly increased. Ideally, it could be achieved using one type of dye molecules (this will eliminate double-labeling), which serves as reporter so perfectly, that in sensing event it switches completely between two ground-state or excited-state forms (Demchenko 2013b). These forms should give separate bands in excitation or emission spectra to produce a dramatic easily recordable change of color. From basic photophysics we may derive that in order to obtain the switching in excitation spectra we need to operate with two or more ground-state forms of the reporter dyes and to couple the sensing event with the transitions between them. In contrast, for obtaining two switchable bands in fluorescence emission spectra, one ground-state form is enough (and preferable), but there should be an excited-state reaction generating new emissive species. This reaction should proceed between discrete energy states, and both the reactant and the product in this reaction should emit strong fluorescence with the shifts in energy. These two cases are illustrated in Fig. 3.15. In the case of ground-state equilibrium the two species are the independent absorbers and emitters, so both their excitation (hν a1 and hν a2 ) and emission (hν F1 and hν F2 ) energies commonly differ. This leads to λratiometric effects in both excitation and emission spectra. In the case of excited-state reaction, the excitation energy hν a1 can be the same but the emission energies (hν F1 and hν F2 ) differ. The λ-ratiometric response is observed only in emission spectra. The excited state with the emission energy hν F2 is populated kinetically and the equilibrium may not be achieved during the lifetime τF .

3.6.1 Generation of Two-Band λ-Ratiometric Response by Ground-State Isoforms The switching of fluorescence signal can be performed between two ground-state forms that may be isomers, different charge-transfer forms or the forms differing in ionization of particular group. If these two forms are fluorescent, they should exhibit different excitation spectra, as it is illustrated in Fig. 3.15a,c. Though the λratiometric recording in excitation is less convenient than in fluorescence and needs two light sources or re-tuning of a single source between two wavelengths, there are different applications of such approach in sensing, particularly when they are based on indication of pH changes or focused on sensing ions. The effects of charge can provide such a strong perturbation of the ground state that could allow switching between the two forms.

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Fig. 3.15 The energy diagrams (a, b) and schematic illustration of the changes in excitation (c) and emission (d) spectra in the cases of ground-state (a, c) and excited-state (b, d) equilibrium between two emissive forms of the sensor. Isobestic in (c) and isoemissive in (d) are the crossing points in spectra, at which the intensity does not change on transition between the two forms of reporter

The Ca2+ -chelating dye Fura 2 (Grynkiewicz et al. 1985) has got great popularity in cellular studies despite its near-UV excitation and low photostability. The reason is the great λ-ratiometric effect on Ca2+ binding modulating the ground-state intramolecular charge transfer (ICT), see Sect. 4.1. There are also many reports on the construction of chemical sensors for other, beside calcium, ions based on ICT mechanism (de Silva et al. 1997). A new seminaphtho-fluorescein platform suggested for ion sensing (Chang et al. 2004) uses the switching between the two ground-state tautomers of the dye that produce different excitation and emission spectra under the influence of the bound ion. Such effect is hard to achieve on recognition of neutral molecules. The dyes highly polarizable in the ground state have found application in the sensing of local electric fields (Callis 2010; Clarke et al. 1995). They belong to the class of electrochromic dyes (see Sect. 5.3) that have got important application in the studies of biomembrane potential in living cells.

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3.6.2 Excited-State Reactions Generating Two-Band Response Within Single Dye in Emission When the effects of two-band switching occur in emission spectra, this offers additional advantages over that in excitation spectra. In this case, for target detection one does not have to change or re-tune the excitation light source, and the λ-ratiometric fluorescence detection can be realized even using the simplest bandpath filters. This advantage can become efficient with the use of dyes possessing strong electron-donor and electron acceptor substituents and exhibiting localized electronic intramolecular charge transfer (ICT) or excited-state intramolecular proton transfer (ESIPT) states (Fig. 3.16), see Sect. 4.1. The excited-state ICT is well described in the literature (Suppan and Ghoneim 1997). It is a basically fast and reversible transition from initially excited A* form to the product B* form. Usually in ICT the low-polar A* state is excited, and the reaction results in the generation of charge-transfer state that is highly polar and strongly interacting with the environment. The shift in excited-state equilibrium to the level of lower energy guides the spectroscopic effect: the stronger is the interaction with the environment of one of the excited-state forms, the stronger will be the shift of correspondent band in fluorescence emission.

Fig. 3.16 The mechanisms of generation of new wavelength shifted fluorescence bands. a FluoA rescent dye A on absorption of light quanta hνA abs emits light quanta hνF from the excited state A* and also exhibits transition to another excited state B* that emits quanta of energy hνB F at different wavelength, usually with a strong red shift. The effect of sensing switches the population of excited-state species between A* and B*, which produces the redistribution of emission intensity between A* and B* emission bands. b The intramolecular charge transfer (ICT). The redistribution of electron density produces the energy change of emitted quanta and observed spectral shifts. c The excited-state intramolecular proton transfer (ESIPT). The change in energy of emitted quanta occurs because of proton transfer along the intramolecular H-bond. Electronic density redistribution also takes place being coupled with proton motion. This results in commonly strongly shifted band in fluorescence emission

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The ICT reaction can often generate the two-band emission in aromatic dyes possessing strong electron-donor and electron acceptor substituents. The possibilities of functional substitutions in some of these dyes are limited, since they may alter dramatically the fluorescence properties. Thus, in bianthryl (the dimer of anthracene) the two bands belonging to initially excited and ICT emissions are well separated and have a different shape with sufficiently high quantum yield. Variations of their relative intensities suggest bianthryl to be a good reporter of polarity and mobility of its environment (Demchenko and Sytnik 1991a, b) that might be used in sensing. However, this dye cannot be functionalized, since any chemical substitution changing the symmetry between two anthracene rings results in disappearance of the ability of switching between the emission bands. The other problem is the poor resolution between normal and ICT bands observed in different dyes. In some of them the ICT state stabilization in polar media is coupled with intramolecular conformational change (twisting) leading to twisted intramolecular charge transfer (TICT) states with higher spectral resolution (see Sect. 4.1). However, significant and often unpredictable quenching effects that are often observed for the forms with separated charge complicate their application. Excited-state intramolecular proton transfer (ESIPT) is one of the most popular response mechanisms that can be generated in organic dyes (Sect. 4.1). In aromatic molecules that contain hydrogen bond donor and acceptor groups located at a close distance, proton can migrate from one group to the other (Fig. 3.16,c) giving rise to a form that emits fluorescence with a substantial and often dramatic shift of spectrum to longer wavelengths (Demchenko et al. 2013; Sedgwick et al. 2018) (see Sect. 4.1). For sensing applications, the molecules exhibiting ESIPT are especially attractive because the switch between the two forms in emission can be modulated by the target. The overall ESIPT reaction is fully reversible, starting from the ground N state and returning to the same state after four-level reaction cycle. Basic events in these reactions can be very fast (subpicosecond), and the needed simultaneous presence of N* and T* bands in emission could be due to two factors (Tomin et al. 2014, 2007): (a) Kinetic. The equilibrium in ESIPT reaction is shifted towards the T* form but the N* and T* forms are separated by energy barrier that makes the reaction slow and comparable in rate with the emission. The barrier appears due to the presence of conformers in configurations unfavorable for the transfer. In protic environments the intermolecular hydrogen bond partners that compete with hydrogen bonds on the ESIPT pathway can be involved. This slows down the ESIPT reaction, and the emission from the N* form occurs in the course of its transition to T* form. (b) Thermodynamic. In this case the ESIPT reaction can remain very fast but the reverse reaction is efficient on a time scale faster than the emission. It allows establishing the equilibrium between the N* and T* forms, so that the two emissions of comparable intensities can be observed. These two cases can be recognized by the difference in fluorescence decay functions (Shynkar et al. 2003), the effects of external collisional quenchers (Tomin et al.

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2007) and also the solvent polarity (Klymchenko and Demchenko Klymchenko and Demchenko 2003) and temperature (Oncul and Demchenko 2006) effects. The identity of long-lifetime components of decays shows that the two forms are in equilibrium. This also means that any fluorescence quenching produces identical decrease of lifetime and intensity of both forms, which allows λ-ratiometric response to be unchanged. In addition to detection and quantitative analysis in sensing, the methods based on λ-ratiometry allow providing rich multiparametric information that includes polarity, H-bonding proton-donor ability and viscosity at the site of location of applied fluorescence reporters, the pH sensing in aqueous media, the sensing of temperature and pressure. These effects depend on the properties of the surrounding medium and may be used as extremely sensitive means for characterizing these properties in the systematic application of fluorescence sensors and probes. One can detect and characterize numerous molecular events based on probe sorption-desorption to surfaces, inclusion into micelles and biomembranes, and attachment to proteins exhibiting conformational changes, self-assembly or aggregation. Such advantages are extremely valuable for microarray technologies (Demchenko 2005a). Among different fluorophores exhibiting ESIPT reaction, the 3-hydroxychromone derivatives (3HCs) are unique. They represent the rare class of organic dyes exhibiting ESIPT, in which the observation of two separate bands in emission can be achieved without involvement of two or more ground-state or excited-state conformers. In 3HCs the proton-donor 3-hydroxyl and proton-acceptor 4-carbonyl groups are attached to a rigid skeleton and coupled by a hydrogen bond. In these dyes the emission is based on a balance between ICT and ESIPT species that occur in dynamic equilibrium. As a result, they exhibit dramatic variation of emission color as a result of ICT-ESIPT reaction (Demchenko et al. 2013). Interplay of these two emissions can be easily observed by λ-ratiometry. By switching between them these dyes demonstrate really dramatic sensitivity of their spectra to polarity (Klymchenko and Demchenko 2003; Klymchenko et al. 2003) and electric fields (Klymchenko and Demchenko 2002b) of their environment and also to intermolecular H-bonding (Shynkar et al. 2004). For more detailed information on their structures and spectroscopic properties see Sect. 5.3. Summarizing, we indicate both the strong and weak points in using molecular sensors based on fluorescence response of single fluorophores. Strong points are: (a) single labeling, so that is much easier to perform and to control the yield and specificity of labeling in chemical and biological systems; (b) easily detectable λratiometric signal that can be realized in all instrumental formats (fluorimeters, microscopes, plate readers, etc.) and (c) internal calibration that is realized on molecular scale, which allows the output signal to be independent of variation of instrumental factors, fluorophore concentration, etc. The weak points are also essential. There are very limited number of fluorophores that allow realizing this type of response, which limits the range of available wavelengths and lifetimes. Despite that, many new developments are observed (Sedgwick et al. 2018). Presently the best in two-band ratiometric response are the 3hydroxychromone dyes exhibiting ESIPT reaction (Demchenko 2006, 2009, 2010;

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Klymchenko and Demchenko 2002a), but the species of this family with excitation wavelengths occupying the range of 380-430 nm are used only. Extension of their emission to red-NIR region was achieved only recently (Kyriukha et al. 2018). The proteins of green fluorescent protein (GFP) family (Subach and Verkhusha 2012) and responsive polymers (Cho et al. 2013) are expected to enrich the range of single-fluorophore ratiometric reporters.

3.6.3 Coupling a Reporting Dye with the Reference In application of two coupled emitters the researcher can choose one of the two basic strategies. First is the introduction of the second fluorophore as the reference (as it was discussed in Sect. 3.2). The reference should not interact with the reporting fluorophore and there can be a broad choice of its location in one of the interacting partners at the periphery of their contact area. The other strategy consists in using actively the interaction between the two fluorophores. In that case more possibilities appear for recording the sensor response in intensity, anisotropy and lifetime domains but the two-band ratiometry in most of these cases is preferential. As it was discussed above, the introduction of reference dye and forming the reporter-reference dye pair allows resolving important problems with intensity sensing (see Fig. 3.5). Here the role of reference is solely to indicate its own presence in particular medium or at particular site and provide robust signal that must be distinguished from that of reporting fluorophore. Therefore, the dye should possess strongly different emission spectrum but of comparable intensity to that of reporter, which could allow determining the analyte concentration in a manner that does not depend (in the required range) on the sensor concentration, using Eq. (3.5). For optimal operation of such system, the direct interactions between the reference and reporter dyes (e.g. leading to PET or EET) that provide modification of dye intensities should be avoided. The requirement on rigid attachment of reference fluorophore to a sensor unit may not be necessary, and its presence in detectable concentrations in illuminated volume is sufficient. An example of the absence of such connection can be the pair of fluorescence probes for detecting of Ca2+ ions in cellular studies. Here two dyes, of similar structure, one responsive to ion binding and the other inert, were incorporated into the cell for λ-ratiometric imaging of ions (Cooley et al. 2013). The problem in such studies is only in potentially different distribution of such dyes in heterogeneous systems (that are the cells), which may lead to incorrect data. The other problem appears in potentially different reaction of spectra between dissimilar dyes on medium conditions (pH, temperature) and the response to dynamic quenching. Also important can be the differences in photostability of the two dyes, since preferential photobleaching of one of the dyes in their pair changes the ratio of intensities at the two observed emission bands.

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3.6.4 Fluorescent Excimers When a molecule absorbs light, its electronic properties change dramatically. It may participate in reactions that are not observable in the ground state. Particularly it can make a complex with the ground-state molecule like itself. These excited dimeric complexes are called the excimers (see Sect. 4.2). Commonly, the excimer emission is quenched, but the lucky exception is pyrene. Its excimer emission spectrum is very different from that of monomer (Fig. 3.17). It is broad, shifted to longer wavelengths and in contrast to monomer it does not contain vibrational structure. These forms are easily resolved in fluorescence spectroscopy. The structured band of monomer is observed at about 400 nm, whereas the long-wavelength shifted spectrum of excimer is located at about 485 nm. The photophysical mechanism of formation of excimers is discussed in Sect. 4.2. Here we note that formation of pyrene excimers is fully reversible, so that a monomer is always excited and an excimer is formed in the excited state. This excited-state complex returns to the state of two monomers after the emission. There are many possibilities to use these complex formations in fluorescence sensing. Just we need to make a sensor, in which the free and target-bound forms of the sensor differ in the ability of reporter dye to form the excimers. Then the fluorescence spectra will report on the sensing event. Designing the excimer-reporting sensor, researcher is limited in selection of reporter dyes. Usually pyrene derivatives are used as excimer formers because of unique property of this fluorophore to form stable excimers with fluorescence spectra and lifetimes that are very different from that of monomers. Other dyes are commonly not useful or less convenient for this application. Double labeling with two monomers is commonly needed and they should be located in such a way that their formation or disruption should be coupled with target recognition event. A conformational change or self-assembly is needed in sensor molecule to couple or to remove apart the dye Fig. 3.17 The fluorescence spectra of pyrene in monomer and excimer forms

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monomers (Yang et al. 2003). In the case of such sensor performance all the benefits of λ-ratiometric sensing become possible.

3.6.5 The Systems with Excitation Energy Transfer (EET) The energy attained by one fluorophore on electronic excitation can be transferred to another fluorophore which is located closely. Such transfer occurs with the loss of energy and this means that the dye that accepts this energy, the acceptor, should emit light at longer wavelength than the dye that donates this energy, the donor. If the switching of fluorescence emission between donor and acceptor is coupled with the sensor response, this allows providing the λ-ratiometric sensing. This effect first theoretically described by Theodor Förster is often called ‘Förster Resonance Energy Transfer’ FRET). Others use the term ‘Fluorescence Resonance Energy Transfer’ with the same abbreviation FRET, which in narrow sense refers only to fluorescence. Throughout this book I use the term FRET when the Förster-type mechanism is specified. If only the fact of transfer is present without reference to its mechanism, the term Excitation Energy transfer (EET) is used in this book. The latter is broader and includes other transfer mechanisms (often unspecified) with the account that the transfer can be observed not only in fluorescence but in other excited-state phenomena. A discussion on the FRET mechanisms will be provided in Sect. 4.4. Here we outline some important issues that allow constructing the sensors with the λratiometric recording. Basically, such application is simple. When the communication between the excited and unexcited molecules exists, we observe that on excitation of the donor its emission is quenched and, instead, emission of the acceptor is increased. When this coupling is absent, the emission of the donor is observed only. The FRET efficiency depends on two major factors: the distance and mutual orientation of donor and acceptor and the overlap of emission spectrum of the donor and the absorption spectrum of the acceptor (Fig. 3.18). For particular case of Förster resonance energy transfer between organic dyes the dependence of transfer efficiency on distance R is very steep on the scale of ~ 5-10 nm (Clegg 1996; Selvin 2000). The EET-based technologies are frequently used in sensing, which is indicated in different parts of this book, and a special discussion on its mechanism can be found in Sect. 4.3. Meantime, their realization usually needs labeling with two dyes serving as donor and acceptor. Selection of these partners can be made among different molecular and nanoscale emitters (Sect. 10.3). Only in rare lucky cases, intrinsic fluorescent group of sensor or target molecules can be used as one of the partners in EET sensing.

3.7 Sensing and Thinking: The Optimal Choice of Fluorescence …

a D

R

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A

b

Decrease of intensity Decrease of life me

Increase of intensity Decrease of anisotropy

Fig. 3.18 Major factors that determine the efficiency of Förster resonance energy transfer (FRET). a The donor-acceptor distance. At their close location, the excitation of the donor results in emission of the acceptor, and if the distance is large, the donor itself will emit. This produces distancedependent switching between two emission bands. b Overlapping absorption and fluorescence emission spectra of two FRET partners. The light-absorbing and emitting at shorter wavelengths fluorophore (donor, D) can transfer its excitation energy to another fluorophore (acceptor, A) absorbing and emitting at longer wavelengths. For efficient transfer, the absorption spectrum of the acceptor should overlap the emission spectrum of the donor (shaded area)

3.7 Sensing and Thinking: The Optimal Choice of Fluorescence Detection Technique Summarizing the results of this Chapter, we derive that there are not so many possibilities in the choice of fluorescence detection method but many requests to use them in different technologies. The choice of fluorescence response mechanism, of responsive dye and of the method of observation is always a compromise in two directions. One is the compromise in choosing between the brightest dyes that are optimal for fluorescence intensity sensing and the ‘responsive’ dyes that are needed for more complicated methods of observation based on the change in lifetime, excited-state complexes formation, band wavelength shifts or two-band fluorescence switching. The other dilemma is between simplicity in measurements and the need for obtaining high information content of response that also depends on the selected

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detection technique. The choice in this respect is rather limited, but it exists. In addition to intensity sensing, one may choose between anisotropy and lifetime measurements and use spectroscopic detection based on spectral shifts or interplay of two bands used in FRET, excimer sensing or sensing by generating the excited-state reaction. Intensity sensing is the simplest technique, but it demonstrates many disadvantages mainly due to the absence of absolute scale and the difficulties in applying the internal calibration. Internally calibrated signal can be provided in anisotropy and lifetime sensing. In principle, this can be done in two-wavelength ratiometric recording with the application of two dyes (excimers and FRET) or a single dye exhibiting ground-state or excited-state reaction. Every one of these techniques needs proper selection of reporter dyes. The two-band λ-ratiometric technique demonstrates extreme simplicity of recording the intensity at two fluorescence band maxima combined with the possibility of obtaining strongly enhanced and, essentially, internally calibrated response (Demchenko 2010, 2013b). In Fig. 3.19 the comparative analysis of different fluorescence parameters regarding the possibility of internal calibration of their readout is presented. The conclusion can be made that the single-channel response allows obtaining the internally calibrated signal only in lifetime sensing. In anisotropy sensing the two (vertical and horizontal) polarizations provide the necessary two channels, and in FRET to fluorescent acceptor and in wavelength-shifting response these two channels are selected as intensities at two wavelengths. In two-band ratiometric sensing, the ratio of intensities at two wavelengths is also obtained, but because the signal comes from

Fig. 3.19 Parameters used in fluorescence sensing and imaging. Only the lifetime sensing recording one parameter does not need self-referencing. In other techniques the self-referencing requires the simultaneous recording of two parameters. The spectroscopic changes (the shifts and generation of new bands in excitation or emission) can be transformed into two-channel λ-ratiometric signal

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a single type of the dye and of the forms emitting at two wavelengths have the same lifetimes, the internally calibrated signal is resistant to any uncontrolled quenching effect. Respond to the following questions and make simple calculations: 1.

Why the wavenumber scale is more correct for presentation and analysis of spectra than the wavelength scale? Convert into wavenumbers 365 nm, 436 nm, 630 nm. Convert into wavelengths 14,820 cm−1 , 19,600 cm−1 , 24,300 cm−1 . Convert into wavenumbers the wavelength shift by 10 nm of fluorescence band at 400 nm and at 800 nm. Convert into wavelengths the wavenumber shift by 200 cm−1 at 12,500 cm−1 and at 24,200 cm−1 . Estimate in cm−1 the bandwidths of 50 nm for the bands located in the blue (450 nm) and red (650 nm) ranges of spectra. 2. Let the reporter dye be attached to the surface of sensor molecule and appear on sensor-target interaction at their contact interface. Count all possible effects that can be used to provide its quenching/dequenching response. 3. Explain how to introduce the reference signal into an intensity sensor to make possible its internal calibration. What output readings should then be obtained and how to calculate based on them the target concentration? 4. What are the possibilities to distinguish static and dynamic quenching? Explain the physical processes behind them. 5. How could you distinguish between two interpretations of quenching effect observed in recording the intensity on exposing your sensor to test sample? (A) The target binds to the sensor producing the quenching. (B) There is no target in the sample but, instead, some substance produces dynamic quenching by collisions with the sensor. How the experiments in the steady state or timeresolved studies can resolve these cases? 6. In anisotropy sensing should the free sensor or its recognition segment necessarily rotate? What rotation rate is acceptable (in comparison with the rate of fluorescence decay)? What other possibilities except providing the sensor, target or competitor rotation, can be used in anisotropy sensing? 7. What is the difference between polarization and anisotropy? Calculate the polarization values for anisotropy r 0 = 0; 0.1; 0.2; 0.3; 0.4. 8. Let a total quenching of fluorescence be observed on target binding, so that intensity goes down from high-level values to zero. What lifetime changes will be observed? Will the lifetime sensing in this case be efficient? 9. Explain, why the measured fluorescence anisotropy is sensitive to light scattering effects. What should be done for reducing these scattering artifacts? Explain also, why this problem may not exist in lifetime sensing. 10. Is a geometry change in a sensor molecule necessary for providing the response in excimer formation? Is this change always needed for response in EET? 11. Explain why and in what conditions the sensor response based on the principle of EET between labeled sensor and labeled competitor will be more advantageous than the intensity sensing with labeling of the competitor only.

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12. What is the dynamic range of λ-ratiometric response based on spectral shifts, on what factors it depends? 13. Suggest the sensor design based on a combination of λ-ratiometric response and EET. 14. Explain the idea of signal enhancement by transforming the observed spectral shift into intensity ratio at specified wavelength positions.

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Guo XQ, Castellano FN, Li L, Lakowicz JR (1998) Use of a long lifetime Re(I) complex in fluorescence polarization immunoassays of high-molecular weight analytes. Anal Chem 70:632–637 Ha T, Tinnefeld P (2012) Photophysics of Fluorescence Probes for Single Molecule Biophysics and Super-Resolution Imaging. Annu Rev Phys Chem 63:595 Hall MD, Yasgar A, Peryea T, Braisted JC, Jadhav A et al (2016) Fluorescence polarization assays in high-throughput screening and drug discovery: a review. Methods Appl Fluoresc 4:022001 Han HD, Kim TW, Shin BC, Choi HS (2005) Release of calcein from temperature-sensitive liposomes. Macromol Res 13:54–61 Hochreiner H, Sanchez-Barragan I, Costa-Fernandez JM, Sanz-Medel A (2005) Dual emission probe for luminescence oxygen sensing: a critical comparison between intensity, lifetime and ratiometric measurements. Talanta 66:611–618 Jameson DM, Croney JC (2003) Fluorescence polarization: past, present and future. Comb Chem High Throughput Screen 6:167–173 Jameson DM, Ross JA (2010) Fluorescence polarization/anisotropy in diagnostics and imaging. Chem Rev 110:2685–2708 Jayaraman S, Biwersi J, Verkman AS (1999) Synthesis and characterization of dual-wavelength Cl–sensitive fluorescent indicators for ratio imaging. Am J Physiol 276:C747–C757 Kapanidis AN, Weiss S (2002) Fluorescent probes and bioconjugation chemistries for singlemolecule fluorescence analysis of biomolecules. J Chem Phys 117:10953–10964 Klymchenko AS, Demchenko AP (2002) Electrochromic modulation of excited-state intramolecular proton transfer: the new principle in design of fluorescence sensors. J Am Chem Soc 124:12372– 12379 Klymchenko AS, Demchenko AP (2003) Multiparametric probing of intermolecular interactions with fluorescent dye exhibiting excited state intramolecular proton transfer. Phys Chem Chem Phys 5:461–468 Klymchenko AS, Pivovarenko VG, Ozturk T, Demchenko AP (2003) Modulation of the solventdependent dual emission in 3-hydroxychromones by substituents. New J Chem 27:1336–1343 Koch M, Perumal K, Blacque O, Garg JA, Saiganesh R et al. (2014) Metal-free triplet phosphors with high emission efficiency and high tunability. International Edition. Angew Chem 53:6378–6382 Koronszi I, Reichert J, Heinzmann G, Ache HJ (1998) Development of submicron optochemical potassium sensor with enhanced stability due to internal reference. Sensors Attenuators B 51:188– 195 Kuimova MK (2012) Mapping viscosity in cells using molecular rotors. Phys Chem Chem Phys 14:12671–12686 Kuwana E, Sevick-Muraca EM (2002) Fluorescence lifetime spectroscopy in multiply scattering media with dyes exhibiting multiexponential decay kinetics. Biophys J 83:1165–1176 Kyriukha YA, Kucherak OA, Yushchenko TI, Shvadchak VV, Yushchenko DA (2018) 3Hydroxybenzo [g] chromones: fluorophores with red-shifted absorbance and highly sensitive to polarity emission. Sens Actuators B: Chem 265:691–698 Lakowicz JR (2006) Principles of fluorescence spectroscopy. Springer, New York Lakowicz JR, Gryczyski I, Kuba J, Bogdanov V (1994) Light quenching of fluorescence: a new method to control the excited state lifetime and orientation of fluorophores. Photochem Photobiol 60:546–562 Lavis LD, Raines RT (2008) Bright ideas for chemical biology. ACS Chem Biol 3:142–155 Lee SC, Heo J, Woo HC, Lee JA, Seo YH et al. (2018) Fluorescent molecular rotors for viscosity sensors. Chem –A Eur J 24:13706–13718 Liang S, John CL, Xu S, Chen J, Jin Y et al (2010) Silica-based nanoparticles: design and properties. In: Demchenko AP (ed) Advanced fluorescence reporters in chemistry and biology II: Molecular constructions, polymers and nanoparticles. vol 9, Springer Series on Fluorescence, Springer, Heidelberg, pp 229–252 McCauley TG, Hamaguchi N, Stanton M (2003) Aptamer-based biosensor arrays for detection and quantification of biological macromolecules. Anal Biochem 319:244–250

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Chapter 4

Photophysical Mechanisms of Signal Transduction in Sensing

In previous chapters we discussed two essential functional components of every fluorescence sensor—the receptors responsible for specific binding of analytes and the units providing the response signal (reporters). The methods of obtaining and analysis of spectroscopic information were presented. Here we focus on the coupling between these functionalities. Such coupling must involve signal transduction resulting in changes on target binding of the reporter emission. The coupling mechanisms can be based on the excited-state reactions, such as electron transfer (intramolecular or intermolecular), intramolecular charge transfer, proton transfer or excited-state energy transfer. Conformational changes in receptor unit coupled with the target binding may generate the reporter signal if they influence these reactions. In the case of several binding sites coupled with response functionality, nonlinear response functions can be obtained and simple logic operations realized. The proper choice of nechanism of signal transduction and reporting in sensing is the subject of general discussion at the end of this Chapter with questions and problems addressed to the reader. The coupling between binding and reporting functional units is needed for providing the transduction of information on the event of target binding into detectable output signal. It is essential that the process of molecular recognition occurs between the groups of atoms of interacting molecules, particles or interfaces in the ground electronic states but fluorescence reporting requires excited states. The latter often exhibit profound changes in electronic structures and their energies. Thus, the coupling mechanisms should exist that connect these two types of events. We call the realization of this coupling the transduction and the molecular or supramolecular structure responsible for that—the transducer. In this Chapter we focus our discussion on the mechanisms of transduction that can be the most easily realized in direct sensing, in which the target-binding and fluorescence signal-generating events are coupled without additional manipulation or reagent addition (Sect. 2.4). All that gives the researchers a variety of possibilities for optimizing all three functions of fluorescence sensor: recognition, transduction and reporting.

© Springer Nature Switzerland AG 2020 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-030-60155-3_4

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Signal transduction Single emitters Protonation/deprotonation Fluorescence/phosphorescence Hydrogen bonding Ground-state charge transfer Excited-state charge transfer Electrochromism Excited-state proton transfer

Double emitters Reporter – reference Monomer – excimer FRET donor – acceptor Quenching in H-dimers Plasmonic enhancement and quenching

Multiple emitters Antenna effect Wavelength converting Lifetime increasing Excitonic enhancement Plasmonic enhancement and quenching Superquenching

Fig. 4.1 Schematic presentation of diversity in signal transduction mechanisms that can be realized operating with single, double and multiple emitters

Different photophysical processes will be considered here (Fig. 4.1). They include intra- and intermolecular processes of electronic charge transfer or the proton transfer. An additional possibility is the coupling of conformational variables and the nanoscale association phenomena with individual and collective response of fluorescence emitters. The ground-state and excited-state transformations are commonly governed by different types of molecular forces, which sometimes allow obtaining multiparametric response from a single dye. Introduction of two fluorophores (similar and differing in optical parameters) and generation of fluorescent nanocomposites expand dramatically the range of applications allowing the energy transfer in different formats. Different means to provide the informative response in excitation and emission spectra using all these individual and composed reporters are extensively discussed elsewhere (Demchenko 2010), and we extend this discussion incorporating new information. Finally we show that the relay mechanisms of signal transduction allow not only providing a simple connection of binding and reporting events, but also performing more complicated logic operations. According to a more general definition, transducer is a device that is activated by a signal from one system and provides this signal (often in another form) to a different system. Therefore, collecting and recording of fluorescent emission and transforming it into electrical signal by the measurement system is also a signal transduction. Such signal transduction on the instrumental level is different in essence; it will be discussed together with proper instrumental tools in Chap. 15. A variety of organic dyes can respond to the changes in their weak interactions with surrounding molecules or groups of atoms. The ground-state interactions generate the differences in both the absorption and excitation spectra (Sect. 3.6). In the ground state, the species displaying multiple light-absorbing states are usually in equilibrium. This allows the spectroscopic response to follow the structural transformations in the studied system and generate the reporting signal. The two ground-state forms of

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the dye reflecting two states of the system and differing only by their intermolecular interactions can be considered as distinct species with their own characteristic excitation and emission spectra (see Fig. 3.15). Different situation is observed when the ground-state interactions are the same but the spectral change on target biding appears as a result of the excited-state reaction. In this case, the new bands belonging to reactant and reaction product forms appear in fluorescence emission spectra only. Other parameters (anisotropy, lifetime) may change also, providing informative signal.

4.1 Basic Signal Transduction Mechanisms: Electron, Charge and Proton Transfer Fluorescent, or in general, luminescent states of molecules or nanoparticles are the electronic excited states. Electrons in these states are very reactive, and this allows starting the reactions that commonly do not happen in the ground states. The most fundamental of them are the electron, charge and proton transfers. These reactions are reversible in a sense that in a cycle [ground-state excitation → excited-state reaction → relaxation to reaction product ground state → transition to initial ground state] the same initial ground-state species are restored. The interest to these reactions from the side of sensing technologies is due to the fact that they allow one-step deactivation of excited state (quenching), the spectral shifts and, notably, the generation of new emission bands belonging to the products of these reactions. These reactions can be modulated by target binding forming non-covalent interactions with the groups of atoms, molecules and surfaces that behave not only as enhancers or quenchers but also as switchers of the excited-state reactions. This allows realizing many different possibilities for signal transduction.

4.1.1 Photoinduced Electron Transfer (PET) Electronic excitation is the gain of energy by electronic structure, which allows its participation in different reactions. Because of that, the excited species can be considered as redox sites that can donate or capture electrons from other sites, being either oxidants or reductants. Photoinduced electron transfer (PET) is the process by which electron moves from one excited site to another site. Important requirements for this reaction are the closeness in space of two sites and matching their oxidationreduction potentials. The need for crossing of electronic wave functions of initially excited and product states determines the distance dependence for this reaction. PET can be facilitated by covalent bonding between donor and acceptor via a short linkage (spacer). In PET systems only one of the states (reactant or product) is commonly lightemissive, and usually it is the initially excited reactant state. When the electron

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+

-

+

-

Fig. 4.2 Signal transduction in the simplest PET-based sensor. In a free sensor molecule the fluorescence is quenched due to efficient electron transfer from donor to acceptor fragments separated by a spacer. Cation binding produces strong electrostatic attraction of electron from the donor fragment preventing the electron transfer. Fluorescence is enhanced

exchange between electronic systems starts from singlet (fluorescent) excited state of the donor, commonly the return is back to the ground state from the non-fluorescent acceptor. Consequently, the reaction leads to complete quenching of fluorescence emission. If the electron donor has a high HOMO (highest occupied molecular orbital) level, its transfer is efficient to acceptor with LUMO (lowest unoccupied molecular orbital) level (Fig. 4.2). The solvent can also participate in this process influencing the energies of correspondent states. In signal transduction systems based on PET, the relay is in fact a spacer that separates the electronic systems of donor and acceptor. The transduction effect on binding the target species can be realized as a change in redox potential of the receptor that suppresses PET reaction giving rise to emission of the donor. Both donor and acceptor can be the fluorescent dyes that may serve as reporters; in these cases also the excited-state energy will be lost as a result of PET. On excitation, the electronic potential of any dye changes dramatically and it may become better electron donor or acceptor than its closely located PET partner. This could lead to PET occurring usually much faster than the emission and that is why the fluorescence can be totally quenched. This process can be exploited in sensor designs of different complexity (Daly et al. 2015; de Silva et al. 2001; Valeur and Leray 2000). Consider the simple case, in which the fluorescent reporter segment is an electron acceptor and the donor is the group of atoms, which is a part of the binding site. Let the target be a metal cation bearing positive charge (see Fig. 4.2). When the target binding site is not occupied, the absorption of light quantum by the reporter makes it a strong electron acceptor. The electron jumps to it from the receptor site resulting in quenching. This state is called the OFF state. When the ion is bound, the situation becomes different. The ion attracts the oppositely charged electron from the receptor, and PET to excited reporter becomes energetically unfavorable. In the absence of PET, the reporter dye

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remains a bright emitter. This is the ON state. The relative fluorescence intensity can be calibrated as a function of ion concentration. Such simple realization of PET has found application mostly in the sensing of ions (Volume 2, Chap. 9). In a typical ion sensor, two electronic systems, one participating in ion recognition and the other used for reporting, are connected by a short spacer (Valeur and Leray 2000). Cation binding produces the strong attraction of electron with the suppression of PET. Different molecular constructs were suggested for sensing anions (Gunnlaugsson et al. 2005). Sensing of neutral molecules in this way can also be made, though with limited success (Granda-Valdes et al. 2000). The schemes that are more complicated allow excluding the target binding from direct influence on PET. For instance, this can be done by influencing the ionization of an attached group (Koner et al. 2007; Tomasulo et al. 2006) and thus extending this transduction mechanism to a much broader range of analytes. PET can occur not only through bonds but also through space, and for this process the most important is the close distance between donor and acceptor. Therefore, in sensing event the donor and acceptor groups can be put together or moved apart due to a conformational change in the sensor (Sect. 4.4). Thus, one more possibility offered by PET mechanism is the association or dissociation of nanoparticles: on association they can play a role of PET donor or acceptor. Noble metal nanoparticles have proved to be the most efficient electron acceptors from different types of fluorescent donors, including semiconductor quantum dots. The latter can be reduced or oxidized at relatively moderate potentials, which tend to vary slightly with the physical dimensions of these nanocrystals (Burda et al. 2005). Therefore, they can be used as efficient electron donors, and this possibility of signal transduction makes them efficient fluorescence reporters. Transition metal cations (such as Cu+2 ) and free radicals quench fluorescence according to PET mechanism. This property can be used for their detection. The PET quenching in conjugated polymers (see Sect. 8.5) deserves special attention. Extremely low amounts of cationic electron acceptors quench the fluorescence of polyanionic conjugated polymer on a time scale shorter than picosecond, so that a greater than million-fold sensitivity (superquenching) is achieved in comparison with the so-called ‘molecular excited states’ (Chen et al. 1999). Nanocrystalline materials such as quantum dots suggest new strategies for optimal exploration of PET phenomenon. Below we will discuss in more detail three important cases of PET that are frequently used in sensing. (a) Electron transfer between the fragments of the same dye molecule. If a single dye molecule contains two localized electronic systems, the one-electron oxidationreduction in the excited state results in quenching. A proper construction of sensor units allows achieving a complete “ON-OFF” sensor behavior (Daly et al. 2015; de Silva et al. 2001). The PET process can be modulated by the target binding at the electron donor or acceptor sites by changing the donor or acceptor strength. Therefore, it is well understood why this mechanism can be applied most efficiently to ion sensing if the ion binding by the ion-chelating group

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is coupled with a π-electronic system. Its application in biomolecular sensing is more difficult since this needs a rather strong electrostatic perturbation of electronic properties. (b) Intermolecular electron transfer. At short distances an electron can be captured by an excited-state fluorophore acting as electron acceptor from a closely located ground-state electron donor, which also results in quenching. The application of fluorescence response based on this principle to DNA beacon technology (Knemeyer et al. 2000) allowed achieving direct homogeneous assay without double labeling of molecular beacons. The role of fluorescence quencher is played by properly located guanosine residue (Heinlein et al. 2003). In sensor proteins, the conformational changes that form or disrupt the PET donoracceptor pair may produce a dramatic change in fluorescence response. In this case, the attached organic dye can serve as a PET acceptor, and intrinsic aromatic amino acids, Trp and Tyr, can be used as the ground-state electron donors (Marme et al. 2003). Based on this principle, the antibody fragments were suggested as versatile molecular sensors (Abe et al. 2011; Jeong and Ueda 2014). Due to PET from Trp residue to attached organic dye, their fluorescence is quenched. Its enhancement is observed on antigen binding because of disruption of Trp-dye contact. (c) Quenching by spin labels. Stable nitroxide radicals are known as strong fluorescence quenchers. Their basic mechanism of quenching is also electron transfer, and its realization also requires a close proximity of quencher to fluorescent dye. But in this case, the fluorophore always serves as electron donor and nitroxide as an acceptor. Such quenching effect is known for a long time (Blough and Simpson 1988), and the covalent spin-labeling of proteins and polynucleotides have been described. Whereas the spin-labeled lipids are frequently used for determining the location of proteins and other fluorescent compounds in the membranes (Chattopadhyay and London 1987), the applications of nitroxide radicals in sensing technologies are still very rare. Meanwhile, regarding the double-labeling methods, they offer some advantages, mainly because of much smaller size of nitroxide group than that of common fluorescent dyes and due to the total “ON-OFF” effect of quenching. In addition to sensing based on proximity, they offer an interesting possibility of sensing based on variation of redox potential of their surrounding (Blough and Simpson 1988), since the reduction of nitroxide radical removes its ability of quenching. Based on this property the method for determination of ascorbic acid has been suggested (Lozinsky et al. 1999). Double (spin and fluorescence) sensing molecules afford various utilization possibilities (Bognar et al. 2006). It is essential to emphasize that in contrast to excitation energy transfer (EET) (Sect. 4.3), in PET quenching the emitting donor and the acceptor should be located at much closer mutual distance, usually forming a direct contact (Heinlein et al. 2003; Jeong and Ueda 2014). PET quenching may also occur on the formation of dye dimers and aggregates and can be the origin of “concentrational quenching” effects

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(Sect. 8.1). In this case it occurs between the unexcited and excited monomers, and this effect can also be used in sensing (Johansson and Cook 2003).

4.1.2 Intramolecular Charge Transfer (ICT) Intramolecular charge transfer (ICT) is, in principle, also an electron transfer. The difference is that ICT occurs within the same electronic system or between the systems with high level of electronic conjugation between the partners, and it has its own characteristic features. The electronic states achieved in this reaction are not the ‘charge-separated’ but ‘charge-polarized’ states. Still they can be the localized states with distinct energy minima (Misra and Bhattacharyya 2018). The two, PET and ICT, states are easily distinguished by their absorption and emission spectra. In PET, the strong quenching occurs without spectral shifts. In contrast, the ICT states are often fluorescent but exhibit the changes of intensities. In addition, their excitation and emission spectra may exhibit significant shifts that depend on the environment. This allows providing the λ-ratiometric recording (see Sect. 3.5). In some cases a switching of intensity between two emission bands, normal (often called the locally excited, LE) and that achieved in ICT reaction, can occur. This is even more attractive for λ-ratiometric measurement. Commonly the ICT states are observed when the organic dye contains an electron donating group (often a dialkylamino group) and an electron-withdrawing group (often, carbonyl). If these groups are located at opposite sides of molecule, an electronic polarization is induced. Because the electron donor becomes in the excited state a stronger donor and an acceptor a stronger acceptor, an electronic polarization can be substantially increased in the excited-state. A created large dipole moment interacts with the medium dipoles resulting in strong Stokes shifts. The change in the medium conditions can produce the LE-ICT switching. Fluorescence from the normal LE state is commonly observed in low-polar solvents and in cryogenic conditions, where the spectra may contain residuals of vibrational structure. At an increase of polarity and temperature the ICT fluorescence appears, which has a broad and structureless long-wavelength shifted fluorescence band. In polar solvents, these shifts become larger because the solute-solvent dipole interactions are stronger. That is why the so-called ‘polarity probes’ (Sect. 5.3) are in fact the ICT performing dyes. In addition, the ICT states are very sensitive to electric field effects and, therefore, to the presence of nearby charges. Hydrogen bonding in the ground and excited states may contribute to stabilization of ICT states increasing the spectral shifts and formation of new bands in emission spectra (Pivovarenko et al. 2000). On interaction with H-bond donor at the acceptor site they shift the spectra to longer wavelengths and on interaction with proton acceptor at the donor site—to shorter wavelengths. Such effects are usually smaller than that on protonation and occur in the same direction (Wang et al. 2002). Carbonyl groups attached to π-electronic systems are known for generating the strongest Hbonding sensors that are sensitive to solvent environment (Shynkar et al. 2004).

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The electron lone pairs on oxygen atoms allow arrangement of two such bonds, the stronger and weaker ones. Their formation is clearly observed as a strong two-step deformation of fluorescence spectra (Pivovarenko et al. 2000) (see Fig. 5.13). This offers new possibilities for λ-ratiometric sensing based on the formation/breaking of intermolecular H-bonds. From this discussion, it is clear that the realization of ICT offers many possibilities. However, the application of this effect cannot be as broad as that of PET, and only organic dyes (and not all of them) can generate an efficient ICT emission (see Sect. 5.3). Nevertheless, within this family of dyes one may find many possibilities for producing fluorescence reporter signal in sensing. They are: (1) Switching between LE and ICT emissions by the direct influence of target charge (Fig. 4.3). This switching can be produced by binding an ion to either electron donor or acceptor site. The processes occurring on interaction of ion-chelation group (as receptor) with a cation are well described (Valeur 2002). When an electron-donor group is attached to receptor, the cation reduces the electrondonating character of this group. Owing to the resulting reduction of electronic conjugation, a blue shift occurs in the absorption spectra together with decrease of the molar extinction. Conversely, a cation interacting with the acceptor group enhances the electron-withdrawing character of this group; the absorption spectrum is thus red-shifted and the molar extinction is increased. The fluorescence

Interac on with the donor group

A

D

+

A

D

S1

Blue shi

S0 Interac on with the acceptor group

D S1

S0

A D

A

+ Red shi

Fig. 4.3 Spectral shifts of organic dyes as the sensors displaying ICT mechanism. The dye demonstrates electronic polarization with formation electron donor and acceptor sites. The sensor response appears as a result of interaction of a bound cation (+) with an electron-donor (D) or electron-acceptor (A) group

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spectra are in principle shifted in the same direction as those of absorption spectra. An anion produces the opposite effect. As a more recent example, the dye exhibiting ICT reaction was suggested as a sensor to fluoride (Yuan et al. 2007). Fluoride binds to electron-acceptor group and switches its emission from that at 500 nm to that at 380 nm. The excited-state charge transfer makes the electron-donor site strongly positively charged. If a cation binds at this site, it exhibits a strong electrostatic repulsion, up to its dissociation. If this electron ejection happens on a time scale faster than the emission, no binding will be detected in fluorescence spectra. Therefore, more efficient will be to locate the cation binding site close to electron acceptor group, where the binding in the excited state becomes stronger. Following the same logic, the sites for anion recognition should be located at the electron-donor sites. A variety of chelator groups have been suggested as the recognition units for ions, such as crown ethers, cryptands, coronands and calix[4]arenes. The electron donor or acceptor groups can be incorporated into these structures. A more detailed information on the use of ICT mechanism in ion sensing can be found in Volume 2, Chap. 9. (2) Coupling of ICT emission with dynamic variables. The ICT states can be stabilized and modulated by the dynamics of surrounding molecules and groups of atoms. The ICT state possesses strongly increased dipole moment and, in the absence of strong interactions with surrounding dipoles, its energy may become energetically unfavorable being higher than that of the LE state. Thus, the LE emission at shorter wavelengths will be observed in low-polar or highly viscous environments, where the strong dipole interactions are absent or cannot form by dipole rotations at the time of emission (see Fig. 3.12). In contrast, in highly polar environments the surrounding dipoles tend to re-organize towards their equilibrium configuration, stabilizing the ICT state. This allows the ICT emission to be energetically favorable and easily accessible kinetically. Because of stronger interaction, a decrease of excited-state energy occurs, and the emission is shifted to longer wavelengths (Fig. 4.4). Thus, an efficient transduction with the detectable change of reporter spectrum can be provided by the change in dynamics of molecules and groups of atoms at the dye location. (3) Increased environment sensitivity of ICT emission. Position of ICT energy level strongly depends on the strength of dipole-dipole interaction that is the major factor determining the effect of solvent polarity. Exploration of this property allows extending the sensing possibilities to detecting nonionic targets. If the target is a neutral compound, its binding can influence the local polarity in the vicinity of reporter dye. This can be easily realized if the testing has to be made in water. Since water is frequently used as a medium for sensing different analytes, it is worth to mention that it is not only a highly polar liquid but also the medium with very rapid (~1 ps) dielectric relaxations. In sensing in water, different coupled processes can be used, such as conformational change altering the environment of reporter dye or screening the dye from direct contact with water.

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D

A

δe

+

D

A

-

Fig. 4.4 The simplified energy diagram showing the influence of molecular relaxations on the energies of LE and ICT states. The ICT states involve motion of partial charge (δe) within short distances generating strong dipoles that can be stabilized in polar media in the course of relaxation (mainly by orientation of surrounding dipoles) resulting in substantial shifts of fluorescence spectra to lower energies (longer wavelengths)

(4) Quenching of intramolecular charge transfer (ICT) states. The intramolecular charge transfer excited states are characteristic for fluorophores that possess strong electron-donating and electron-accepting groups, and strong quenching of their IST emission can often be observed in polar media and especially in water (Bhattacharyya and Chowdhury 1993; Yatsuhashi et al. 1998). Different factors, such as transition of the dye to low-polar environment can induce fluorescence enhancement in these systems. The explained above general principles of efficient usage of ICT mechanism in sensing is best demonstrated on the example of designing the Zn+2 ion sensor (Sumalekshmy et al. 2007). The two compounds, whose formulas are presented below (Fig. 4.5), one containing two protons (1) and the other one is substituted by fluorine atoms (2). These compounds generate strong shifts in absorption and fluorescence spectra on the binding of zinc ion, and this allows precise λ-ratiometric recording. Compound 2 demonstrates 1:1 ligand-Zn+2 binding mode and yields a dissociation constant of K d = 2.4 μM. This compound is strongly asymmetric, which allows an increase of ICT upon excitation, generating in the excited state a strong dipole moment. In this configuration, the metal ion binds to the acceptor rather than to the donor binding site, so that the ion binding increases (but not decreases) the charge-transfer character of the excited state resulting in substantial shifts of spectra to longer wavelengths. Such increase, instead of commonly observed quenching in polar media, makes the Zn-bound state even more intensive in emission. This example demonstrates that the general principles work and can be applicable to a broad range

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a

Wavelength, nm

Fluorescence

Absorbance

b

Wavenumber, 103 cm-1 Fig. 4.5 The ICT-based sensor for zinc ions (Sumalekshmy et al. 2007). a The basic sensor molecule (1) and its fluorinated derivative (2). b Absorption and fluorescence emission spectra for the titration of compound (2) with zinc ions in micromolar concentration range in methanol. Addition of zinc ions results in the absorption band shifted from 339 to 362 nm, and fluorescence spectra from 441 to 497 nm. Quantum yield grows from 35 to 71%

of donor-acceptor fluorophores modified with tailored chelating sites for selective sensing of charged targets. The transition from LE to ICT emission is not always detected as the appearance of a new band. However, in some aromatic dyes possessing strong electron-donor and electron acceptor substituents the ICT band is clearly distinguished showing this excited-state reaction as the transition between two localized states (El-Kemary and Rettig 2003; Rurack 2001; Yoshihara et al. 2003). The ability of these dyes to generate the two-band emission is an extremely valuable property for the design of λ-ratiometric reporters. This approach was used for the first time by the group of Roger Tsien in the development of their famous Ca2+ binding probes Fura 2 and Indo 1 (Grynkiewicz et al. 1985). These dyes serve as good examples of the influence of target binding on the ground-state intramolecular charge transfer (ICT) behavior of stilbene-like dyes. The ion binding results in redistribution of absorbed energy between long and short-wavelength excitation bands. The appearance on Ca2+ binding of a new shortwavelength band at 405 nm (490 nm for a free dye) is the result of IST reaction that occurs on interaction of chelated Ca2+ ion with the electron-donor nitrogen atom.

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In principle, if the excitation spectra are so different, the fluorescence spectra should be different too. But only in the case of Indo-1 the two-band response is observed also in fluorescence. The story is that in the excited states of these dyes due to redistribution of electronic density there appears the positive charge close to the bound ion. This results in ion ejection, and only the ion-free form remains present displaying fluorescence spectrum in emission. In contrast to other molecular Ca2+ sensors, in Indo-1 the electrostatic repulsion in the excited state between nitrogen atom and the bound ion is not sufficient for its ejection from the binding site and this interaction generates a new band in emission. The remarkable property of the dyes demonstrating ICT is their response to applied electric field. When the dye interacts with closely located charged group or with array of these groups (such as in biological membranes), this interaction changes the energies of electronic transitions and results in spectral shifts. This phenomenon is caused by the direct interaction of the ground- and excited-state dipoles with the applied electric field and is known as electrochromism (also called as Stark effect) (Bublitz and Boxer 1997). It allows designing the so-called molecular voltage sensors (Demchenko and Yesylevskyy 2009). Their maximal response should be observed when the dye exhibits substantial change of its dipole moment μ  on electronic excitation, which implies a substantial redistribution of the electronic charge density. This effect also depends on the polarity of the probe environment (dielectric screening reduces this effect) and on its orientation with respect to this field. Specially designed dyes can be applied for recording the electrostatic potential in biomembranes (Demchenko et al. 2009).This effect produces spectral shifts that are commonly observed in excitation spectra with recording of variations of fluorescence intensities at its two slopes. The more convenient for detection are the shifts in fluorescence spectra, but for these dyes such shifts are much smaller (Minta et al. 1989). Below we will see that the excited-state intramolecular proton transfer (ESIPT) reaction can dramatically amplify the electrochromic effect.

4.1.3 Twisted Intramolecular Charge Transfer (TICT) When the dye molecule consists of two or several fragments, the electronic conjugation between them can change during the ICT reaction (Fig. 4.6). This occurs by changing the planarity between the electron-donor and acceptor fragments, since the planar structure produces the best π-π electronic conjugation, and this conjugation is broken. Such disruption of planarity usually occurs with twisting around single bond connecting donor and acceptor fragments. The appearance of localized IST emission is well studied in many systems, and correspondent states got the special name twisted intramolecular charge-transfer (TICT) states (Grabowski et al. 2003). Occurrence of this reaction depends on two major factors. Polarity of the medium decreases the energy of TICT state and thus provides the driving force for its stabilization. The other factor is the fluidity of dye environment that allows or not allows rotation of molecular fragments. These facts suggest the use of TICT dyes as both

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LE TICT

Non-emissive decay

Fluorescence

Fluorescence

Absorption

S1

S0 Fig. 4.6 The Jablonski diagram showing twisted intramolecular charge transfer (TICT) dynamics. Upon S0 → S1 electronic excitation to the LE state the ICT reaction proceeds resulting in intramolecular twisting around the single bond connecting donor D and acceptor A. Non-emissive decay becomes the most important channel of decay to the ground state resulting in quenching of the TICT emission

polarity and viscosity reporters. Intramolecular mobility can be modulated by covalent modifications of the dyes and the changes of their configurations, which suggests many possibilities to generate the sensor response. Different dyes synthesized for these applications have got the name molecular rotors (Haidekker et al. 2010; Lee et al. 2018). The switching from LE to TICT state producing spectroscopic response can be used in sensing. The problem with application of such dyes in sensing is in significant quenching of their strongly Stokes-shifted TICT emission (Sasaki et al. 2016). Therefore their plain application was found only in intensity sensing (Haidekker et al. 2010). With the introduction of reference dye (see Sect. 3.2) they can be used in λ-ratiometric mode (Haidekker and Theodorakis 2016). Also, since the quenching is of dynamic nature, the time-resolved sensing (see Sect. 3.4) can be applied (Kuimova 2012). The major application areas of dyes performing TICT reaction are in probing the polymerization dynamics and mapping the viscosities in living cells (Lee et al. 2018). The most popular though very expensive method of studying microviscosity in cytoplasm and membranes in living cells is fluorescence lifetime imaging (Sarder et al. 2015). The dyes possessing this mechanism of response are prospective for molecular sensing as the reporters that can be immobilized in contact areas.

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4.1.4 Intramolecular Excited-State Proton Transfer Excited states of dye molecules differ from correspondent ground states by the ability to donate proton to the medium or to accept proton. Proton is a particle with a positive charge, so the protonation or deprotonation of a molecule neutral in the ground state makes a charged one in the excited state (Tolbert and Solntsev 2002). Molecules that increase their acidity and more easily loose the protons in the excited states are called ‘photo-acids’, and those that increase their basicity are called ‘photo-bases’. These reactions occur with the participation of solvent. In aqueous solutions two or more forms based on protonation equilibrium can be observed with their shifts as a function of pH. In many organic molecules both proton-bound and proton-dissociated forms are strongly fluorescent and demonstrate different positions of their spectra (Arnaut and Formosinho 1993), so if these effects are coupled with the sensing events, they are easily recorded. Thus the ionization of attached groups (usually aromatic hydroxyls) may change dramatically the spectroscopic properties of organic dyes leading to the appearance of new bands in absorption and emission spectra. The variation of their excitation spectra reflects the ground-state proton dissociation. Since the acidity of dissociating hydroxy groups is commonly much higher in the excited than in the ground state, the pH range of sensitivity of this group attached to aromatic moiety can be dramatically shifted to lower pH in emission spectra (Davenport et al. 1986; Laws and Brand 1979). This allows providing the λ-ratiometric recording in an extended pH range. Presently the most extensively used dyes are the benzo[c]xanthene derivatives, such as C-SNAFL1 (Mordon et al. 1995; Whitaker et al. 1991). The strongly red-shifted second band in emission spectra due to deprotonation of hydroxy group in the excited state can respond to local shift in association-dissociation equilibrium at the site of target binding and can be efficiently used in sensing technologies. One of examples of such developments is the molecular sensor for anions based on switching in protonationdeprotonation equilibrium in a designed coumarin dye mediated by anion binding (Choi and Hamilton 2001).

4.1.5 Excited-State Intramolecular Proton Transfer (ESIPT) The sensor response can be very efficient when the same molecule contains both proton-donor and proton-acceptor groups in a close proximity and connected by Hbond. Then the excited-state intramolecular proton transfer (ESIPT) reaction can proceed. It does not require protic environment and may occur in any solvent, in solid matrix, even in vacuum. The strict requirement is only on the structure of molecule exhibiting ESIPT (Formosinho and Arnaut 1993). Proton-donor group is almost invariably a hydroxyl (Demchenko et al. 2013) and rarely the amino group (Chen et al. 2018). The basic proton acceptor must be either heterocyclic nitrogen atom or the oxygen in the form of carbonyl. The two groups are the hydrogen-bonded

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in the ground state. They form in the excited state a pathway for proton transfer. The excitation leads to dramatic redistribution of electronic density in the fluorophore, so that the proton donors become stronger donors and acceptors—stronger acceptors. Hence appears the driving force for ESIPT (Demchenko et al. 2013; Kwon and Park 2011). The absorption of light quantum by these dyes leads to normal excited-state form (N*), which converts into the excited photoproduct, the so-called phototautomer (T*), by the translocation of proton. The alteration of the electronic configuration in T* state is recognized by fluorescence spectrum strongly shifted to longer wavelengths. The T* relaxation (i.e., the fluorescence emission plus the decay via radiationless channels) leads to the ground-state tautomer (T) that is unstable and undergoes a fast back proton transfer with the recovery of N species, closing the reaction cycle (Fig. 4.7). In many systems, ESIPT is very fast and irreversible on fluorescence lifetime scale and therefore it produces a single ESIPT band in fluorescence spectrum, which

hN

h T

Fig. 4.7 The four-level diagram for a typical excited-state intramolecular proton transfer (ESIPT) reaction. This reaction occurs between the proximate proton donor and acceptor groups connected by a hydrogen bond in the same photoexcited molecule. A number of radiative and nonradiative processes accompany this reaction. The correspondent rate constants k are shown. The 3-hydroxyflavone derivatives were chosen as examples. R and R’ are the hydrogen atoms or their electron-donor or electron-acceptor substituents

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lacks switching ability needed for sensing. However, there are several dye families with a very attractive property of producing two bands in emission, one belonging to initially excited LE state, and the other—to the product of ESIPT reaction. Such dyes have been found among hydroxyphenyl -benzothiazole, -benzoxazole and benzimidazole derivatives. Their two-band switching, dependent on pH and binding of ions, can potentially be explored in sensing (Henary et al. 2007). Switching between normal and ESIPT emissions can be achieved by changing aggregation state of the dye. For instance, 2(2’-hydroxyphenyl)benzoxazole (HBO), a typical dye of this group, forms dispersed aggregates in water exhibiting green ESIPT emission, whereas the dye solution demonstrates blue normal emission (Huang et al. 2006). It must be noted that on a general scale these dyes did not find many applications, mainly because of the presence of several ground-state and excited-state forms (such as rotational isomers) and of strong quenching of some of these forms. However, with proper design these properties can be overcome. In Sect. 5.3 we describe the family of 3-hydroxychromone (3HC) dyes that possess the property to observe normal and ESIPT emissions as two well-resolvable and intensive fluorescence bands. The great interest to these dyes from the side of sensing technologies is due to easy switching between the two forms in response to variations of their weak intermolecular interactions with the environment. In accordance with predictions of Kasha (Kasha 1986), the observed ‘normal’ emission is in fact the emission from ICT state that is separated from ESIPT state by a small energy barrier (Demchenko et al. 2013). The power of these dyes is in the fact that directly, without any side reactions, they combine ICT and ESIPT mechanisms to report on the change of weak interactions with their environment by intensity variations of two bands with different colors in their emission. Azacrown-substituted analogs of these dyes were tested as ion sensors, and the two-wavelength λ-ratiometric response was observed both in excitation and in emission spectra (Roshal et al. 1998, 1999). Smart derivatives of these dyes have found important applications in molecular and cellular studies (Demchenko et al. 2013; Yesylevskyy et al. 2006). The dyes exhibiting ESIPT are very attractive as mediators of sensor signal and its transformation into generated new band in fluorescence spectrum. Here the two spectrally resolvable excited states are connected by the excited-state reaction that can produce emissive species with dramatic (often by 100 nm and more) long-wavelength shifts of fluorescence spectra. Switching between these emissions is a very sensitive indicator of the change in polarity. The electrochromic effect can be also coupled with the ESIPT reaction, which allows displaying it as the ratio of intensities of two bands in emission spectra (Klymchenko and Demchenko 2002).

4.1.6 Future Directions Photoinduced electron transfer can be considered as the most general photophysical mechanism of signal transduction in fluorescence sensing. It can be realized with all fluorescence and luminescence emitters including all kinds of emitting nanoparticles

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(see following Chapters) and in this sense can provide the optimal way of communication between inorganic and organic worlds in the form of exchange of electrons. Usually this exchange results in complete quenching, and different configurations of participating PET donors and acceptors find application as the response elements in sensing. Being so easily achieved and so broadly observed, the PET effect is well-realized only in intensity sensing format (Sect. 3.2), since the quenched states cannot provide reference for time-resolved, anisotropy or wavelength-shifting measurements. There is an active search for PET sensors with dual (LE and PET) emissions, which is possible in principle but very difficult to achieve. In contrast, ICT has a basic ability to generate new bands in emission or produce strong shifts in fluorescence spectra, but this mechanism is available only to organic dyes. Moreover, these dyes should possess certain very necessary elements of structure, such as electron donor and acceptor groups separated in space for providing a high dipole moment when they are excited. It is known that fluorescence in such systems can be strongly quenched in highly polar media, so the design of the best fluorescence reporters based on ICT (or, probably, on TICT) remains for the future. Protonation-deprotonation of fluorescent dyes has found straightforward application in pH sensing, and it can be used on a broader scale when it is coupled with other reactions induced by target binding in water. Signal transduction with the aid of ESIPT is free from this limitation; the informative signal can be obtained in different solvents and upon modulation of solvent/sensor environment. This prospective trend is highly promising for further development.

4.2 Excimer and Exciplex Formation When a molecule absorbs light, its electronic properties change dramatically. It may participate in reactions that are not observable in the ground state. Particularly it can make a complex with the ground-state molecule like itself. These excited dimeric complexes are called the excimers. Excimer emission spectrum is very different from that of monomer; it is usually broad, shifted to longer wavelengths and it does not contain vibrational structure. Particularly this is the case of excimers of pyrene. An interest to pyrene excimers is in the fact that emission spectrum is easy to observe because it is very different from that of the monomer. The interplay of intensities at two selected wavelengths, λ1 and λ2 , with their change in converse manner and can be easily detected (see Fig. 3.17). These two bands in emission are well separated and allow λ-ratiometric measurements. The molecules different in structure can also make complexes with the excitedstate molecule. These complexes are called exciplexes (the term excimer comes from excited dimer and the exciplex from excited complex). Some exciplexes are fluorescent (Dinnocenzo et al. 2019), but usually their emission is quenched. The formation of excimers and exciplexes is reversible: on decay to the ground state they break apart into initial species and they may be formed again on excitation. In order to make

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a sensor, we need the free and target-bound forms to differ in the ability to form excimers and exciplexes. Then the fluorescence spectra will report on the sensing event.

4.2.1 The Dyes Forming Excimers and Exciplexes The choice of dyes forming excimers with attractive spectroscopic properties is not large. Pyrene derivatives are preferred by many researchers due to very distinct fluorescence spectra of the monomers and excimers. The structured band of monomer is observed at about 400 nm, whereas that of excimer is broad, structureless and long-wavelength shifted; it is located at ~485 nm. Both forms possess the same excitation spectrum in the near-UV, which is not convenient for cellular studies due to high autofluorescence in this range. But long lifetimes (~300 ns for monomer and ~40 ns for excimer) allows easy discriminating in time-resolved experiment the background fluorescence emission (the latter usually does not extend longer than 3–5 ns). Unfortunately, there are no other fluorophores with such unique properties. The best performing exciplexes are also based on pyrene derivatives (Shen et al. 2019). It has to be noted that the formation of excimers and exciplexes requires close location and proper orientation between the partners (for excimer the arrangement of cofacial sandwich between two heterocycles rich in π-electrons is usually needed). Since these interactions appear in the excited state and do not exist in the ground state, the complexes cannot be distinguished in excitation spectra, and both forms can be excited at the same wavelengths. If the excimer is not formed, in fluorescence spectra we observe emission of the monomer, and upon its formation there appears characteristic emission of the excimer.

4.2.2 Nonemissive Dimers With the exception of pyrene and several other aromatic heterocycles, the excimers are nonfluorescent, thus on their formation we observe fluorescence quenching. The quenching of emission is commonly observed in the exciplexes that are formed between aromatic and polar organic compounds. The organic dyes can also form associates in the ground state known as H-aggregates. Their properties are discussed in Sect. 8.2. Important to stress here is their efficiency to release without emission not only their own excitation energy by immediate relaxation to the ground state but also the energy transmitted from other fluorophores by excitation energy transfer (EET) mechanism (see Sect. 4.3), which results in concentration quenching. This is unpleasant fact for those researchers that want achieving the highest density of labeling the macromolecules and making the dye-doped nanoparticles. It can turn to a pleasant story for those who can benefit from these facts designing efficient sensors.

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4.2.3 Application in Sensing Technologies

Fluorescence intensity

In chemical sensing, a number of molecular constructs have been suggested that employ excimer formation. The idea was to change on the target binding the groundstate configuration of sensor molecules in such a way that on excitation they can form/break excimers but only in the presence of target. However, in such applications the double labeling is needed, which is facilitated by the fact that the two partners are of the same structure. The transduction mechanism is also not easy to achieve. The excimer formation requires close location and proper orientation between the partners, specifically, the arrangement of cofacial sandwich between two heterocycles rich in π-electrons. Therefore, molecular recognition between interacting partners (e.g. macromolecules) should be coupled either with molecular assembly (each of these partners containing the monomer), or with the conformational change in one of them, bringing together or forcing apart these aromatic groups. Thus, for obtaining the sensor for the silver ions a construction was suggested based on a pyrene-functionalized heterocyclic receptor. It forms an intramolecular sandwich complex via self-assembly induced by silver ion. This results in a dramatic increase in fluorescence intensity of the excimer and a correspondent decrease of monomer emission (Fig. 4.8). The intensity ratio of excimer and monomer emissions (at 462 and 378 nm) is an ideal measure of Ag+ ion concentrations (Yang et al. 2003). Application of molecular recognition constructs based on excimers is also popular in the sensing of neutral molecules, such as glucose (Yu and Yam 2009). Commonly, two pyrenyl groups are attached to macromolecular sensor or they label two molecules assembled on a sensor unit, but the multiple binding can be also realized (Duhamel 2012). Performance of excimer sensors has been improved on

Wavelength, nm Fig. 4.8 The structure and fluorescence emission spectra of pyrene derivative (1) forming excimers in the presence of silver ions (Yang et al. 2003). Addition of these ions in concentrations 0, 4.0, 10, 15, 20, 40, 75, 150, 300 μM results in decrease of intensity of the normal emission with typical bands at 378 and 397 nm (excitation 344 nm) and the appearance of a red-shifted structureless maximum centered around 462 nm, typical of pyrene excimer

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incorporation of excimer-forming reporter dyes into cyclodextrin cavity. Excimerforming pyrene-conjugated oligonucleotides can be used for the detection of DNA and RNA sequences (Mahara et al. 2002) in complex biological fluids (Huang et al. 2011). Such ability to form excimers coupled with the conformational change in pyrene double-labeled flexible oligo- or polynucleotides is actively explored in DNA hybridization techniques and recognition of specific mRNA sequences (Fujimoto et al. 2004; Yamana et al. 2008). Oligonucleotide conjugates bearing two pyrene residues at their 5’-phosphate terminals exhibit excimer fluorescence intensity that is highly sensitive to duplex formation: the binding to their DNA and RNA targets leads to 10-fold increase of fluorescence. It depends linearly on the concentration of target DNA and permits quantification of DNA in solution. Pyrene dimers with flexible linkers were also suggested for double-stranded DNA detection (Yang et al. 2009). A photostable pyrene derivative 1-phenylethynylpyrene that exhibits redshifted fluorescence with excimer emission at 500–510 nm was suggested for nucleic acid labeling (Prokhorenko et al. 2009). Aptamers (see Volume 2, Sect. 5) that use two pyrene substituents can be efficiently applied for detecting specific proteins in complex biological fluids (Pu et al. 2005). Being incorporated into thermosensitive polymer, the exciplex forming pyrene groups can serve as molecular thermometers (Chandrasekharan and Kelly 2001). These results demonstrate good prospects for the design of molecular sensors with monomer-excimer λ-ratiometric response. Of some value for sensing technology is also the application of dyes demonstrating the transition from dark excimers to fluorescent monomers. Thus, the anionmediated disaggregation of the excimer formed of a cationic macrocycle resulted in fluorescence enhancement (Yang et al. 2019).

4.2.4 Comparison with Other Fluorescence Reporter Techniques Special requirements exist for the application of excimer and exciplex sensing techniques. The double labeling is commonly needed (with the exception if the target itself can form exciplex with the dye incorporated into sensor). We have also to keep in mind that the monomer and excimer (or exciplex) are independent emitters and that any non-specific influence of quenchers may be different for two forms. The researcher is limited in selection of reporter dyes. Pyrene derivatives are used as excimer formers because of unique property of this fluorophore to form stable excimers with fluorescence spectra and lifetimes very different from that of monomers. Other dyes are commonly not useful or less convenient for this application. And finally, a conformational change or self-assembly is needed in sensor molecule to couple or to remove apart the dye monomers on target binding groups (Yang et al. 2003), If these requirements are properly addressed, the user can benefit from advantages of this technique. With the observations at two emission wavelengths, one can achieve the two-channel sensor operation (λ-ratiometry) that will be independent of sensor

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concentration and allow self-calibration of fluorescence response. Time-resolved sensing and lifetime-based imaging can be easily provided due to long lifetimes of pyrene derivatives in both forms.

4.3 Excitation Energy Transfer (EET) Two or more dye molecules with similar excited-state energies can exchange their energies due to dipole-dipole resonance interaction between them. One molecule, serving as the donor of excited-state energy, can absorb and transfer it to the other molecule, the acceptor. The latter can be excited by this transferred energy and emit light (Clegg 1996; Schneckenburger 2019; Selvin 2000). When such communication exists between excited and unexcited molecules, we observe that on excitation of the donor its emission is quenched and, instead, emission of the acceptor is augmented. When this coupling is absent, the emission of the donor is observed only. This approach is frequently used in sensing. Meantime, its realization usually needs labeling with two dyes serving as donor and acceptor. Only in rare lucky cases, intrinsic fluorescent group of sensor or target molecules can be used as one of the partners in EET sensing. The popularity of excitation energy transfer is explained not only by an immense range of possibilities for constructing the advanced molecular and nano-scale sensing devices, but also by a variety of fluorescence detection methods that can be used. EET can be monitored by intensity changes (quenching of fluorescence of the donor and enhancement of the acceptor) and also by the change of polarization and lifetime of fluorescence emission (see Chap. 3). Variation of inter-fluorophore distances and their spectroscopic parameters can modulate EET within the sensor device from its complete absence to highest probability of occurrence. In addition, communication between fluorophores allows adjusting the parameters of emission: the positions of spectra, their polarizations and lifetimes. The basic long-distance mechanism of energy transfer was first described by Theodor Förster and the whole phenomenon is often called by his name (Förster resonance energy transfer, FRET). We will use excitation energy transfer (EET) as a general term and FRET when the Förster resonance mechanism is specified.

4.3.1 Physical Background of the Method The FRET effect depends strongly upon the distance between donor and acceptor. If the distance is short and one of the partners (donor) is excited, it can transfer the electronic excitation energy to the other partner (acceptor), whereby resulting by the latter to emit fluorescence. FRET has to be distinguished from the effect of re-absorption of the light quanta emitted by one dye by the other dye molecule: reabsorption depends on geometry of the system but FRET does not. Even at a relatively

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long distance (nanometers), the dyes may interact through space by the dipole-dipole resonance interactions, and the resonance provided by this dipole coupling allows the energy to migrate from donor to acceptor without emission. FRET can take place if the emission spectrum of the donor overlaps with the absorption spectrum of the acceptor (Fig. 3.18) and they are located at separation distances within ~1–10 nm from each other. Its occurrence can be recognized by the decrease of intensity of the donor emission band and the correspondent increase of the acceptor emission, if the latter is not quenched. Other fluorescence parameters (anisotropy and lifetime) may change on transfer. The efficiency of energy transfer E can be defined as the number of quanta transferred from the donor to the acceptor divided by all the quanta absorbed by the donor. According to this definition, E = 1 − F DA /F D , where F DA and F D are the donor intensities in the presence and absence of the acceptor. Both have to be normalized to the same donor concentration. If the time-resolved measurements are used, then the knowledge of donor concentration is not required, and E = 1 – / , where and are the average lifetimes in the presence and absence of the acceptor (Wu and Brand 1994). The energy transfer efficiency in FRET exhibits a very steep dependence on the distance separating two fluorophores, R:   E = R06 / R06 + R 6

(4.1)

Equation (4.1) determines this steep distance function for observed intensity ratios of donor and acceptor emissions. Here R0 is the parameter that corresponds to a distance with 50% transfer efficiency (the Förster radius), expressed in Ångstroms. It is characteristic for a given donor-acceptor pair in a particular medium. Figure 4.9 illustrates how the dynamic range of FRET effect variation depends on donoracceptor distance for different R0 values. One can observe that these distances are comparable with the dimensions of many biological macromolecules and of their complexes (Clegg 1996; Selvin 2000). The transfer efficiency depends strongly on the quantum yield of the donor in the absence of acceptor Φ D and on the overlap of fluorescence spectrum of the donor with absorption spectrum of the acceptor (see Fig. 3.18) expressed as normalized spectral overlap integral J:   R06 = 9.78 × 103 κ 2 n −4 Φ D J

(4.2)

Here n is the refractive index of the medium between donor and acceptor. The spectral overlap integral J is a function of the fluorescence intensity of the donor, F D , and molar absorbance of the acceptor, εA , as a function of wavelength, normalized against the total donor emission: 

 J=

FD (λ)εA (λ)λ4 dλ

FD (λ)dλ

(4.3)

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R

R0 (Å)

D-A distance (Å)

Fig. 4.9 Dependence of the dynamic range of Förster resonance energy transfer (FRET) on the donor-acceptor (D-A) distance for different R0 values given in Ångstroms. The dotted lines delineate the regime of maximum sensitivity of response to distance variation for each D-A pair with different R0 , which is commonly between 0.7 and 1.5 R0 . Redrawn from (Kapanidis and Weiss 2002)

In Eq. (4.3) the integration should be made over the whole spectrum. κ2 is the factor that depends on relative orientation of donor and acceptor dipoles; it can assume the values from 0 to 4. The lowest value will be if they are oriented perpendicular and the maximum value corresponds to parallel and aligned dipoles. For random orientations of donor and acceptor, κ2 = 2/3 if they move rapidly on fluorescence time scale and κ2 = 0.475 if they are rigidly fixed. Thus, the transfer efficiency depends on relative orientation of donor and acceptor dyes. If lifetime is long and rotations of segments containing reporter dyes are fast, then the approximation κ2 = 2/3 is justified. In the case of rigidly fixed donor and acceptor, the situation is less definite, and the estimation of donor-acceptor distances may lead to great errors. In designing the FRET-based sensors there are the cases when even at short distances FRET does not occur. Such a case was described on interaction of conjugated polymer (donor) with the double-helical DNA containing dye ethidium bromide as an acceptor. Rigid structure of this complex and orthogonal orientation of transition dipole moments prevented detection of DNA hybridization by FRET (Xu et al. 2005b). Nevertheless, in practical sense, variations of both the distance and orientation may substantially increase the dynamic range of fluorescence response. Attentive reader must note that the Förster-type energy transfer mechanism considers weak dipole-dipole interactions between donor and acceptor dyes that operates at a distance. What happens if the donor and acceptor are close and interact

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stronger, up to overlapping their electronic orbitals? In this case the energy is transferred due to a double electron exchange mechanism (the Dexter energy transfer) within the molecular orbitals of the donor and the acceptor. The electronic coupling leading to energy transfer requires significant orbital overlap. Therefore, close interaction between the excited donor and the acceptor ground state is necessary. This type of transfer is often called “through-bond energy transfer”, in which a strong departure from the Förster-type energy transfer mechanism (FRET) is observed (Andrews et al. 2011; Fan et al. 2013). The Dexter-type of transfer can be involved in different cases ambiguously attributed to the Förster mechanism (Tanner et al. 2018). Whereas the Förster-type energy transfer is able to occur over very large distances, the Dexter energy transfer dominates with much greater rates at short distances; it falls off exponentially as the distance increases and commonly requires close contacts between the partners. Specific excitonic effects appear in regular associates of similar molecules, even the dimers, leading to quite characteristic spectra of J and H aggregates (see Sect. 8.2). If different dyes are coupled electronically, the requirement of spectral overlap between donor and acceptor dyes is lifted, which allows the fluorescence band shifting on a broader wavelength scale (Gong et al. 2012). Thus, in addition to discussed above variations of relative distances and spectral overlaps, other mechanisms of generating the sensor response can be activated. They deserve focused study and exploration.

4.3.2 Applications of EET Technology Modulation of donor-acceptor distance offers a lot of possibilities (Fan et al. 2005). Many constructions designed for fluorescence sensing with EET-based reporting explore strict stoichiometry between EET donor and acceptor groups that are provided by either covalent linkage between them (often on a protein or oligonucleotide matrix) or by formation of intermolecular complexes. In these cases the change in distance and orientation between interacting partners can report on the sensing event. This can be achieved when both donor and acceptor fluorophores belong to the sensor and a conformational change in the sensor on target binding causes the changes of their relative distance. Alternatively, donor and acceptor can belong to different interacting partners. One of them can label the sensor and the other the target or its analog (in displacement assay), and upon their binding the two fluorophores come to close proximity. Since detectable switching between donor and acceptor can be achieved at separation distances up to 5–10 nm (see Fig. 4.9), the distance variations within this limit are explored. For instance, if the donor and acceptor are connected with a spacer, the switching between acceptor emission and donor emission can be arranged by extending the chain length (Harriman et al. 2009). Such range of distances is comparable with the dimensions of many biological macromolecules and of their complexes. Therefore many constructions can be realized for coupling the response with these changes. Typical analytical experiments involving EET pairs are depicted in Fig. 4.10. The conformational change in double labeled

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a D

D

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A Target

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Fig. 4.10 Typical applications of EET from excited donor (D) to fluorescent acceptor (A) in the studies of intermolecular interactions. a The changes of relative distances between molecules or their fragments (e.g. due to conformational change in one double labeled interacting partner). b The change in distance between interacting partners, each of them is labeled. c Detection of nonfluorescent target in substitution assay with labeled donor and competitor

sensor (Petitjean and Lehn 2007), enzymatic splitting of covalent bond between two labeled units (Gershkovich and Kholodovych 1996, Takakusa et al. 2002) and substitution of labeled competitor in a complex with the labeled molecular sensor (Xu et al. 2005a) are the illustrative examples. Two labels can be located in one of interacting partners indicating its conformational change, in both partners indicating their close contact and in one of the partners and the competitor. Another possibility that is no less important is the modulation of donor-acceptor spectral overlap for obtaining the sensor response (Kikuchi et al. 2004; Yu and Ptaszek 2013) (Fig. 4.11). The responsive photochromic and pH-sensitive dyes (as well as any dyes changing their absorption spectra as a result of change in their ground-state interactions) can be used as EET acceptors producing the reporter signal. Modulation of EET response at a fixed donor-acceptor distance by changing the absorption spectrum of the acceptor (see Fig. 4.11a, b) may have important advantages in fluorescence sensing and imaging. In organic dyes, the absorption spectra shift on the formation of the charge-transfer complexes and, more efficiently, on their transition between pH-dependent protonated and deprotonated forms (Povrozin et al. 2009; Yao et al. 2007). The large spectral separation and high emission intensities of these forms in the visible allow generating strong sensor signal (Takakusa et al. 2003). The composites assembled of quantum dot donor and covalently attached squarene dye demonstrate such ability (Snee et al. 2006). In sensor design, the effect of target binding can be coupled either with pH change or with pKa shifts of smart EET receptors.

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D

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bF

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Fig. 4.11 Generation of sensor response by modulating the EET donor-acceptor spectral overlap. a The absorption spectrum of the acceptor shifts reducing the overlap, which activates the donor emission. b The acceptor fluorophore is irreversibly or reversibly destroyed with the appearance of donor emission. c The EET gating. The spectral overlap is initially negligible that does not allow EET to proceed and the appearance of intermediate connecting the spectral overlaps between donor and acceptor activates EET

Modulating or even removing acceptor absorption can be made in different ways. One is to provide its photobleaching when the EET pairs are formed by highly photostable donor and easily photodegradable acceptor. The map of target distribution can be obtained when the two images are taken at the wavelength of donor excitation before and after the acceptor is bleached (Jares-Erijman and Jovin 2003; Van Munster et al. 2005). There are also the possibilities to eliminate EET by removing the acceptor absorption in a reversible way with photochromic dyes as the acceptors. They can be switched by light between two states reversibly: one state with the excitation band overlapping emission band of the donor and the other, in which this band is lacking. This allows realizing photochromic EET (pcEET) (Giordano et al. 2002). Dithienylethene can serve as such acceptor. It can switch to the colorless open form under the action of visible (green) light and switch back to closed light-absorbing form under the action of ultraviolet light. This switching influences dramatically the fluorescence intensity of the donor. Only several absorbed photons are required for such switching in contrast to ~104 –106 photons needed for irreversible photobleaching. In this technique, quantum dots can serve as EET donors (Díaz et al.

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2012). Some members of GFP family can exhibit light-activation and can be used as photochromic EET acceptors (Bourgeois and Adam 2012). Thus, it is possible to switch reversibly the EET effect between “ON” and “OFF” states without any chemical intervention, just by light. Another interesting possibility that can be used in sensing is the ‘EET-gating’, when the adding or removing of the intermediate in the multi-step EET process modulates the fluorescence response by changing the link between donor and acceptor (see Fig. 4.11c). The following literature example illustrates this possibility (Wang et al. 2004). It was shown that the excitation of cationic water-soluble conjugated polymer results in inefficient EET to ethidium bromide dye intercalated within the bases of double-stranded DNA. When fluorescein label is attached to one terminus of the DNA, the link between donor and acceptor appears and an efficient EET starts to be observed from the polymer through fluorescein to ethidium bromide. These experiments show that the proximity and conformational freedom of fluorescein provide an EET gate to dyes intercalated within DNA. The conjugated polymer donor possessing strong absorbance also serves for amplification of this emission. Thus we observe that when the donor and acceptor are in proximity but their spectral overlap is insufficiently small for the transfer, then an introduction of the third partner that serves an acceptor to primary donor and donor to terminal acceptor results in the appearance of efficient transfer. Different realizations of this idea were reported (Aneja et al. 2008). The possibility of modulation of EET response by spectral shifts in donor and acceptor can be illustrated by ion binding as the target. This response depends on the site of attachment of the ligand binding site—to donor or to acceptor (Guliyev et al. 2009). When a fluorophore responsive to ion binding is located at the acceptor site, an efficient chemosensor can operate with the advantage of its strong Stokes-shifted emission (Fig. 4.12a). In this case, the observation of donor emission is not needed and the dual emission response or response in any other fluorescence parameter to target binding can be provided by the acceptor. Here the advantage is a stronger separation of excitation and emission bands and broader selection of excitation and emission fluorophores. In the case when the target-responsive unit is located at the donor site (Fig. 4.12b), the acceptor is just a passive emitter and informative signal comes from the perturbation of fluorescence of the donor on interaction with the target. The variability of fluorescence parameters that can be achieved in both cases is beneficial for multiplex sensing.

4.3.3 Observing New Developments EET is increasingly popular in the studies of recognition between macromolecules and in the construction of biosensors based on this recognition. It operates with two reporter dyes (EET donor and acceptor), which allows introducing the geometry parameter into fluorescence sensing and observing the sensing effects based on the

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Fig. 4.12 Two different designs for excitation energy transfer (EET) coupled ion sensing (Guliyev et al. 2009). a The ligand binding and correspondent fluorescence response occurs at the EET acceptor site and the donor plays a passive role. The informative signal comes from emission of the acceptor. b The ligand binding and correspondent fluorescence response occurs at the donor site. The ligand binding changes the excitation spectrum of the donor observed at the emission wavelength of the acceptor

changes in distances and orientations of the partners. Their excited-state energies modulating their spectral band positions also produce strong influence on this effect. The fact that the fluorescence response depends strongly on the properties of both donor and acceptor and on their distance and orientation allows many possibilities for its modulation and adaptation to the desired conditions. Two labels can be located in one of interacting partner indicating its conformational change, in both partners indicating their close contact and in one of the partners and the competitor. A variety of donor-acceptor pairs of organic dyes were selected for EET applications (Fan et al. 2013). Genetically encoded indicators driven by the EET mechanism have been developed and became reliable tools for sensing inside live cells (Sanford and Palmer 2017; Xue et al. 2016). Being a general photophysical phenomenon, EET is displayed on nanoscale level between dye aggregates, semiconductor quantum dots, upconverting nanoparticles, carbon dots, etc. (Dos Santos et al. 2020; Teunissen et al. 2018). The applications of these systems extend to studies of living cells and tissues and further to clinical diagnostics (Qiu and Hildebrandt 2019). In the following Chapters, the EET-based technologies and, particularly, the use of the most requested FRET mechanism will be discussed in many aspects. New effects

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appear when the fluorophores form the nanoscale assemblies with mutual reversible migration of energy. Light harvesting, superenhancement and superquenching can be achieved in these systems. The broad-scale manipulation with emission wavelength, anisotropy and lifetime is possible with nanoscale assemblies of molecular emitters where there are possibilities of EET gating and directing by light. We demonstrated how still without incorporating these collective effects and operating with only two parameters on which FRET depends—distance and spectral overlap—one can get very interesting solutions for sensing. Exploration of collective effects (see Sect. 8.1) opens new dimensions. A strong move is observed from monitoring single events to simultaneous detection and analysis of multiple analytes, the EET-based multiplexed techniques (Kaur and Dhakal 2019).

4.4 Signal Transduction Via Conformational Changes There are many possibilities to incorporate conformational changes triggered by target binding into the process of signal transduction. Many such changes in flexible molecular structures occur with low activation energies, so the switch between two conformations can proceed on target binding. Those of them that cannot occur in the ground states can sometimes be observed in the excited states, where the activation barriers for rotations around certain bonds are dramatically reduced. Thus, the responding sensors involving both large-scale and small-scale conformational changes can be constructed. The excited-state electron transfer and energy transfer on molecular level are the distance and orientation dependent. Therefore the change in the distances and orientations between the partners in these reactions can be used as the tools in signal transduction.

4.4.1 Excited-State Isomerism in Small Molecular Reporter Dyes There are several classes of dye molecules that are highly stable in the ground state but can easily isomerize in the excited state. One of classical examples for that is stilbene. In a trans-form it is strongly fluorescent, but in solutions that allow its rapid isomerization into cys-stilbene its fluorescence is very weak. Based on this fact, one can make a sensor, in which the target binding will influence the stilbene isomerization. Such possibility can be suggested based on results obtained with the anti-trans-stilbene antibodies (Simeonov et al. 2000). In a complex with antibodies it exhibits bright emission indicating that its isomerization is suppressed. For our purpose, the antibody may serve a prototype of a target to show the efficient modulation of response on its binding. The other feasible idea is to modify stilbene with target-recognition moiety and use such stilbene-antibody associate as a sensor.

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Following this way a sensor for mercury ions was constructed (Matsushita et al. 2005). Stilbene fluorescence is bright but becomes strongly quenched by these ions. A new type of excited-state dynamics of organic fluorophores that is associated with dramatic spectroscopic changes was discovered recently (Zhang et al. 2015). It was demonstrated that molecules belonging to a class of N,N-disubstituteddihydrodibenzo[a,c]phenazines (DPACs) possessing a bent conformation in the ground state demonstrates planarization in the excited state, similar to butterfly motion stretching its wings. Such motions can be observed as a function of time and results upon excitation at ~350 nm to shift of fluorescent band to as much as ~610 nm. It was shown that such strong effect is due to significant extension of π-electronic system. The decrease of blue and increase of red emission can be seen as a function of solvent viscosity but does not depend on polarity. Therefore these dyes were suggested to use for studying microviscosity in living cells (see Volume 2, Chap. 15). Efficient application in sensing technologies is ahead, in view of emission response in a bright change of color.

4.4.2 Conformational Changes in Conjugated Polymers In Sect. 8.5 we will review the properties of conjugated polymers as fluorescence reporters and demonstrate that their response can be based on two unique features, ‘superquenching’ and the dependence of fluorescence emission on chain length and conformation (Kaloni et al. 2017; Thomas et al. 2007). The modulation of chain conformation on target binding has found many applications, especially in imaging (Feng et al. 2012) and detection of nucleic acids (Volume 2, Chap. 11). Here we stress that the chain conformation produces dramatic impact on the properties of π-conjugated system that influences the fluorescence, since it determines the conjugation length (Pecher and Mecking 2010). The conformation-dependent fluorescence of conjugated polymers was shown by many researchers (Barbara et al. 2005; Wang et al. 2016, 2017). It is important that these polymeric molecules allow many different modifications with the attachment of side groups, and these groups may determine the conformational state of the backbone and thus the fluorescence signal. Of special interest are the modifications with the charged side groups that transform the low-polar polymers into polyelectrolytes and allow target binding based on electrostatic interactions. With proper modification of side groups, these polymers can specifically recognize charged molecules, such as ATP (Li et al. 2005). But the most significant effect is recorded on binding to regular protein fibrils that appear in some pathology, such as Alzheimer’s disease. These pathological protein aggregates exhibit β-sheet structure, and the polymer binding to them produces remarkable changes in their spectra (Herland et al. 2008). The binding of conjugated polymer with cationic substituents to DNA is based on electrostatic interaction and depends on the DNA conformation. On binding to DNA, the polymer has to change its conformation and thus generate the sensing signal. This methodology found application in DNA hybridization assays, in which

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conjugated polymer plays a role of ‘hybridization transducer’ (Dore et al. 2006). It was successfully applied to nucleic acid aptamers as sensors (Ho and Leclerc 2004). Since single-stranded DNA (ss-DNA) can specifically bind to various targets, including complementary ss-DNAs, ions, proteins, drugs, and so forth and on this binding the oligonucleotide probe often undergoes a conformational transition, the range of application of this methodology can be very broad.

4.4.3 Conformational Changes in Peptide Sensors and Nucleic Acid Aptamers In the mechanism of recognition between flexible peptides and nucleotides and their well-organized targets there is nothing artificial. A great number of ‘natively unfolded’ proteins were discovered in recent decades, which even required revising the paradigm on interactions of proteins with their substrates and ligands. It became clear that the ‘lock-and-key’ and ‘induced fit’ mechanisms should be complemented by the mechanism of ‘induced and assisted folding’ (Demchenko 2001). Many cases were found, in which the natively unfolded protein on its specific interaction with another protein or nucleic acid exhibits the unfolding → folding conformational change to form a stable and well organized complex (Fig. 4.13a).

Fig. 4.13 Illustration of mechanisms of ‘induced and assisted folding’. a Unfolded protein folds upon interaction with the other folded protein used as template. b Two unfolded proteins assist each other to attain folded conformations. In both cases, specific recognition occurs on the level of groups of atoms. It is sequentially enforced in the course of folding

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There is a special class of proteins, called molecular chaperones, which, interacting with unfolded proteins, provide their transition into the folded state. Moreover, examples were found of interacting partners, so that both of them are unfolded, and upon interaction, they find a common pathway to the folded states (Fig. 4.13b). In both cases, the interactions between the partners can be very specific, starting from their initial contacts. The whole process follows strict thermodynamic and kinetic rules and involves millions of decisions of the chains for abortion or for re-enforcements of contacts. Therefore, the formed complex is also very specific, satisfying the requirement for molecular recognition. The strategy to make virtually any protein the sensor with signal transduction based on target-induced folding was suggested (Kohn and Plaxco 2005). Rational genetic engineering can destabilize the protein structure in such a way that it becomes naturally unfolded. Binding the target should make the folded state thermodynamically more stable and induce the unfolding-folding transition. These results make a fair background for understanding how the unfolded peptide selected from the library without regular structure and at the absence of formed binding site, can serve as a very specific binder. The specificity of interaction appears if the design allows a strong coupling of the chain folding and recognition. Such a profound change in conformation of recognition unit provides many possibilities for signal transduction in sensing. Different fluorescence reporting mechanisms can be adapted to such systems. Meantime, one precaution has to be made. Modification with fluorescent dye can be a strong intervention into the process of folding. Therefore, such modification has preferably to be made in a site that has a minimal involvement into this process. Alternatively, the modification should be made on the step of selecting the prospective receptors from a combinatorial library, so that the selection has to be made with the account of folding and recognition properties of peptide-dye conjugate. Zinc finger presents a classical example of transforming the designed peptide into a sensor. It is derived from small (25–30 residue) protein domain that selectively binds zinc ion with a dissociation constants in the picomolar range. In the absence of zinc, the peptide is unfolded and it folds to coordinate the Zn ion (Fig. 4.14). The primary coordination sphere involves two His and two Cys residues. The chemical synthesis of this peptide with incorporation of fluorescence reporters was easily performed. In this case, the signal transduction does not involve the interaction of reporter with coordinated zinc and can be provided by the dyes that change their environment or interact between themselves and report based on EET or PET mechanisms (Walkup and Imperiali 1996). Therefore many modifications are possible to improve the performance of such sensors. The conformational adaptation of receptor on forming the complex with the target can be realized in sensing aptamers. The selection, synthesis and operation of nucleic acid aptamers will be described in detail in Volume 2, Chap. 5. Here we focus on mediation and generation of fluorescence signal coupled with conformational change in these sensor molecules. Many aptamers (especially with a short sequence) are without or with only small segments of regular structure in their free states in

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Fig. 4.14 The zinc-induced folding of zinc finger peptide. The conformational change in peptide is detected with the environment-sensitive dye. In unfolded state (left) the dye is exposed to water and indicates its position in a polar environment. On the folding of peptide triggered by zinc ion its environment changes to low-polar (right) with a strong spectral shift

solutions and the target binding is coupled with their ordering into well-defined threedimensional structures (Hermann and Patel 2000; Stojanovic and Kolpashchikov 2004). This suggests many possibilities for providing fluorescence response with realization of principle of direct sensing. An interesting possibility for signal transduction can be found with aptamers. Since they can reorganize and form on target binding the double-helical structures, this process can be detected by the dyes that can specifically intercalate into folded double helical segments of this structure (Chi et al. 2011). The response to their conformational change can also be detected with the aid of cationic conjugated polymer (Ho and Leclerc 2004).

4.4.4 Molecular Beacons (Hairpin-Like Structures) Molecular beacons (the other name ‘molecular hairpins’) are the direct molecular sensors designed for detection of specific nucleic acid sequences based on the formation of complementary double helix structures. Nucleic acids are widely known for their ability to form Watson-Crick double helical structures that are stabilized by hydrogen bonds between complementary base pairs. Formation of these structures is strongly sequence-specific. The double helix can be formed between two different chains with complementary structures. Being a single chain it can fold to itself to form a double helix involving the segments that contain complementary sequences. This allows making sensors from single-chain nucleic acids that fold into a specially designed structure containing two structural components, a loop and a stem (Fig. 4.15). They can be considered as a special case of aptamers, with specificity to recognize the nucleic acid sequences.

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Loop

+ Stem

Molecular beacon

Target DNA/RNA sequence

Molecular beacon – target duplex Fig. 4.15 Schematic representation of the generation of fluorescence signal by the hybridization of nucleic acid sequences with molecular beacons. A molecular beacon is a hairpin-shaped nucleic acid with the sequence of the loop complementary to the sequence in the target nucleic acid. In the absence of target, the two dyes, donor (D) and acceptor (A), that can be a fluorophore and a quencher, are located closely and interact. When the loop hybridizes with a target sequence, the stem is disrupted and the two labels move apart generating a fluorescence response of the donor

The loop is the recognition element containing complementary nucleic acid sequence to the target sequence that has to be detected in a pool of many different oligonucleotides. The stem is formed of two complementary sequences that fold to themselves to flank the loop. Hybridization of the loop part with the target DNA or RNA sequence generates a conformational change in this structure, its stretchingout. As a result, the chain segments forming stem disconnect and move apart. This motion is detected being translated into fluorescence signal. Since the nucleic acid sequence of the loop can be made complementary to that of any nucleic acid target, molecular beacons can be used as a generic technology for recognition of specific nucleic acid sequences (Tyagi and Kramer 1996). The distance between 3’ and 5’ ends, being very short in folded beacon, dramatically increases on target binding. So, the fluorescence reporter signal can be generated based on recording the disconnection between stem segments. The dye can be attached to one of these chain terminals or to both of them. There are several possibilities to generate the reporter signal. 1. The increase of distance between two beacon ends can be used in detection by EET to non-fluorescent acceptor (quencher). The introduction of two labels,

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donor and acceptor, is needed in this case. In the sensing event (hybridization with complementary sequence) the separation between two dyes increases, causing fluorescence enhancement. This approach was explored in a number of works (Marras 2006) 2. More advantageous is the detection technique in which both EET donor and acceptor are fluorescent. This possibility was realized with the use of cyanine dyes Cy3 and Cy5 (Ueberfeld and Walt 2004). When the donor and acceptor dyes are in close proximity, an emission of the acceptor is observed. Upon hybridization, this emission disappears and emission of the donor is enhanced. It was found that the ratio of donor-acceptor intensities is independent of the amount of the probe and provides a quantitative measure of the free target concentration. 3. The quenching of fluorescence signal is achieved by photoinduced electron transfer (PET) quenching (see Sect. 4.1). In PET quenching, the emitting dye and the quencher (electron donor or acceptor) should be present in stem structure at a very close distance from each other (Heinlein et al. 2003), in contrast to much larger distance (of several nanometers) that is needed in EET sensing. Since in the folded state of molecular beacon the distance between two chain terminals is very short, a complete quenching can be achieved, so that fluorescence enhancement appears on hybridization. The advantage of this technology is in the possibility to avoid second labeling. The nucleic acid guanosine base can play this role of PET quencher of complementary dye in beacon construction. 4. One of hairpin ends can be immobilized on metal surface (Strohsahl et al. 2007). The other terminal contains the dye, the fluorescence of which becomes quenched due to contact with the surface (also due to PET mechanism). Fluorescence enhancement will be observed on hybridization. The sensing DNA or RNA oligonucleotide can be successfully substituted by artificial oligomers—peptide nucleic acids (see Volume 2, Chap. 11). In their structures the negatively charged ribose or deoxyribose phosphate is replaced by an uncharged N-(2-aminoethyl)-glycine scaffold, to which the nucleobases are attached via a methylene carbonyl linker (Brandt and Hoheisel 2004). The target recognition by these beacons is less sensitive to ionic strength and their fluorescence is not affected by DNA-binding proteins. The principle of molecular beacon acquires more and more general importance. An interesting result on its application in detecting proteins has been reported (Thurley et al. 2007). The peptide beacon was synthesized that uses the ability of a designed peptide to change its conformation between target-bound and target-free forms with fluorescence reporting based on double labeling. In another development, a short peptide beacon was suggested for detecting nucleic acids (Wu et al. 2012). Two pyrene units were appended to a synthesized construct. On their intercalation into DNA double helix, the contact between them is disrupted with disappearance of excimer and appearance of monomer band in emission. Such DNA sensor was shown to enter spontaneously into the living cells. The strength in EET technologies is seen when they are coupled with conformational changes on sensor-target association. As an example, a single-stranded

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dual fluorescently labeled DNA molecule that adopts a stem-loop conformation with the close proximity of EET donor and acceptor dyes can be constructed (the DNA hairpin). It exhibits a conformational change on hybridization with an increase of the dye-to-dye distance and the appearance of a new band belonging to the acceptor (Ueberfeld and Walt 2004). We can also mention the re-arrangement of subunits in the sensor protein (Adams et al. 1991) and a conformational change within the subunits. Since the distance dependence of EET is very steep, an efficient switching between the two emissions can be achieved.

4.4.5 Proteins Exhibiting Conformational Changes Flexibility of protein structures coupled with target recognition is used in different sensing technologies (Ha and Loh 2012; Plaxco and Soh 2011). These structures were optimized in evolution for performing biological functions, which requires highly selective ligand binding. As the target recognition units, they can display different modes of conformational change: rigid-body domain movement, limited structural rearrangement, global fold switching, and folding–unfolding. Such changes can be induced by target binding. There is an active search for the most efficient coupling of recognition with sensor response in the case of fluorescence sensing. The major problem here is to transform the act of ligand binding to the measurable response signal. This task is not easy, even in the cases of ligand-binding proteins that exhibit global hinge-bending motions of domains that are compact rigid elements of their structures. The analysis of intra- and inter-domain dynamics of 157 ligand-binding proteins (Yesylevskyy et al. 2006) allows formulating the necessary conditions, which should be satisfied in order to couple the intramolecular dynamics of biosensor exploring the transfer of signal from the binding site to the reporter group. This can only be achieved if (a) the protein undergoes significant conformational change upon the ligand binding; (b) the intra-domain conformational changes are small in comparison with the relative motion of domains and (c) the strength of the interdomain contacts is significant in the ligand-bound conformation and very small in the ligand-free conformation. This technique can be applied to the candidate proteins to see if such conditions are fulfilled for them. It can allow minimizing the possible design errors caused by the choice of the protein, which is not suitable for the design of sensors, where the reporter group is not in contact with the ligand. An algorithm was developed, which allows physically consistent simulations of slow large-scale protein dynamics that is based on subdividing the protein into the hierarchy of the rigid-bodylike clusters (Yesylevskyy et al. 2007). A closed conformation, starting from the open one, can be predicted in agreement with their known crystallographic structures. Such techniques are promising in deciphering the character of protein motions involved in the signal transduction from the ligand binding site to the fluorescence reporter. It was shown in (Yesylevskyy et al. 2006) that the periplasmic ligand-binding proteins and calmodulin are the most probable targets for the biosensor design. Being

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Fig. 4.16 The change in conformation in maltose-binding protein (a) and phosphate-binding protein (b) from an apo state (left) to a ligand-bound state (right) by a bending-twisting motion of the N domain (green) and C domain (blue) about the hinge region (cyan) (Tainaka et al. 2010). A fluorescent reporter group (orange) to monitor ligand binding has been attached to an allosteric site or a peristeric site, respectively

composed of two well separated domains, they undergo large hinge-bending motion upon ligand binding. In the open ligand-free state the domains are far from each other, while in the closed ligand-bound state the domains become tightly packed. The ligand is often captured in the cleft between the domains. One of such proteins, the Maltose binding protein (MBP) (Fig. 4.16) is the most successful sensor protein, which is extensively studied and widely used in saccharide sensing (de Lorimier et al. 2002). Its mutated forms are a part of active efforts in glucose sensor development (Fonin et al. 2014). Many genetic mutants of MBP were produced to modulate specificity and affinity in target recognition and to provide signal transduction and reporting (Gilardi et al. 1994; Medintz and Deschamps 2006). In particular, a competitor displacement assay was suggested for maltose binding, in which the competitor was the labeled β-cyclodextrin derivative (Medintz et al. 2003). The use of quantum dot in a pair with organic dye allowed to use distance-dependent interactions between them resulting in PET quenching or EET (Medintz and Deschamps 2006). Located distantly in open conformation, they become close neighbors in a closed conformation. A different construction using conformation-dependent EET efficiency from ruthenium complex to quantum dot was also found efficient (Raymo and Yildiz 2007). In contrast, the application of environment-sensitive dyes did not result in expected spectral shifts and the significant response was obtained in intensity and lifetime only (de Lorimier et al. 2002; Fonin et al. 2014; Pickup et al. 2013). This may be the result of strong quenching, hiding the spectral sensitivity, see Fig. 3.14.

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Calmodulin is the calcium binding protein that, possessing a rather flexible conformation, changes it dramatically upon the binding of four Ca2+ ions (Fig. 4.17a). Tsien and coworkers have designed a genetically encoded calcium sensor comprising four components: calmodulin, a polypeptide named M13 that binds the calciumbound form of calmodulin, and two fluorescent proteins selected as EET donor and acceptor and serving as reporters on conformational change occurring on ion binding (Fig. 4.17b). This change results in the appearance of fluorescence emission of EET acceptor with easily observable spectral shift. The small number of these successful cases is an indication of difficulties in extending of this principle of sensing on other ligand-binding proteins. Therefore an interesting strategy was suggested to decrease stability of a protein up to partial unfolding by inducing some structural changes, thus allowing the target to induce the

Fig. 4.17 The cell calcium sensor obtained by protein engineering. a Calmodulin in open ligandfree conformation (yellow) and closed ligand-bound conformation (semitransparent blue) are aligned by the first domain. The ligand Ca2+ ions are shown as red balls. The motion of the second domain induced by the ligand binding is clearly visible and indicated by arrow. b The “chameleon” that changes the fluorescence spectrum in response to Ca2+ ions binding. It is composed of calmodulin (responding to ion binding by conformational change) and green (GFP) and yellow (YFP) fluorescence proteins that are fused to N and C terminals of calmodulin via calmodulin-binding peptide M13 (Miyawaki et al. 1997). The binding of Ca2+ ions induces the conformational change that brings together the EET donor and acceptor domains resulting in switching from the donor to acceptor emission

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switching to folded form with correspondent spectroscopic response (Ha and Loh 2012; Plaxco and Soh 2011). Successful application of sensors based on protein molecules exhibiting conformational changes allows avoiding direct contact of fluorescence reporter with the target but depends critically on a position of right reporter at a right place. An example is the response of two-color ratiometric dye to the conformational change of enzyme inhibitor α1 -antitrypsin with elastase (Boudier et al. 2009). The 3-hydroxychromone (3HC) dye was attached to a residue located distantly from the binding site but sensitive to conformation change of enzyme on binding the inhibitor. Ratiometric response of the dye is the result of change in its interaction with molecular environment at its binding site reporting on the complex formation.

4.4.6 Prospects in Exploration of Conformational Changes Conformational changes in the receptor molecular unit together with implementation of reporting mechanism based on electron, proton or energy transfer provided by fluorescence reporters allow realizing the principle of direct sensing (see Sect. 2.5). Based on these changes we are able to obtain direct response to target binding in the form of changing the parameters of emission. Depending on the system design, it is possible to select conformational isomerizations in the reporting dye or the changes in conformation of conjugated polymers with their strong effects on electronic conjugation between the fragments of dye molecule or the monomers in a polymeric structure. The folding reactions in polypeptides and ologonucleotides selected as the best binders from great libraries offer a dramatic progress in sensing technologies. These flexible sensors should be selected for being committed to be folded only in complexes with the targets. Though they are structureless or possess only a small amount of three-dimensional structure, they can be selected to contain all geometrical and bonding features for correct target recognition. Sensing by ‘induced folding’ must find many applications as a simple, universal and economical way to address many targets. Molecular beacons are a specialized version of aptamers that realize transition from intramolecular (hairpin) to intermolecular (complex) folded state of oligonucleotide. Detecting response to this change is easy and can be provided in a number of ways. And finally, regarding the ligand binding proteins and their genetic versions, their conformational changes are very specific, as specific as their target recognition. Time will show if a generic methodology will be developed on their basis.

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4.5 Smart Sensing with Logic Operations Sensing and computing are always connected and both exhibit dramatic advancement in miniaturization leading them to molecular level. In information technology, miniaturization is responsible for outstanding progress seen in recent decades. But it still remains to be based on macroscopic concepts and its realization in silicon-based devices, the reduction of size of which is approaching limit. The next and definitely ultimate limit of miniaturization is that of molecules, since the molecules are the smallest bodies in which information transfer can be achieved in the form of directed translocation of charge. Information inputs and outputs on molecular level can be provided by optical means, and this makes an additional strong connection between sensing and computing (Daly et al. 2014; De Silva 2016; Romieu 2015). Thus, will it be helpful to examine the background of optical sensing from a computer science viewpoint? The target could be the input, the emission—the output, the sensor serving as the processor device, and the excitation as the trigger and the power supply.

4.5.1 Why Logic Operations in Fluorescence Sensing? In previous chapters we always implicitly assumed that with the sensor response function being of any shape, the transduction function (that connects the number of formed complexes with response that they produce) is always linear (or weighted linear as seen in Sects. 3.3 and 3.4 for anisotropy and lifetime sensing). This means the existence of strict proportionality between the amount of bound target and the detected signal. However, the transduction mechanism can be more complicated and operate instead of one, with a number of input parameters, analyze their combinations and interferences. Speaking in the language of information technology, such analysis can include logic gates operations (Andréasson and Pischel 2010; Erbas-Cakmak et al. 2018; Magri et al. 2007). The introduction of the language of information science into sensing technology, if it is not a mental exercise, should have some important reason. The reason is such, that using this language we can design and classify the sensors with nonlinear and multiparametric operation. For instance, if we need in a given system to obtain the response to the presence of one analyte on the background of the presence of second analyte and the absence of third analyte, in a traditional way we have to apply three separate sensors and then analyze three output signals. The results will contain extra information and it will be difficult to analyze. But we are, in principle, able to devise molecular or supramolecular construction that will do that itself and provide us an already processed single output, such as depicted in Fig. 4.18. Molecule presented in Fig. 4.18b is the logic gate operating according to the design principles of modular photoinduced electron transfer (PET) systems (Magri et al. 2006). More specifically, it possesses a “receptor 1-spacer 1-fluorophore-spacer 2-receptor 2-spacer 3-receptor 3” format. The receptors incorporated in the design

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a Receptor 1 Receptor 2

AND

Receptor 3

Receptor 2

b Receptor 1 Receptor 3

Fluorophore

Fig. 4.18 Schematic presentation of sensor performance with molecular AND logic gate. a The sensor consists of several (shown 3) types of target binding sites. On signal transduction step a logic operation is performed to provide an output signal reporting on a designed combination of occupations of these sites. b An example of such sensor performing AND logic operation (Magri et al. 2006). This molecular construction produces the highest fluorescence output when all three receptors (sodium ions, zinc ions and protons) are saturated with their ligands

are benzo-15-crown-5 ether as a receptor 1 for Na+ , tertiary amine as a receptor 2 for H+ , and phenyliminodiacetate as a receptor 3 for Zn2+ . An anthracene moiety is used as the fluorophore. The three spacer units between adjacent receptors and the fluorophore are methylene spacers. They minimize the distance between the receptors and the fluorophore, facilitating conditions for efficient PET, notably if there are folded conformers. Such construction delivers the highest fluorescence output when all three receptors are occupied with their respective inputs, i.e., sodium ions, protons and zinc ions. This arrangement and many others presented later in the literature (Andréasson and Pischel 2010; Erbas-Cakmak et al. 2018; Magri et al. 2007) demonstrate the possibility of deriving a single optical output signal from a complex analytical situation, such as simultaneous sensing of pH, glutamate and zinc concentrations in

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neuron terminals (Hettie et al. 2014). This approach might indeed lead to a “lab-on-amolecule” with interesting future implications for multiparametric sensing. Different complex operations become possible on this pathway, including design and operation of chemical sensors with non-linear and modulated performance. Some of molecular and nanoscale devices can already perform logic operations of remarkable complexity.

4.5.2 Logic Operations on Molecular Scale The logic gate operations on molecular scale are formally the same as that performed in digital computing (Magri et al. 2007), and basically similar operations can be performed by the sensors. They are based on Boolean algebra, which is in the background of computing dealing with 0 and 1 binary code. With a single input (e.g. analyte binding) and single output (e.g. switch-on of fluorescence intensity), only two operations are possible. They are: YES when input is 0 or 1 and output is the same and NOT, which is the opposite: when input is 0 or 1, the output is 1 or 0. Thus, the simple PET system (see Fig. 4.2) can already serve as a logic gate. The PET-based luminescent switches may be triggered by various chemical and physical stimuli: protons, metal cations, anions, different organic molecules and nanoparticles. With two inputs (such as two different targets bound to two different sites), more complicated logic operations are possible. When both inputs are 1 and 1, AND operation gives output 1, OR operation gives 1 and NOR operation gives 0. When both inputs are 0 and 0, AND operation gives 0, OR operation gives 0 and NOR operation gives 1. If, in contrast, the first input is 1 and second input is 0, then AND operation gives 0, OR operation gives 1 and NOR operation gives 0. In INHIBIT logic gate, if both inputs are either 0 or 1, the output will be 0, and if they are different, the output will be 1. Detailed description of molecular basis of these operations can be found in the literature, and they are typically explained on examples of ion binding to fluorophoreattached chelating groups (Andréasson and Pischel 2010; Szacilowski 2012). OR is the operation that can be achieved with a sensor receptor without selectivity in input stimuli, e.g. when it does not recognize between the binding of two or several different ions producing the same output. Molecular AND logic gates can be constructed of two receptors and a fluorescent unit linked covalently with both receptors. In contrast to the OR gate, in AND gate the fluorescence should be switched on only when both receptors bind correspondent substrates. Implementation on molecular scale of other functions is more complicated and can involve combination of receptors and/or reporters. For instance, molecular-scale implementation of an XOR gate requires a chemical system, which responds to two different stimuli in a complex way: any of these two stimuli should switch on the gate, while concomitant presence of both triggering molecules should leave the system in the OFF state. This gate is in the core of complex logic systems: binary comparators and arithmetic units (half adder, half subtractor, full adder, etc.). Design

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of molecular structures that could be such signal transformers is sometimes a difficult task. Meantime, with different and multi-target inputs and also the combination of emitters and of the mechanisms of signal transduction, such functions can be realized.

4.5.3 Exploration of Basic Signal Transduction Mechanisms Photoinduced electron transfer (PET) has proved to be the efficient mechanism in performing basic logic operations (De Silva 2016; Magri 2018) because it can be easily realized in small-molecular organic donor-spacer-acceptor systems. There are many examples using this photophysical process for molecular-level implementation of single-input logic gates such as YES and NOT. Molecular versions of double-input logic gates such as AND, OR, NOR, NAND, INHIBIT, and XOR were realized. A prototypical number-processing system can attain a half-adder function. Presented in Fig. 4.18 is an example of molecular construction with AND logic gate. Many possibilities appear when a nanocomposite is assembled with luminophores excited at different wavelengths and, being connected by EET, emitting on different wavelength and time scales. Thus, quantum dots were combined with the longlifetime terbium complexes (Tb), a near-IR Alexa Fluor dye (A647), and selfassembling peptides (Claussen et al. 2013b). Such composite based on time-gated EET demonstrates the functioning of a combinatorial and sequential logic device. Upon excitation, the Tb-QD-A647 EET-complex produces the time-dependent photoluminescent signatures from multi-EET pathways enabled by the capacitorlike behavior of the Tb. This system allows manipulating with the dye/Tb inputs by variation of excitation wavelength and time-gating signal collection. Fluorescent output is converted into Boolean logic states to create complex arithmetic circuits including the half-adder/half-subtractor, 2:1 multiplexer/1:2 demultiplexer, and a 3-digit, 16-combination keypad lock. The key element of this nano-device is the Tb luminophore that, with its millisecond lifetime, serves as a memory element capable of “storing/carrying” information, while directly sensitizing the QDs and indirectly sensitizing any downstream EET acceptors attached to the QD (Claussen et al. 2013a). Application of long-lifetime emitting lanthanides expands dramatically the toolbox for constructing new logic elements (Pu et al. 2014).

4.5.4 Prospective Applications in Sensing and Imaging Technologies Design of logic gate elements are closely related to the development of multiplexing (many inputs) and multiparametric (many outputs) sensor technologies and expand their possibilities. Applying single type of sensor units, direct detection of two or even

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three analytes at once becomes possible. Moreover, a response can be programmed based on concentration ratios of several analytes with established threshold, above which the output signal is released. The suggested constructs are mainly based on the products of synthetic organic chemistry. They are made by assembling several ion recognition sites with two or more organic heterocycles that provide the signal transduction and response. This transduction is based on the basic mechanisms that were discussed above, PET, ICT and EET. It was shown that efficient signal processing can be achieved with one or several fluorophores and electron donors or acceptors coupled with ion binding sites (Pál et al. 2019; Straight et al. 2007). Even the combination of ion chelating group and pH-titrated group may be surprisingly efficient for providing two inputs (concentrations of ions and of protons). In the construction of logic gates, special attention has started to be paid to photochromes (Gust et al. 2006). They are organic molecules that are isomerized by light between two stable forms. They can be covalently linked to other dyes, so that the changes in their properties resulting from photoinduced isomerization can be used to switch the electron and energy transfer on or off. With the aid of photochromes the problem of not only the transformation but also of the storage of information on molecular level can be resolved. Other developments of molecular logic gates include designing the constructs involving DNA (Okamoto et al. 2004), deoxyribozymes (Stojanovic and Stefanovic 2003), peptide nucleic acid (PNA) (Cheah et al. 2005), DNA aptamers (Yoshida and Yokobayashi 2007) and cationic conjugated polymer/DNA assemblies with ethidium bromide as intercalating reporter dye (Zhou et al. 2005). For connecting the elements performing logic functions, different types of ‘molecular wires’ were developed. They can explore the excitonic conduction mechanism that can be realized in conducting polymers (Yashchuk et al. 2000). In connecting the functional elements, these neutral (electron + hole) quasi-particles can display a one-way conductivity. Realizing the multi-channel outputs in ‘molecular computing’ requires from fluorescent reporter to be present in several recognizable emissive states (PerezInestrosa et al. 2007). This problem, in principle, can be resolved with color-changing fluorescent dyes based on different excited-state processes (see above, Sect. 4.1). Interestingly, smart molecules allow not only computing but also protecting the information on the molecular scale (Margulies et al. 2007). By exploiting the principles of molecular Boolean logic, a molecular device can be developed that mimics the operation of an electronic keypad lock, e.g., a common security circuit used for numerous applications, in which access to an object or data is to be restricted to a limited number of persons. The ‘password’ should include not only the proper combination of the inputs but also the correct order by which they are introduced. It was shown that different ‘passwords’ can be coded by a combination of two chemical and one optical input signals, which can activate, separately, blue or green fluorescence output channels from pyrene or fluorescein fluorophores. The information in each channel is a single-bit light output signal that can be used to authorize a user, to verify authentication of a product, or to initiate a higher-level process.

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Such smart sensors are highly needed in cellular research. Tunable fluorescent molecular logic gates with applications to neuronal imaging focus on the studies of neurodegenerative diseases (Hettie et al. 2014). The three-input AND molecular logic gates are based on the coumarin-3-aldehyde scaffold and designed to give a turn-on fluorescence response upon the co-release of glutamate and zinc ions from secretory vesicles via exocytosis. There are attempts to expand the utility of digital logics concept to processes outside optical input and optical or electronic output. As their result, a two-module molecular de-multiplexer was synthesized, in which the output is switching between near-IR emission and photochemical generation of cytotoxic singlet oxygen (ErbasCakmak et al. 2013). In the neutral form, the designed compound fluoresces brightly under excitation at 625 nm. However, the addition of acid moves the absorption bands of the two modules in opposite directions, resulting in an effective reversal of excitation energy transfer direction with suppression of fluorescence and a concomitant upsurge of singlet oxygen generation. Since the switching occurs exactly in the same pH range as the difference in intrinsic pH between normal and cancer cells, under the same excitation conditions the cancer cells can be selectively destroyed by singlet oxygen action, whereas the cells in the surrounding healthy tissue may rest without damage and fluoresce brightly. The logic operation with drug release at the output was suggested (Yan et al. 2018). Realizing such ideas on switching between diagnostic and therapeutic modes is highly needed in theranostics (see Volume 2, Chap. 17). And the fact that this can be done on the level of composite organic molecule allows making very optimistic predictions. Thus, we observe that organic molecules, nanostructures and their compositions allow not only transduction of sensor output signal into emissive form but also are able to provide its non-linear transformation, simulating the operation of elementary digital computational devices. There is a possibility to go deeper and to request from designed molecules to perform smart logic operations, serving as logic gates. The excited-state reactions of electron, proton and energy transfers offer such possibility. The fact that these reactions are triggered by light, allow optical output and can be coupled by design in different ways may allow solving important problem of intelligent transformation of reporter signal already on molecular or nano-scale level. As a result of these complicated multiparametric logic operations, an already processed output signal can be obtained. No doubt, digital logics will be a common language in these developments. The term ‘lab-on-a-molecule’ is ready for use to coin the multifunctional and multiparametric sensors (Daly et al. 2014), although the real distance to their practical use is not so short. Many of the fluorescent molecular device designs, e.g. based on PET switching principle were established for small molecules. They can serve as starting points for achieving new steps of complexity and perfectness on nanoscale (Claussen et al. 2013a).

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4.6 Sensing and Thinking. How to Optimize Photophysical Transformations for Generating the Output Signal? Summing up, we list the basic mechanisms of signal transduction that operate in fluorescence sensors coupling the target binding and fluorescence (luminescence) response. Photoinduced electron transfer (PET) can operate also at short distances between localized electronic systems. It can be both intramolecular and intermolecular. Intramolecular transfer requires separation between donor and acceptor fragments by only one or two single bond bridges, and intermolecular transfer needs direct contact between them. All these mechanisms are basic and universal, but for their realization they need proper molecular structures, that can be the most efficiently provided by organic dyes. Very short on the scale of distances is the intramolecular charge transfer (ICT). It can operate within π-conjugated electronic system and is able to transform directly the weak interaction energy of target binding into the spectral shifts. Elementary act in intramolecular proton transfer (ESIPT) occurs at even shorter distances (proton is much heavier than electron and its wavefunction is shorter), but in organic dyes this transfer may be coupled with different reactions, such as conformational isomerizations, making-breaking of hydrogen bonds and ICT. The 3-hydroxychromone family of dyes is unique among well-described systems, in which ESIPT is coupled only with ICT, and this allows obtaining strong and adjustable two-band ratiometric response. These basic phenomena (and their frequently observed coupling) can generate wavelength-dependent switching of fluorescence intensity and provide the response in a broad dynamic range. The excited-state energy transfer (EET) commonly operating by mechanism of Förster resonance energy transfer (FRET) can extend to greater distances, up to 8–10 nm. It is much more universal and can be realized with all kinds of emissive molecules and particles as the donors and all kinds of light-absorbing acceptors if the conditions regarding distance, orientation and spectral overlap are satisfied. Conformational isomerizations add a new dimension to signal transduction. They offer many possibilities for coupling the target binding with the response. They are especially important in the cases of large and neutral target molecules, the binding of which does not produce the perturbation sufficient for direct modulation of IST, PET or ESIPT. Target-induced aggregation of nanoparticles is an additional possibility of detecting the large-size targets. Thus, the mechanisms of response are few, but innumerable possibilities to realize them. Please, think on the approach that you would choose to address sensing metal cations, organic acids, neutral small molecules (glucose?), large protein or DNA molecules. Target size, charge and ‘hot spots’—all these parameters may be important, which needs the proper choice of basic response mechanism. The ability of molecular sensors to perform elementary logic operations opens new horizons for exploration and promises new advanced products. This function can be realized even with small organic molecules possessing several recognition

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sites and several transduction mechanisms modulating the response. Such molecules are the prototypes of ‘lab-on-a-molecule’ devices. For operating such device, do we need something else? Excitonic J and H organic dye aggregates, semiconducting and upconverting nanocrystals, luminescent few-atom metal clusters, plasmonic nanoparticles and carbonic nanostructures, will they become building materials of molecular devices with multiparametric inputs and outputs? These materials will be discussed in other chapters of this book. Now more questions are addressed to readers for checking the understanding and for testing their creative minds. 1.

2.

3.

4.

5. 6. 7. 8.

9.

10.

11. 12.

13.

Explain the relation between photoinduced electron transfer (PET) and fluorescence quenching. Does PET always lead to quenching? Is quenching always the result of PET? What is the distance dependence for PET? What other factors determine PET efficiency? Suggest several examples for using these factors in the construction of sensors. Based on what criteria would you differentiate ICT and PET? Suggest the sensor designs that can be realized with PET but cannot with ICT and vice versa. What methods of fluorescence detection shown in Fig. 3.4 are applicable for the detection of response based on PET and what on ICT? How LE and ICT emissions depend on solvent polarity? Can the switching between them be induced by changing the temperature? If yes, in what direction is this change and what is its mechanism? What are the differences in requirements for observation and in reaction mechanisms between intermolecular and intramolecular proton transfers? Let the distance between donor and acceptor on interaction with the target change from 5 to 10 Å and R0 is 50 Å. Will this sensor be efficient? Explain, what are the relative advantages and disadvantages in application of EET with the use of fluorescent or non-fluorescent acceptor. Let all the conditions for optimal EET observation be satisfied but the donor is quenched on interaction with the target. Will this system work as efficient EET sensor? Let donor and acceptor in EET be identical dye molecules. Can EET be efficient between them? If yes, on what conditions? Propose the construction of sensor based on this principle. Make order in the sensing mechanisms as a function of distance. Which of them operate at close contact, which allow separation of several groups and which on larger distances? What is the induced folding of macromolecules? How can it be used in sensing? Can molecular beacons be immobilized on a surface? What transduction mechanisms can be used when molecular beacon is in solution and when it is immobilized? Try to suggest the structures alternative to molecular beacons performing the same function—hybridization with single strand DNA with the reporting based on conformational change.

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14. What are the advantages and disadvantages of maltose binding protein as a scaffold for the design of various sensors? What are the optimal detection methods to be used with this protein as the sensor: (a) with single labeling? (b) with double labeling? 15. Explain the concept of molecular computing. If a molecule has several ionizable groups that protonate in different pH ranges with difference in fluorescence response, can it perform a logic operation? 16. Analyze the inputs, the outputs and the transduction mechanisms in operation of sensor depicted in Fig. 4.18. What is the role of EET? 17. Can molecular logic gate provide two or more different outputs? In what parameters of fluorescence emission you would prefer to get it?

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Chapter 5

Organic Dyes and Visible Fluorescent Proteins as Fluorescence Reporters

A variety of fluorescent and luminescent materials in the form of molecules, their complexes and nanoparticles are available for implementation as response units into sensing and imaging technologies and we discuss their general properties that are essential for their applications. Organic dyes were the first and remain to be the most popular emitters used for these purposes. The labeling and responsive properties of the major classes of these dyes are reviewed here. Major emphasis is provided on those dyes that possess reporting functions and can be used in direct sensing technologies. The dyes demonstrating sensitivity to polarity, local electric fields, hydrogen bonding and other factors are considered. The dyes important for particular applications, such as near-IR emitting, two-photonic and photochromic dyes as well as those demonstrating phosphorescence and delayed fluorescence are also overviewed. Examples of their successful developments for sensing in protein and nucleic acid science are presented. Natural fluorescent proteins are synthesized spontaneously as the gene products inside the living cells and they stimulated synthesis of synthetic analogs. The final section “Sensing and thinking” will be accompanied by questions and problems addressing to the readers. Organic dyes are the most commonly used fluorophores in sensing and imaging. Their advantages are not only in easy availability and low price, but most importantly, in versatility of their applications. Whereas all types of fluorescence emitters can be used for labeling and can respond to external stimuli by changing the emission intensity and lifetime, organic dyes demonstrate much richer means of response, including detectable changes in anisotropy and strong shifts in excitation and emission spectra. These dyes can display ground-state and excited-state reactions leading to the generation of new emission bands that allow convenient and very sensitive λ-ratiometric detection. They offer tremendous possibilities in applications due to immense variations of their structures and diversity of their spectroscopic properties. In the past, the field of organic dye synthesis was strongly stimulated by the needs of color photography and dye laser technologies. These fields lost their actuality with the development of electronic digital photography and solid-state lasers, so that the booming implementation into molecular probe and sensor technologies became the © Springer Nature Switzerland AG 2020 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-030-60155-3_5

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major field of application of organic dyes. The amount of newly synthesized dyes grows exponentially, and it is not possible to describe in a small section of a book even the most important of them. Therefore, we will concentrate on their general properties that are essential for sensing applications and describe several of their classes that are frequently used. Many original publications, reviews (Gonçalves 2008; Grimm et al. 2012; Lavis and Raines 2008; Wysocki and Lavis 2011) and online resources will be of help as a general introduction to the researcher. Addressing the field of fluorescence sensing and imaging, we introduce the classification of organic dyes into two categories—those used for labeling and those used for reporting. With this concept, we consider different dye families focusing on their ability to perform the excited-state transformations, including the family of fluorescent proteins. Additionally, we highlight the dyes extremely efficient in certain application areas. Those are phosphorescent, two-photonic, photoswitching and near-IR emitting dyes. The organic fluorophores formed spontaneously within fluorescent proteins inside living cells and their synthetic analogs will be briefly overviewed.

5.1 Fluorescent Organic Molecules and Their Characteristics 5.1.1 General Properties of Organic Dyes The number of fluorescent synthetic organic products is so great that a researcher can easily select the proper dye corresponding to particular need in terms of spectroscopic properties and chemical reactivity. Due to enthusiastic efforts of Dick Haugland (Haugland and Larison 1992) and other researchers (Tsien and Waggoner 1995) already in 1990th the systematic record of a broad range of then existing fluorescent dyes was made, and this provided a strong background for the development of sensing and imaging field. Within the same fluorophore families, a broad variety of analogs were synthesized. With synthetic means, it became possible to modulate the spectroscopic properties by electron-donor and electron-acceptor substitutions (Gonçalves 2010) and to endow them with different specificity and reactivity for covalent labeling. Due to their small size and the ability of incorporation with minimal perturbation into biological structures (e.g. biomembranes, proteins or nucleic acids), organic dyes are ideally suited as both molecular labels and reporters. Organic dyes, the size of which is commonly less than 1 nm, are characterized by the presence of delocalized π-electrons forming discrete energy states. Absorption of light quanta and their emission occurs as electronic transitions between these states. The wavelengths of their absorption and emission occupy the whole visible range extending to near-IR. The increase in the number of π-electrons and in electronic conjugation between their molecular fragments usually shift the fluorescence spectra

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Fig. 5.1 The measurements of light absorption and fluorescence spectra in solution with fluorescein as example. Horizontal arrows show the light pass through monochromator M and through the sample to record the absorption spectrum. The fluorescence spectrum is usually collected at the right angle after passing the second monochromator. Vertical arrow indicates the fluorescence excitation wavelength that is usually selected closely to absorption band maximum. The major parameters derived from absorption and fluorescence measurements are listed

to lower energies, longer wavelengths (Levitus and Ranjit 2011). Figure 5.1 illustrates the measurements of light absorption and fluorescence emission spectra with fluorescein as an example and indicates major parameters that are used to characterize them. Major parameters that are used to characterize light absorption and emission spectra were introduced and described in Sect. 3.1.3. Regarding organic dye molecules, their molar absorbance is relatively high in view of their small size. The spectra rarely show the resolved vibrational structure and usually are seen as broad smooth bands both in absorption and in emission. Their fluorescence quantum yields can vary dramatically from almost zero to nearly 100%. Accordingly, the brightness, which is the molar absorbance multiplied by quantum yield, proves to be variable (Lavis and Raines 2008). Some of the basic parameters of organic dyes are listed in Table 5.1 that may help the reader with a brief navigation of their properties and comparing them with those of other luminescent emitters discussed throughout this book. Organic dyes offer tremendous diversity of structures, chemical reactivities and spectroscopic properties, which is the great advantage of these fluorescent materials. The dye photostability can create important problems in some applications (discussed in the next section). Some of these features can be improved by encapsulating them into polymeric or macrocyclic host molecules (Dsouza et al. 2011) and nanoparticles (see Sect. 8.4). The great number of fluorescent organic dyes used in sensing and imaging applications reflect the fact that the ideal dyes simply do not exist, and the researcher

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Table 5.1 The properties of organic dyes used in fluorescence sensing technologies Parameter

Property

Size

0.5–2 nm

Absorption spectra

Variable and often narrow, asymmetric and sometimes with vibronic structure, sensitive to intermolecular interactions

Molar absorption, ε

Variable, usually in the range 103 –105 M−1 cm−1

Emission spectra

Broad (~40–70 nm half-width) and asymmetric, position may exhibit strong sensitivity to intermolecular interactions

Stokes shifts

Variable, commonly ~10–100 nm

Excited-state lifetime, τF

Short, ~0.5–5 ns (with few exceptions)

Fluorescence quantum yield, 

Variable, strongly dependent upon the medium strongly variable and uncorrelated with ε

Two-photonic cross-section, α

Strongly variable, dependent on the position of absorption band

Fundamental emission anisotropy, r 0

Variable, up to 0.3–0.4

Photostability

Variable to poor

Chemical resistance

Variable and often low

Availability of chemical modification/functionalization

Variable to high

Cell toxicity

Variable, often low. High phototoxicity may be observed in some cases

has to compromise some properties for making other properties ideal for a particular application. Therefore, the first question to respond is on a particular role that the dye has to play in the sensing process. It could be the labeling of a sensor, of a competitor or of a sample pool containing the target (the dye indicates its location or attachment to a particular structure). These are the simplest applications, to which many dyes are well suited. If the reporting function is needed (the parameters of dye emission should be changed on interaction with the target), then additional criteria should be introduced. As it will be shown below, fluorescent dyes can be classified into two broad categories: no responsive (used for labeling) and responsive (used as probes and reporters in molecular sensing). The first operates based just on their presence in particular medium or at particular site. Second, in contrast, should respond to different stimuli required in sensing and probing by the most significant change of fluorescence parameters. Ideally, this response should not depend on dye concentration.

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5.1.2 The Photostability of Organic Dyes Photostability is one of the most important parameters that determine the usefulness of organic dyes in different applications (Demchenko 2020). It is the dye ability to withstand many cycles of excitation and back relaxation to dark ground state without irreversible damage. Organic fluorophores always demonstrate limited photostability compared to inorganic materials. Hopefully, photobleaching is seen to be the least probable among different photochemical events. The reactions leading to photobleaching are very fast but the probability of any of them is very low, roughly one million times lower than that of light emission. Since the kinetics of this process is separated from the emission kinetics by so many orders of magnitude, it is only the irreversibility that allows following it by temporal decrease in the amount of absorbing and emitting species. Therefore, the factors that influence photobleaching must be quite different from those influencing the emission. They involve chemical transformations occurring very fast, but with very low probability. If organic dyes are used in sensing technologies that do not require exposure to intensive light, their photostability may not be a great problem. It may not be so important in common spectroscopic studies, in which the narrow excitation slits and fast recording of spectra are used, as well as in flow cytometry and microplate reading where the sample remains in the laser beam for a short time. However, when high light intensities are used or the light quanta from single molecules are collected, the emission of only millions of photons that one can extract from a sample before irreversible decay is not enough. Image generation and single-molecular tracking need highly photo-resistant fluorophores in their favorable media. In these applications, a low fluorescence signal reducing the signal-to-noise ratio cannot be compensated by an increased fluorophore concentration or high intensities of incident light. However, no general solution can be found here because photodegradation can follow different oxygen-dependent and oxygen-independent mechanisms that can extend over very broad ranges and depend on many factors (Demchenko 2020; Eggeling et al. 1999). The quantum yield of photobleaching b is a quantitative measure of dye photostability. It is defined as the ratio of the number of photobleached molecules to a total number of absorbed photons. It is determined in kinetic experiments using spectrofluorimetry (Corredor et al. 2006) and spectrophotometry (Belfield et al. 2004a). For low-concentrated solutions, a method based on the time-dependent changes in fluorescence intensity is thought to be the most accurate, and for highly concentrated solutions a method recording the temporal changes in the absorption spectra offers higher accuracy. The fluorescence method is preferable for two-photon excitation conditions (high intensity pulsed-laser irradiation) due to relatively low efficiency of two-photon absorption in comparison with the correspondent one-photon case. Important for different applications of fluorescence is the average number of photons emitted before a dye molecule bleaches (Eggeling et al. 1999). It can be described as the ratio between the b of the dye and its quantum yield of fluorescence.

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Typical photobleaching quantum yields for organic dyes, if they are studied in solvents at relatively low excitation intensities, are observed on the level of 10−4 – 10−5 (Demchenko 2020; Zheng and Lavis 2017). Among the least photostable are the coumarin dyes and fluorescein, with b ~ 10−4 . Thus, before decomposing, these molecules can survive between 104 and 105 excitation-emission cycles (Tsien and Waggoner 1995). Therefore, when dissolved in water, with b ~ 3 × 10−5 the average fluorescein molecule before being permanently bleached emits 30,000– 40,000 photons. Commonly, the studied dyes are compared as being more or less photostable than fluorescein. Relatively low photostability is also of fluorescent proteins. More photostable are rhodamines and BODIPY dyes, for which b varies between 10−6 and 10−7 , which is related to their low probability to convert to triplet states. The extraordinarily small values of ~10−8 can be found for dye molecules as impurity centers in a molecular crystal or on incorporation into polymer matrix. Such variability in orders of magnitude suggests the presence of different mechanisms of photobleaching that can be realized in different extent depending on the dye structure. What are these mechanisms? One of the most important of them is the dye degradation in reaction with singlet oxygen 1 O2 * and other reactive oxygen species (ROS) (Vogelsang et al. 2008; Widengren et al. 2007). These highly reactive forms of oxygen are produced by sensitization of the ground-state triplet oxygen 3 O2 (3  g ) by the excited triplet-state fluorophores. The latter state is achieved in the reaction of 1 S1 → 3 T1 intersystem crossing after the fluorophore excitation to a singlet excited state 1 S1 . This mechanism first suggested by Michael Kasha (Demchenko et al. 2014) has got many confirmations (Fig. 5.2).

Fig. 5.2 Generation of photodestructive singlet oxygen 1 O2 * in a photochemical reaction. The photosensitizer dye is excited to the 1 S1 state and when it exhibits intersystem crossing (ISC) to the 3 T state it can either generate phosphorescence or transfer the excitation energy to the ground-state 1 triplet oxygen 3 O2 to result in the excited 1 O2 * state. The latter can emit phosphorescence in the near-IR region but being highly reactive, it is able to activate different photochemical processes. One of them is an irreversible photooxidation of the dye

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Thus, by removing oxygen or suppressing its diffusion in the system can we achieve ideal photostability? There are numerous observations that it is not enough and that a second pathway for the chromophore degradation must exist (Alamiry et al. 2015). It involves reactive radical intermediates that appear on photoionization of the dye when it is excited to the first (1 S1 ) or higher (1 Sn ) excited states and is more efficient in triplet 3 T1 and 3 Tn (n > 1) states due to their stronger reactivity. In the case of oxygen-independent pathway, the application of so-called “antifading agents” suppressing the generation of free radicals, produces positive effect (Vogelsang et al. 2008). Photobleaching under the two-photon excitation may lead to enhancement of the photobleaching rates by several orders of magnitude at comparable photonemission yields (Belfield et al. 2004b; Dittrich and Schwille 2001), which is also explained by photooxidation and free radical formation. The general scheme depicting different possible steps of dye photodegradation is presented in Fig. 5.3. The irreversible photochemical reactions may start already in the 1 S1 state (step 1) and lead to other destructive transformations during its lifetime. Reducing the 1 S1 lifetime may not be a good idea due to the loss of fluorescence quantum yield. But its reduction may be coupled with a useful photochemical transformation, such as the excited-state energy transfer to fluorescent or non-fluorescent acceptor (Basu et al. 2018; Strack 2018). Also, since superradiance, as in J-aggregates (Bricks et al. 2017), or plasmonic enhancement (Willets et al. 2017) reduces the 1 S1 lifetime, it may increase the photostability. During the 1 S1 lifetime, the intersystem crossing to 3 T1 state may proceed. Suppressing the transition to 3 T1 state (step 2, see Fig. 5.2) would reduce the probability of all destructive reactions starting at this step. This can be achieved by proper dye design, which requires eliminating the factors favoring ISC, such as the presence of heavy atoms in fluorophore or in its environment (Mukherjee and Thilagar 2015; Solov’ev and Borisevich 2005). When the conversion to 3 T1 state is still efficient, it is the long lifetime together with higher reactivity that is responsible for the dye irreversible reduction or oxidation generating radical ions. The lifetimes of 3 T1 – 3 Tn states can be reduced on step 3 by using the triplet state quenchers (Zheng et al.

Fig. 5.3 Schematic representation of different steps in organic dye excited-state transformations leading to their photodegradation. The possibilities of blocking these steps are depicted

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2016). Since the reactive triplet states generate irreversible photochemical events and the most important of them is the production of singlet oxygen and other reactive oxygen species with their destructive action (see Fig. 5.1), the suppressing of their production is important (Aitken et al. 2008) (step 4). And finally, highly destructive photoionization reactions and free radical generation, step 5, that proceed both in singlet and triplet excited states, must be avoided or suppressed. A combination of oxidative and reductive species may be efficient on this step (van der Velde et al. 2014). In general, the dyes exhibiting susceptibility to excited-state electron transfer reactions demonstrate low photostability, whereas cyclic aromatic hydrocarbons, such as pentacene or perylene, in which the excitation does not lead to strong electronic charge separation, are the most photostable (Rieger and Müllen 2010). This is not a good story for developing the direct sensors (discussed in Sect. 2.5), since the mechanism of response of many of them is based on electron-transfer quenching or on intramolecular charge transfer induced spectral shifts. Within the family of related compounds, some regularities were found in connection with their structures. The increase in number of delocalized π-electrons leading to fluorophore elongation and to red-shifting of electronic spectra is known to decrease the photostability. This fact was detected among cyanines (Diaspro et al. 2006; Tsien and Waggoner 1995) and the dyes of BODIPY family (Wittmershaus et al. 2001). In contrast, the increase in oxidation potential increases the dye photostability. Such correlation was found within the series of BODIPY dyes (Cui et al. 2007). Important also to indicate that the flexibility of dye molecular structure can greatly reduce the photostability, thus making the structure rigid improves it (Song et al. 2004). With the account of these regularities, new generations of dyes have been synthesized, in which modifications of already existing fluorophores are introduced. The presently popular series of Alexa dyes (see Fig. 5.5) were obtained by rigidification of structure and addition of sulfonate groups to a series of common dyes (PanchukVoloshina et al. 1999). It was also reported that thiolated polyenic chromophores, exhibiting large optical nonlinearity, possess excellent photostability (Cheng et al. 2008). Addition of an aryl group at C-8 position of the pyrromethene chromophore of BODIPY increases the photostability (Mula et al. 2008). The result of these substitutions may be the protection of labile groups from interaction with active forms of oxygen. Also, the insertion of cyano group into specific position in the central polymethine chain of merocyanine dyes demonstrates its improvement due to reduction in reactivity with singlet oxygen (Toutchkine et al. 2007). Dramatic increase of photostability was achieved by inserting the substitutions into the central part of symmetric near-IR carbocyanine dye (Samanta et al. 2010). The low photostability of 3-hydroxyflavones can be improved by substituting central oxygen into nitrogen to make 3-hydroxyquinolones (Yushchenko et al. 2006b). The simple replacement of oxygen by nitrogen atom in the dicyanomethylene-4H-pyran (DCM) moiety creates a new family of fluorophores with a significant photostability (Li et al. 2015). The photostability of cyanine-styryl dyes of the indole-quinolinium type that are used for DNA staining can be substantially improved by structural modifications

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(Bohländer and Wagenknecht 2013). Fluorination generating the electron accepting groups results in strong dye improvement, which is seen in coumarins (Sun et al. 1998) and in BODIPY dyes (Huynh et al. 2016). These features are characteristic for near-IR absorbing heptamethine cyanine dyes (Funabiki et al. 2016). Thus, the targeted inclusions of protective groups in the sites potentially sensitive to photodamage, the attachment of the triplet quenchers, of fragments that exhibit redox properties and suppress the formation of reactive oxygen species illustrate the range of these possibilities (Van Der Velde et al. 2016; Zheng and Lavis 2017).

5.1.3 Protection of Dyes with Macrocyclic Hosts A valuable utensil for protection of organic dyes from chemical and photochemical degradation and to suppress their intramolecular motions is their inclusion into macrocyclic hosts of cyclodextrins and cucurbiturils (Fig. 5.4). This is also the way to provide their solubility in aqueous media and to prevent their aggregation (Dsouza et al. 2011). The remarkable success of this approach was demonstrated in the cases of formation of rotaxane-type encapsulation of cyanine (Buston et al. 2000; Yau et al. 2008) and squaraine (Arunkumar et al. 2005a; Patsenker et al. 2010a) dyes into the cyclic saccharide α-cyclodextrin cavity. In this mechanically interlocked molecular architecture, the cyclodextrin is tightly threaded round the central part of the dye. Another type of partners in the dye photoprotection are cucurbiturils, which are the water-soluble macrocyclic molecules composed of glycoluril units (Koner and Nau

Fig. 5.4 Macrocyclic hosts for fluorescent dyes. a The cyclic saccharides cyclodextrins. The spatial structure of β-cyclodextrin (β-CD) is shown. b The general structure of cucurbiturils and the model of spatial structure of cucurbit[7]uril (CB7). c The reaction of incorporation of a squaraine dye into CB7 host (Patsenker et al. 2010a)

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2007). The strong stabilizing effect of these host molecules is due to low polarity of their inner cavity and efficient screening of the dye reactive sites from solvent water (Mohanty and Nau 2005). Thus, a strong improvement was reported for BODIPY complex formation with the macrocyclic host cucurbit[7]uril (Gupta et al. 2017). It was stated that molecular encapsulation is an attractive supramolecular strategy because it is inherently flexible and does not necessarily require the time-consuming synthetic processes (Arunkumar et al. 2005b). Indeed, In addition to increased photoprotection, the incorporation into cyclodextrin and cucurbituril cavities improves other attractive properties of the dyes, such as their fluorescence quantum yield, solubility in water and acid-base behavior (Nau and Mohanty 2005; Patsenker et al. 2010a; Sayed and Pal 2016).

5.1.4 New Discoveries and Improvements The general requirements for fluorescence emitters were discussed in Sect. 3.1. What of them are the most important for sensing and imaging applications and deserve the efforts of researchers for their improvement? The answer depends on the goal to be achieved. For every particular application, the dye parameters have to be optimized in many directions and as it often happens neither empirical knowledge nor the aid of quantum mechanics calculations may be a good help. Despite of that, we observe very rapid developments in several directions. We overview them in short. Targeted modifications within the existing fluorophore families. Most efforts are directed to increasing the brightness, which with a given absorbance is the increase of fluorescence quantum yield (Lavis and Raines 2008). This provides the directions to suppress the non-emissive losses that may exist due to intramolecular vibrations and rotations and also to conversion to triplet state. The development and introduction of Alexa Fluor dyes was one of the most successful projects of Molecular Probes (Haugland 1996, 2005). The dyes Alexa 350, Alexa 430, Alexa 488, Alexa 532, Alexa 546, Alexa 568, and Alexa 594 were obtained by rigidification of structure and addition of sulfonate groups to coumarin derivative AMCA, Lucifer Yellow, fluorescein, Rhodamine 6G, tetramethylrhodamine or Cy3, lissamine Rhodamine B, and Texas Red dyes. Their excitation/emission spectra are similar to the parent dyes, and the numbers in the Alexa names indicate the approximate excitation wavelength maxima in nm. Thus, the excitation and emission wavelength ranges of Alexa dyes cover the entire spectrum from ultraviolet to red with the retention of such properties of parent compounds as high molar absorbance. An increase in brightness and insensitivity to pH in the 4–10 range was achieved (Panchuk-Voloshina et al. 1999). The introduction of negatively charged sulfonic acid groups also makes these molecules more water-soluble and decreases their inherent tendency to form aggregates. This also allows performing their conjugation with biological macromolecules in the absence of organic solvents. The modified dyes are less subjective to self-quenching, and their photostability is even higher

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(Panchuk-Voloshina et al. 1999). Following this trend, several companies suggest their well-developed products (Fig. 5.5). Another important development is the replacement of N,N-dialkylamino groups in the dyes that exhibit twisted intramolecular charge transfer (TICT) with fourmembered azetidines, creating the so-called Janelia Fluor (JF) dyes (Grimm et al. 2015). It was shown that in rhodamine derivatives in which TICT is suppressed, an increased quantum efficiency, longer fluorescence lifetimes, and twice higher photostability were achieved. Fluorination generating the electron accepting groups results in strong dye improvement, which is seen in coumarins (Sun et al. 1998) and in BODIPY dyes (Huynh et al. 2016). Novel photostable polyfluorinated cyanine dyes exhibit significantly reduced aggregation in aqueous media, enhanced fluorescence quantum yield, greater resistance to photobleaching upon direct irradiation, and reduced reactivity towards singlet oxygen (Renikuntla et al. 2004). Thus, the vast collection of small-molecule fluorescent probes is derived from a modest set of basic scaffolds that were known in many other applications beyond sensing. Hybrids made by fusing existing fluorophores. Several aims can be achieved on this way. First is the possibility of increasing the π-conjugation that allows enhancing the absorbance and shifting the spectra to longer wavelengths. The other trend is to induce the intramolecular charge transfer and excitation energy transfer (EET) for increasing the Stokes shift (Harriman et al. 2013). In the designed hybrids, the fluorophores can be made more functional (Karmakar et al. 2015). Hybrid dyes based on rhodamine fluorophore allowed extension to near-IR region (Wang et al. 2019a). Of interest are the naphthalene-BODIPY (Yang et al. 2015) and anthracene-BODIPY conjugates (Aotake et al. 2015), as well as squaraine-BODIPY heterodimers (Lambert et al. 2015) and BODIPY fused with cyanine dyes (Zatsikha

Fig. 5.5 Fluorescence emission spectra for Alexa Fluor dyes (from ThermoFisher Scientific catalog)

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et al. 2013). Thus, BODIPY fluorophore is central in many of these developments (Zatsikha et al. 2015). Discovery of new fluorophore families. The magnitude of organic dyes is not limited to derivatives belonging to small number of their existing families. Sometimes, a happy chance appears to discover a basic fluorophore that can originate a new family. Such story happened with the discovery of Seoul-Fluor dyes (Kim et al. 2008). Their core skeleton, 9-aryldihydropyrrolo[3,4-b]indolizin-3-one, was identified in an effort to synthesize novel polyheterocycles as the novel drugs using a privileged substructure-based diversity-oriented synthesis strategy (Kim et al. 2015). Possessing similar to fluorescein quantum yield, the core product demonstrated much larger Stokes shift, higher photostability and insensitivity to pH in a broad range 4–9. Within a single molecular framework, simply by changing the substituents at just two variation points of the fluorophore, it became possible to achieve the tunability covering a full visible-color range and environment sensitivity through intramolecular charge transfer processes. Discovery of green fluorescent protein (GFP) and its analogs and establishment of their fluorophore structures stimulated many research activities in designing their synthetic analogs. They will be discussed in Sect. 5.5. Combinatorial approach. An interesting comparison was made between two approaches for improvement of fluorescent dyes, targeted synthesis based on rational design and combinatorial approach operating with large libraries (Kim and Park 2010). It was stated that whereas targeted approach is efficient to rationally study the photophysical properties of known fluorescent core skeleton, the combinatorial approach requiring the selection within the dye libraries is able to discover or develop novel fluorescent probes. Complementing the traditional targeting approach, it can bypass at least three central obstacles to the discovery of new dyes for probing and sensing based on their rational design (Finney 2006): (a) Complexity of underlying photophysical phenomena. The excited-state processes are different from those occurring in the ground states and not so strongly related to the chemical structure. They involve multiple relaxation phenomena. Because of that, the fluorescence parameters are hardly predictable using quantum chemical calculations. (b) The still common one-molecule-at-a-time dye synthesis together with its purification and characterization is low productive. (c) For the evaluation of dye properties, a labor-intensive characterization needs to be used that involves detailed spectroscopic analysis and comparison with the known standards. The combinatorial approaches offer solutions to overcome these difficulties (Finney 2006; Vendrell et al. 2012). Optimization can be achieved by exploring the structural space around the known core fluorophore, keeping the line along the needed properties. This approach simplifies the synthetic challenge and generates many related compounds for comparative analysis. Then, the multitude of products can be evaluated in the simplest way, disregarding most of the quantitative aspects. This can be done in a high-throughput manner using fluorescence plate readers. Any

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interesting “hits” can be then characterized in more detail. A number of solid-phase and solution-phase synthetic procedures are now well developed, and their application shows good results (Vendrell et al. 2012). Though discovery of completely new fluorophores with this approach is not addressed, this allows rapid optimization of many properties of known fluorophores together. Thus, through a comparison between targeted synthesis and combinatorial approach in the study of fluorescent organic compounds, we can conclude that the targeted synthesis with rational design and systematic modification using combinatorial approach are complementary to each other for the discovery of novel, small molecules with desired photophysical properties (Kim and Park 2010).

5.1.5 Labeling and Sensing—Two Basic Methodologies With regard to applications, we need to draw distinguishing line between ‘labeling’ and ‘sensing’ functions of fluorescence emitters because these functions are completely different (Fig. 5.6). The role of ‘labels’, ‘tracers’ or ‘tags’ is solely to indicate their presence in particular medium or at particular site (often achieved by their specific binding), so that their own concentration displayed as fluorescence intensity is the source of information. Regarding this function, they do not differ from radioactive, NMR-active or electron-dense labels. In the case of labeling the

Fig. 5.6 Two trends in application of fluorescent materials in sensing and imaging. The labels should be insensitive to any parameter changing the fluorescence emission. In contrast, the fluorescence reporters participating in sensor functions should provide the changes of fluorescence parameters in response to particular stimuli in the broadest possible dynamic range

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output signal should be proportional to fluorophore concentration. For achieving that, spatial separation between free and target-bound sensor may be needed. Therefore, they are used to identify and quantify the target on microarrays, microscopy images, chromatograms, electrophoregrams and other systems with spatial separation. There is a broad selection of fluorescent species for labeling. Their major parameters that have to be optimized are the brightness and photostability, together with convenient conditions for excitation and emission. Quite a different function of fluorescent materials is their serving as ‘probes’, ‘sensors’ or ‘reporters’. They should respond to the changes of their molecular environment (probes) or to the target binding (sensors) by the change of parameters of their emission and therefore they should satisfy often special and more stringent requirements. For providing informative response, their fluorescence emission should be switchable and coupled with target recognition events. Here, the brightness and photostability are equally important, but the optimized parameters should be those that allow their variation in the broadest possible range. Responding to sensing event, they should provide the output signal independent of their own concentration and the factors intervening with sensing. There is a general principle. If the electronic density is delocalized over the whole fluorophore, which is characteristic of resonant or mesomeric dyes, such as fluoresceins, rhodamines, and cyanines, the spectral sensitivity to environment is small. In contrast, the dyes exhibiting polarization of electronic structures that changes on optical excitation can be potent responsive fluorescence reporters. Thus, both types of dyes, for labeling and sensing, are useful in fluorescence-based technologies, but in different ways. For obtaining the highest brightness and highest contrast and also for optimal protection against various quenching and bleaching effects or the perturbations by interactions with the medium, one has to choose the ‘labels’. Alternatively, if we wish, together with the high brightness, to attain the highest dynamic range of response in terms of variations of intensity, lifetime or of the wavelength of emission, we choose the ‘sensors’ or ‘probes’. In their design, we need to activate some photophysical mechanisms of response (ESIPT, EET, static or dynamic quenching, isomerizations, formation of exciplexes, etc.), as it was discussed in Chap. 4. This limits dramatically the choice of fluorophores that must possess desired features. In the sections that follow, we will discuss different molecular-size fluorescence emitters that can perform these tasks. Nanoscale format of fluorophores offers many additional possibilities for efficient applications. They will be discussed in the next chapters.

5.2 Organic Dyes Optimal as Labels and Tags There are several classes of dyes that conform in the best way to the criteria for optimal labels and tags. As it was stated above, a great variety of these dyes derive from only several parent fluorophores by a variety of chemical substitutions. Below, we discuss the properties of some of these parent dyes. The excellent review (Fu

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Fig. 5.7 The structures of fluorescein (a) and rhodamine 123 (b) and normalized absorption (black) and fluorescence (red) spectra of rhodamine 123 in methanol (c)

and Finney 2018) analyzes the sites of these substitutions and their spectroscopic outcome. High quantum yield of fluorescence emission in solutions can be achieved in the case of resonant or mesomeric dyes, such as fluoresceins, rhodamines, BODIPY and cyanines. Their skeleton is rigid, which allows minimizing the vibration-related energy losses, and their electronic density is delocalized over the whole fluorophore. Because of the absence of strong charge asymmetry, their interaction with polar environment is small and this is the reason why we observe their small Stokes shift and low spectral sensitivity to the environment. They can be efficiently used if a ‘responsive’ function is not needed. Synthetic chemistry suggests many improvements in their properties. The ‘classical’ dyes together with their most efficient improvements are discussed below. Presented in Fig. 5.7 structures of fluorescein and rhodamine 123 and the spectra of the latter are typical in this respect. Fluoresceins. The spectra of parent fluorescein dye are a part of design in Fig. 5.1. The dyes of fluorescein family are widely used for labeling in the form of amineand SH-reactive derivatives. They possess relatively high absorbance, εM ≈ 70,000– 90,000 cm−1 M−1 , high fluorescence quantum yield (even in water) and aqueous solubility. Additionally, due to the fact that their excitation band maximum matches closely the 488 nm spectral line of the most popular argon-ion laser, these dyes are very well accepted in confocal laser-scanning microscopy and flow cytometry applications. Meantime, fluorescein dyes possess many essential drawbacks. They demonstrate low photostability and also the presence of pH-sensitivity of fluorescence spectra with significant reduction of intensity below pH 7. They are subjective to strong concentration-dependent quenching that does not allow obtaining high density of labels. Photostability and pH-sensitivity of fluorescein can be finely tuned by chemical modifications. Its derivative Oregon Green behaves in a more attractive way. The fluorinated analogs of fluorescein in the conditions of high degree

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of labeling are quenched to a much lesser extent. They are more photostable and pH-insensitive in the physiological pH range (Haugland 1996). Rhodamines. These dyes (see Fig. 5.7) are very attractive for applications due to their high fluorescence quantum yields (even in water) and aqueous solubility. Their high molar absorbance (εM ≈ 85,000 cm−1 M−1 , for Rhodamine 123), which together with high quantum yield makes them very bright emitters for green range of the visible spectrum. Just like fluoresceins, rhodamine dyes are considered as a popular choice of the dyes for labeling. Their excitation band maxima are located at about 520 nm, which is close to the 514 nm spectral line of the argon-ion laser, making them both popular and important in the studies involving confocal microscopy and flow cytometry. In different electronic energy transfer (EET) schemes, they are often used as the excitation energy acceptors. Selected as an example, rhodamine 123 is a cationic green-fluorescent dye with good solubility in water and the ability to permeate the living cells. When used in biological labeling, it is sequestered by active mitochondria without cytotoxic effects. Because of its positive charge, its binding to mitochondria depends on membrane potential of this organelle. Due to its small Stokes shift, this dye is self-quenched when accumulates in mitochondria in high concentrations and thus provides a ‘slow’ response to its membrane potential (Plasek and Sigler 1996). New modifications of rhodamine were developed recently. Particularly important are Si-rhodamines (Koide et al. 2011) and carborhodamines (Kolmakov et al. 2010). Variation of the functionality in rhodamine skeleton results in new near-IR dyes with improved brightness and photostability. These dyes are particularly useful for super-resolution fluorescence microscopy. Remarkable is the relatively high photostability of rhodamine dyes. Their b determinations resulted in a broad range of values between 2 × 10−6 and 2.5 × 10−5 (Eggeling et al. 1999). These values are superior in comparison to many common fluorescent dyes. BODIPY dyes. If someone looks for ideal not responsive dye for labeling, it can be found among boron–dipyrromethene BODIPY dyes (Fig. 5.8). These dyes demonstrate very rapid developments showing attractive advantages in different applications (Banuelos 2016; Kowada et al. 2015; Ulrich et al. 2008). In contrast to many other fluorescent dyes, BODIPY molecules are uncharged. They are insensitive to solvent polarity and pH. They display sharp, intense absorption bands with molar absorption coefficients of about 80,000 M−1 cm−1 and fluorescence quantum yield approaching 100% (even in water!). Their absorption and emission spectra show good mirror symmetry with unusually small Stokes shifts. Their radiative lifetime is quite long, typically being about 5 ns. They allow various modifications modulating their photophysical properties (Cui et al. 2007; Fu and Finney 2018). Depending on the substitution pattern, these dyes can emit light in a broad wavelength range starting around 500 nm and going to the near-IR region (Lu et al. 2014). The application of these dyes is extremely versatile. They are used to produce fluorescent conjugates of proteins, nucleotides, oligonucleotides and dextrans, as well as to prepare fluorescent enzyme substrates, fatty acids, phospholipids, lipopolysaccharides, receptor ligands and polystyrene microspheres. Due to their relatively long

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Fig. 5.8 The BODIPY (4,4-difluoro-4-bora-3a,4adiaza-s-indacene) dyes (Haugland 1996). a The structure of the parent fluorophore. b Normalized fluorescence emission spectra of (1) BODIPY FL, (2) BODIPY R6G, (3) BODIPY TMR, (4) BODIPY 581/591, (5) BODIPY TR, (6) BODIPY 630/650 and (7) BODIPY 650/665 dyes in methanol

lifetime (~4 ns and longer), they are particularly useful for fluorescence polarizationbased assays. They are far more photostable than fluorescein, which makes them more attractive for biological imaging (Hinkeldey et al. 2008; Mula et al. 2008). Success has been achieved in designing the highly photostable near-IR BODIPY emitters, particularly by fusing two aromatic molecular units (Yu et al. 2015). Acrydine and ethidium dyes. These cationic dyes are frequently used for staining double-helical DNA. They bind with a stoichiometry of one dye per 4–5 base pairs of DNA, and the binding mode is an intercalation between the nucleic acid bases with little or no sequence preference. Intercalation is the noncovalent inserting of molecules between the base pairs of the DNA duplex almost without sequence preference, so that the dye molecule is held stacked perpendicular to the helix axis. Ethidium bromide (Fig. 5.9) and its analogs exhibit approximately 30-fold enhancement of emission intensity on their binding. Screening of fluorophore moiety from the quenching effect of water is in the origin of such enhancement. Cyanine dyes generically consist of a π-conjugated system based on a polymethine chain linking two nitrogen-containing heterocycles (Levitus and Ranjit 2011), see Fig. 5.10. The length of this chain determines the spectral range of absorption and emission: with its increase by one vinylene unit (CH=CH) the spectra shift to longer wavelengths by about 100 nm. This allows tuning the absorption and emission spectra in very broad ranges, extending to near-IR. These dyes are characterized by very high molar absorbance that for the near-IR dyes may reach 200,000–250,000 M−1 cm−1 (Bricks et al. 2015). The versatility of their structure is increased by possibilities of substitutions in the main chain and by variation of terminal groups (Levitus and Ranjit 2011). Their common disadvantage is a small Stokes shift, short lifetime (commonly less than 1 ns) and the presence of positive charge.

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Fig. 5.9 The structure of ethidium bromide (a) and its absorption and fluorescence spectra on binding to double-stranded DNA (b)

Due to significant quantum yields and the ability to assemble into highly fluorescent J-aggregates (Bricks et al. 2017), these dyes have found many applications, starting from photography to laser media and biological imaging (Mishra et al. 2000). Their potential for chemical sensors is great (Sun et al. 2016). The shortest monomethine dyes, which include the asymmetric cyanines thiazole orange (TO), oxazole yellow (YO), or dimers of both TO and YO (TOTO, YOYO) are used for staining nucleic acids; they do not fluoresce in solution but possess intense fluorescence when are bound to their minor groove. The new wave of interest to them has emerged due to their capability to provide imaging in the near-IR range 700–1000 nm, which is the only range of relative transparency of human and animal tissues. Polymethine cyanines include the frequently used dyes Cy3 and Cy5 (see Fig. 5.11) that possess increased chain length and are known for binding nucleic acids with much greater affinity. Cy3 can be maximally excited near 550 nm and emits orange light, whereas Cy5 has a narrow excitation peak at 650 nm and produces an intense signal in the far-red region of the spectrum. Their remarkable brightness is due to a high molar absorbance that exceeds 105 M−1 cm−1 . The extraordinary strong increase in fluorescence intensity upon binding to nucleic acids is a result of rigid fixation of their trans-conformation (Tatikolov 2012). In some of them, such increase of quantum yield can be 100-fold, from less than 1 to nearly 100%. Polyfluorination allows reducing the aggregation of these dyes in aqueous medum and enhancing

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Fig. 5.10 The thiacyanine dyes with variable chain length. a The structure. b The influence of chain length (n) on the absorption spectra in methylene chloride. The lengthening of the chromophore leads to a regular bathochromic shift of the band maxima. The spectral effect per vinylene group, V, is traditionally called ‘the vinylene shift’

Fig. 5.11 Cyanine dyes Cy3 and Cy5 that are popular in DNA labeling. a The structures of Cy3 and Cy5 dyes. R are the sites of substitutions that are used in covalent labeling. b The absorption (blue) and fluorescence (red) spectra that have been normalized to the same intensity for comparison purposes (Haugland 2005)

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their fluorescence quantum yield and photostability (Renikuntla et al. 2004). They are frequently used in DNA and RNA microarray hybridization techniques. Squaraines (Fig. 5.12) are increasingly important dyes for sensing and imaging due to their exceptionally high brightness in the red—near-IR region (Xia and Wang 2017). Their narrow and symmetric absorption maxima are found between 630 and 670 nm and fluorescence emission maxima are between 650 and 700 nm. Their molar absorbance reaches the value of 330,000 M−1 cm−1 together with relatively high quantum yield. Their unique aromatic four membered ring system is formed by condensation of squaric acid with two electron-rich moieties. Two aromatic or heterocyclic structures can be conjugated directly with the central ring or attached through the spacer; they can be different. This donor-acceptor-donor (D-A-D) architecture gives rise to the unique photophysical properties of squaraines. They are highly photostable but their chemical stability is low due to strong electron deficiency of central ring. Also, they are poorly soluble in most of the solvents. For increasing the solubility and protection the central ring from nucleophilic attacks their inclusion into different macrocyclic complexes was realized (Patsenker et al. 2010a), see Fig. 5.4. Thus, the representative parent compounds are characterized by rigid structures and small polarization of electron density. This allows their minimal participation in the excited-state reactions and achieve their highest brightness (Lavis and Raines 2008). If such reactivity is required, it can be realized by substitutions and modifications of parent structures. As we will see below, for achieving large Stokes shift and desired excited-state reactivity, some requirements are just the opposite—a strong electronic polarization is needed and, sometimes, induction of vibrations and rotations of molecular segments (Ren et al. 2018).

Fig. 5.12 Typical dyes of squaraine family. The structure (a) and the absorption (blue) and fluorescence (brown) spectra (b) of the parent compound

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5.3 The Dyes Providing Fluorescence Response Here, we concentrate on the dye properties that are required for performing fluorescence response in sensing systems (see Fig. 5.6). They should optimally correspond to detection techniques described in Chap. 3 and allow achieving the broadest dynamic range and highest sensitivity of detection. Only in sensing based on anisotropy (polarization), the role of the dye reporter can be passive to respond to rotations of large molecules or their fragments. In detection based on EET, the response is coupled to variation of the distance between two dyes or overlap of their spectra. In all other cases, the dyes have to respond to formation/breaking of specific or nonspecific noncovalent interactions with their neighboring molecules or groups of atoms. Among the reactions that make organic dyes so sensitive is the intramolecular charge transfer (ICT) that allows responding by spectral shifts to the changes in polarity of their environment. For instance, in the dye Nile Red, the absorption spectrum ranges between hexane and water from 480 to 590 nm, and the variation of fluorescence spectra between these solvents reaches 130 nm, from 530 to 660 nm (Jose and Burgess 2006). The other important reaction that provides the strongest shifts of fluorescence spectra is the excited-state intramolecular proton transfer (ESIPT) (Demchenko et al. 2013). In a series of substituted 3-hydroxychromone derivatives, the dynamic balance between ICT and ESIPT states that generate switchable well-resolved emission bands can be achieved. They can be strong indicators of polarity and molecular-scale electric fields (Demchenko 2006; Demchenko et al. 2003). Formation of stable excimers (complexes between unexcited and excited fluorophores) is characteristic of pyrene. This reaction changes dramatically fluorescence spectrum (from 400 nm for monomer to 450 nm for excimer) and lifetime (~300 ns for monomer and ~40 ns for excimer) and is frequently used in fluorescence reporting (Yang et al. 2005). The formation of excimers requires close location and proper orientation between the partners, therefore the sensing is commonly coupled with conformational changes in the sensor unit that bring the pyrene units together (Kadirvel et al. 2008). Based on organic dyes, many types of molecular sensors were developed: the pH sensors based on photodissociation of protons from aromatic hydroxyls (Whitaker et al. 1991), the molecular rotors that sense local viscosity (Haidekker and Theodorakis 2007), the temperature sensors based on excimer formation (Chandrasekharan and Kelly 2001), the pressure sensors based on oxygen quenching of triplet state (Hochreiner et al. 2005), the ion sensors based on switching between charge-transfer states (Grynkiewicz et al. 1985). There are several classes of dyes exhibiting electrochromism and sensitive to local electric fields (Klymchenko and Demchenko 2002; Kuhn and Fromherz 2003). Due to their small size and the ability of incorporation into biological structures (e.g. biomembranes or double-stranded DNA) with minimal perturbation, the organic dyes are ideally suited as molecular sensors.

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5.3.1 Special Requirements for Fluorescence Reporters Here we analyze the special requirements for the dyes with reporter functions. They are the following: 1. Strong variation in one or several fluorescence parameters described in Chap. 3 that can be quantified. The response can be provided by formation of specific complexes of the dyes (e.g. with the quenchers), by formation of their dimers (e.g. excimers) or aggregates, by pH changes. Alternatively, they can participate in interactions that can be described by such variables as polarity, viscosity, hydrogen-bonding ability. 2. Strong discrimination in response to measurable parameter against the response to other parameters that influence the spectra (e.g. polarity probes should not be highly sensitive to H-bonding and the reverse). Alternatively, the possibility of multiparametric analysis of complex response may be provided (Klymchenko and Demchenko 2003). 3. Insensitivity of response to different interfering factors, such as impurities in the solvents, ions, temperature, pH, etc. if these factors are not the targets of assay. Generation of reporting signal from responding dyes may involve different signal transduction mechanisms that were described in Chap. 4, and the dye properties should be optimally selected for realizing these mechanisms. Responding to intermolecular interactions, these dyes allow explorng many possibilities: to characterize the properties (polarity, proticity, microfluidity) of the binding sites and their changes in the course of binding, to follow the binding-dissociation kinetics, to estimate the changes of molecular distances, etc.

5.3.2 Fluorophores in Protic Equilibrium. pH-Reporting Dyes Many fluorophores conjugated with proton-dissociating chemical groups (phenolic hydroxyl, carboxylic and sulfonic acids) are used as fluorescent pH probes (Han and Burgess 2009). The ionization of attached groups may change dramatically the spectroscopic properties leading to the increase of fluorescence intensity or to the appearance of new bands in absorption and emission spectra. The dyes displaying these bands of high and comparable intensities can be used for λ-ratiometric sensing. Since the acidity of dissociating groups is commonly much higher in the excited than in the ground state, the pH range of sensitivity in emission spectra can be dramatically shifted to lower pH (Davenport et al. 1986; Laws and Brand 1979). This allows providing the λ-ratiometric recording in an extended pH range. Presently, the most extensively used dyes are the benzo[c]xanthene derivatives, such as C-SNAFL-1 (Mordon et al. 1995; Whitaker et al. 1991). The variation of their excitation spectra reflects the ground-state proton dissociation. The pH-dependent

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appearance of strongly red-shifted second band in emission spectra is due to deprotonation of hydroxyl group in the excited state. The fact that the pH-dependent change in ionization of the dye functional groups is local and can respond to the shift in association-dissociation equilibrium of macromolecules without the change of medium pH, can be efficiently used in sensing technologies.

5.3.3 Hydrogen Bond Responsive Dyes The presence of H-bond donor and acceptor groups coupled to aromatic structures possessing π-electrons offers new possibilities for sensing based on the formation/breaking of intermolecular H-bonds. Generation of fluorescence reporting signal based on modulation of intermolecular hydrogen bonding could be a very important possibility for fluorescence reporting. It is known (de Silva et al. 1997) that the formation/disruption of these bonds is an important mechanism of fluorescence quenching. Enhancement of fluorescence in protic environments was also reported (Uchiyama et al. 2006), but it is a rare phenomenon. The most important issue is the possibility of λ-ratiometric detection. The intermolecular hydrogen bonds can influence the intramolecular charge-transfer (ICT) character of the excited states with the correspondent spectral shifts. These effects are usually smaller than that on protonation and occur in the same direction (Wang et al. 2002). In excitation spectra on interaction with H-bond donor at the acceptor site, one can observe the shifts of spectra to longer wavelengths and on interaction with proton acceptor at the donor site—to shorter wavelengths. The fluorescence spectra on such interactions may shift strongly to longer or shorter wavelengths, correspondingly. Carbonyl groups attached to π-electronic system are known as strong sensors of intermolecular H-bonding (Shynkar et al. 2004). Serving as H-bond acceptors, they can be found in many organic dyes. The electron lone pairs on oxygen atoms allow arrangement of two such bonds, the stronger and weaker ones. Their formation is clearly observed in fluorescence spectra (Pivovarenko et al. 2000) and can generate strong λ-ratiometric signal. As we can see in Fig. 5.13, addition of millimolar amounts of alcohol to aprotic solvent produces dramatic changes in these spectra resulting in the loss of fine vibrational structure. Higher amounts of protic molecules result in further transformation of the spectra and the spectral shifts.

5.3.4 The Environment-Sensitive (Solvatochromic and Solvatofluorochromic) Dyes Interacting with the environment the dye molecules may change their interaction with the environment. This interaction may influence both ground-state and excited-state

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0 md/l

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0.049 0.179 0.561 1.424 6.172

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Wavelength (nm) Fig. 5.13 Hydrogen bonding effects in fluorescence spectra of ketocyanine dye 2,5-bis[4-N,Ndimethylaminobenzylidene]cyclopentanone-1. The spectra are obtained in toluene (the narrow structured spectrum at shorter wavelengths) on the addition of various concentrations of methanol (Pivovarenko et al. 2000)

energies and thus produce the spectral shifts. The shifting in absorption spectra is commonly called solvatochromism, and of fluorescence spectra—solvatofluorochromism. The molecular reporter information can be generated when in the course of sensing event the dye changes its interaction with the local environment (Sect. 3.5). The polarity-responsive properties are already present in amino acid tryptophan, which is a natural constituent of many proteins (Demchenko 1986). But small values of these effects together with inconvenient UV excitation do not allow its extensive use in fluorescence sensing. In contrast, there are fluorophores that provide record values in solvent-dependent spectral shifts in the visible range. Like tryptophan and more than tryptophan, they have to change their dipole moment on excitation that changes their interaction with surrounding molecules and results in the shifts of fluorescence spectra. This concept was introduced by Gregorio Weber and the first dyes that found many applications, mostly in the studies of structure, dynamics and interactions in proteins and biomembranes, were the naphthalene sulfonate derivatives such as ANS and TNS (Fig. 5.14). A number of dye derivatives used for covalent labeling of proteins (Dansyl, Acrylodan, NBD, IAEDANS, etc. (Haugland 1996)) exhibit strong polarity-dependent shifts. They were developed for the labeling of proteins. Nowadays, they are used on an extended scale, for covalent labeling to amino or SH groups of any molecular sensors.

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Fig. 5.14 The mechanisms of generation of spectral shifts in the case of ICT and the structures of typical environment-responsive dyes: 2,6-ANS (6-(phenylamino)-2-Naphthalenesulfonic acid); 2,6-TNS (2-(p-toluidinyl)naphthalene-6-sulfonic acid); Nile Red; Prodan (6-propionyl-2dimethylaminonaphthalene) (a) and the solvent-dependent fluorescence spectra of Nile Red in indicated solvents (b)

The dyes Nile Red and Prodan (Fig. 5.14) are of latter generation demonstrating increased solvatochromism (Kucherak et al. 2010b). The background of this effect is intramolecular charge transfer (ICT) between two distant but electronically coupled functional groups of atoms, one serving as electron donor and the other as electron acceptor (see Sect. 4.1.2). They are integrated into π-electronic chromophore system and shift its electronic density creating strong dipoles. Prodan is one of the most solvent-sensitive among commonly used probes operating on the principle of solvent-dependent band shift. Its absorption spectrum is observed at 350–370 nm and fluorescence spectrum ranges from 416 nm for toluene to 505 nm in methanol (Fig. 5.14). The dyes, containing the H-bond proton acceptor groups, such as carbonyl group of Prodan, exhibit additional spectral shifts. It occurs in the same direction as the increase of polarity, to longer wavelengths. In Prodan, almost a half of this shift is due to hydrogen bonding (Balter et al. 1988). Water

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molecules are among the strongest proton donors, so the sensing effect can be coupled with the change in dye hydration. The strong solvent sensitivity of this molecule is connected with disadvantages, such as dramatic fluorescence quenching in all protic media. Recent developments resulted in obtaining Prodan analogs with superior properties. It was found that isomers of Prodan with different positioning of the donor and acceptor group in the naphthalene core exhibit much stronger solvatofluorochromic properties (Benedetti et al. 2012). Its naphthalene core was also substituted with aromatic hydrocarbons possessing increased electronic conjugation. Substitution by anthracene resulted in Anthradan, which led to the shifts of both excitation and emission spectra to the red by about 100 nm with retention of high sensitivity to polarity and hydrogen bond formation in protic environment (Lu et al. 2006). New probes on the basis of fluorene (Kucherak et al. 2010a) also demonstrated red-shifted absorption, an absorption coefficient (43,000 M−1 cm−1 ), twice as large, and a manifold larger two-photon absorption cross section (∼400 GM) compared to Prodan. Much stronger polarity-dependent shifts of spectra are due to twice as large transition dipole moment (14 Debye units). The strong increase of fluorescence quantum yield was also reported. Very high quantum yield in both polar and apolar solvents together with strong solvatofluorochromy was found for Prodan analog with pyrene core (Niko et al. 2013). These novel derivatives with superior spectroscopic and solvent-sensitive properties are expected to totally substitute Prodan in molecular probing studies. Phenoxazone dye Nile Red, is the typical fluorescent dye with remarkable ability of solvent-dependent spectral shifts both in absorption and fluorescence spectra. Its absorption spectrum ranges from 480 to 590 nm between hexane and water, and variation of fluorescence spectra between these solvents reaches 130 nm, from 530 to 660 nm (see Fig. 5.14). Such strong shifts occur due to generation of ICT states that are stabilized on interaction with polar media. Dramatic decrease of quantum yield of Nile Red in water is thought to be due to formation of aggregates, and the introduction of polar substituents increases not only the solubility but also the quantum yield (Jose and Burgess 2006). Derivatives of this dye have been synthesized for covalent labeling of proteins and peptides (Karpenko et al. 2014), also inside the living cells (Prifti et al. 2014), and labeling of phospholipid membranes with strong response to variations of their structures have been demonstrated (Kucherak et al. 2010b). The design and application of solvatofluorochromic dyes that can be used for λ-ratiometry (Sect. 3.5) have frequently been reviewed (Benedetti et al. 2013; Boens et al. 2012; Vendrell et al. 2012). The dyes of 3-hydroxychromone family are known to exhibit the excited-state intramolecular proton-transfer (ESIPT) reaction, which results in two-wavelength switching, as will be discussed below. Introduction of the dialkylaminophenyl group in position 7 changes the orientation of the excited-state dipole moment in opposite direction and abolishes ESIPT completely. The excited ICT state demonstrates superior solvatofluorochromic properties (~170 nm emission shift between heptane and DMSO) compared to Prodan (Fig. 5.15). Methylation of the 3-OH does not change

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Fig. 5.15 Novel strongly solvatofluorochromic 3-hydroxychromone derivatives (Giordano et al. 2012). Absorption spectra in THF (dashed line) and fluorescence spectra (solid lines) of compounds 7AHC, 7AMC in comparison with Prodan (excitation at 370 nm). The values in arrows are the magnitudes of solvent-dependent shifts in passing from heptane to DMSO

this effect and leads to improved photostability. These probes can be efficiently used in biomembrane studies (Giordano et al. 2012).

5.3.5 Electric Field Sensing (Electrochromic) Dyes The request for sensing the electric fields at the distances of molecular dimensions is great. The electric field sensitive dyes can respond to electrostatic potential at interfaces, to biomembrane potential, surface potential of protein molecules or nanoparticles, etc. They can be efficiently used in those technologies, in which the sensing effect is coupled with relocation of nearby located charge. Their response is based on electrochromism (also known as Stark effect), which is the phenomenon of changing the energies of electronic transitions resulting in shifts of electronic (absorption and fluorescence) spectra under the influence of electric field (Bublitz and Boxer 1997). The electrochromic dye senses the integrated electric field at the site of its location whatever this field is applied externally in a macroscopic device (e.g. inside a capacitance) or internally, on a molecular level, by a nearby charge. This allows some averaging and integration of produced field effects. The ‘mesoscopic’ approach considering the fluorophore as a point dipole and electric field as a vector F that averages all the fields influencing the fluorophore in the medium with efficient dielectric constant εef can be used for the description of this effect in the simplest dipole approximation (Bublitz and Boxer 1997). The direction and magnitude of the shift, νobs , is proportional to the electric field vector F and the change of dipole moment associated with the spectroscopic transition μ: 

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       hνobs = − 1/εe f · μ ·  F  cos(θ ),

(5.1)

where θ is the angle between μ  and F vectors. It follows that in order to show maximal sensitivity to electrostatic potential, the dye should exhibit substantial change of its dipole moment μ  on electronic excitation, which implies an essential redistribution of the electronic charge density. Furthermore, it should be located in low-polar environment (low εef ) and oriented parallel (cos θ = 1) or anti-parallel (cos θ = −1) to the electric field vector. It is essential to note that electrochromism (the response to external electric field) and solvatochromism (the response to dielectric interactions with molecular environment) are based on the same physical mechanism (Liptay 1969). Practically, this means that the effects of dipoles chaotically surrounding the dye in a polar liquid (effects of polarity) are basically not distinguishable from that of dipoles arranged in organized ensemble and generating the electrostatic potential. The difference stems from stringent demands on the dye location and orientation with respect to applied field. Therefore the correlation of spectroscopic effects with the applied field strength is possible only if the dye location and orientation relative to the field are fairly known. In applications, the electrochromic effects are of value for the studies of electrostatic effects at interfaces, such as Langmuir–Blodgett films (Tsuchida et al. 2001) and biomembranes (Clarke 2010; Demchenko and Yesylevskyy 2009). In experiments with the charged interfaces, the high structural anisotropy and the possibility of locating the fluorophore in an anisotropic way in opposite orientation to the interface, allows observing strong response based on this mechanism. The effects that do not depend on dye orientation should be attributed to the effects of polarity and those that show such dependence should be due to electrostatic potentials. The request of optimizing the electrochromic effect resulted in selecting the most efficient dyes. They should be the ICT dyes with the largest relocation of electronic charge. Such are the voltage-sensitive styryl and naphthyl styryl dyes. Among the best known electrochromic dyes are the 4-dialkylaminostyrylpyridinium derivatives with electron-donor and electron-acceptor substituents at the opposite ends of their rodshaped aromatic conjugated moieties (Gross et al. 1994). They exhibit the following excited state reaction modulated by applied electric field (Loew 2011), as shown in Fig. 5.16. The fluorescent signal on the change of dipole moment in such dyes in response to the change of electric field is extremely fast but rather modest in magnitude. These shifts are best observed in excitation spectra, where the intensities at their slopes can be recorded in ratiometric manner, whereas the shifts in fluorescence spectra are much smaller and quantitatively less reliable due to the involvement of dielectric relaxations in the excited state. Strong electrostatic effects can be recorded in this way in biomembranes and their phospholipid analogs (Gross et al. 1994), in which the charged groups, adsorbed ions and oriented dipoles of lipids form very strong electric field gradients (Demchenko and Yesylevskyy 2009) that can be modulated by different factors with clear observation of response of incorporated dyes. Therefore,

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Fig. 5.16 Translocation of charge on the electronic excitation of 4-dialkylaminostyrylpyridinium dye di-ANEPPS (above). The excitation spectra demonstrate the electric field induced spectral shift. The correspondent change of fluorescence intensity can be recorded at different excitation wavelengths (below). The relative change in fluorescence, F/F, is shown as a percentage change if different detection wavelengths are selected. When incorporated into cell membrane, this shift indicates the difference in membrane potential between its polarized and depolarized states

it can be expected that electrochromic dyes will find applications in sensing devices that involve charged interfaces or surfaces of nanoparticles. Styrylpyridinium dyes are popular as electric field probes, and different their versions can be found in the literature (Jin et al. 2006; Millard et al. 2004; Vitha and Clarke 2007; Wuskell et al. 2006). Meantime, a critical analysis reveals that further progress requires the development of probes that do not possess a large ground-state charge asymmetry that may allow their desired location and orientation and also allow quantitative λ-ratiometric recording in fluorescence emission. Below, we will see that 3-hydroxychromone dyes, and particularly 4 -(dialkylamino)-3-hydroxyflavones, fit better to this requirement. Thus, three major effects, polarity, electric fields and hydrogen bonding can induce strong shifts of fluorescence spectra. They can be used for probing the properties of different media (liquids, solids, and their interfaces), which will be discussed in Volume 2. Here, we stress that if the sensing event is coupled with the change of molecular environment of reporting dye, this event can be recorded as a fluorescence response if the dye is incorporated into sensor molecule in such a way that its interactions with the environment change as a result of conformational change in the sensor or due to direct contact with the target.

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5.3.6 Supersensitive Multicolor λ-Ratiometric Dyes The working principle in operation of these dyes is the excited-state intermolecular proton transfer (ESIPT) coupled with intramolecular charge transfer (ICT) (Demchenko et al. 2013). This intramolecular reaction that can be modulated by intermolecular forces allows observation of two bands in emission and switching between them as a function of small intermolecular interactions exhibited by the dye (Fig. 5.17). The designed organic dyes belonging to the family of 3hydroxychromones (3HCs) satisfy these requirements in the best way (Demchenko 2006; Demchenko et al. 2003; Klymchenko and Demchenko 2003). Due to rigidity of their skeleton, 3HCs (and their derivatives 3-hydroxyflavones, 3HFs) are the dyes in which the observation of two separate bands in emission is not due to the presence of two or more excited-state structural isoforms, which produce the two-band spectra in some other dyes. The second (long-wavelength) band is generated due to the ESIPT reaction proceeding along the intramolecular hydrogen bond. As shown for the ESIPT reaction in 3HC derivative 4 -dialkylamino-3-hydroxyflavone (Demchenko et al. 2013), the reaction proceeds between highly dipolar and strongly solventinteracting N* form and the tautomer form T possessing much more symmetric O ... H-O O

O-H ... O

ESIPT

OH O

hν F

hνF

3HC

1.0

O OH

O MeO

O MeO

O

O

N

3HBN(m) O

O

O OH

OH O 3HFN

O

O

3HFN(m)

N

N O

OH O 3HFN-L

O OH

O 3HBN-L

0.4 0.2 0.0 1.0

b

0.8 3HBN-L

0.6

O 3HBN

O

3HFN(m)

N

OH

OH

a 3HFN-L 3HFN

0.6

OH

3HB

N

3HF O

O

Flourescence intensity

O

0.8

0.4

3HBN 3HBN(m)

0.2 0.0

450

500

550

600

650

700

750

Wavelength (nm)

Fig. 5.17 Two series of 3-hydroxychromone dyes based on dialkylamino-substituted phenyl or benzofuran at position 2, both exhibiting dramatic variation of λ-ratiometric response to polarity of molecular environment (Klymchenko et al. 2003b; Yesylevskyy et al. 2005). Their twoband fluorescence spectra of flavones 3HFN, 3HFN-L and 3HFN(m) in chlorophorm (a) and benzofurylchromones 3HBN, 3HBN-L and 3HBN(m) in toluene (b). Excitation at 420 nm

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charge distribution. Therefore, the sensitivity of two N* and T* forms to all types of intermolecular interactions is different, resulting in strong variations in positions and intensities of correspondent fluorescence bands. Structural modifications of the parent fluorophore allow variation of spectroscopic properties of these dyes in very broad ranges. In Fig. 5.17 the dyes of two series, based on 3-hydroxyflavone (left) and 2-benzofurylchromones (right) are shown being arranged in line with the increase of their excited-state dipole moments shifting the range of λ-ratiometric sensitivity to lower polarities, The choice of dyes for obtaining optimal response in particular environments can be complemented by the dyes of other series, in which naphthofuranyl (Klymchenko et al. 2001) or thiophenyl (M’Baye et al. 2007) groups are attached in position 2 of chromone heterocycle. Moreover, the compounds, in which oxygen in position 1 is substituted by a group containing nitrogen (3-hydroxyquinolones) were synthesized. They possess unique sensitivity to variation of intermolecular hydrogen bonding (Yushchenko et al. 2006a). The two-color fluorescence switching from a single dye exhibiting an excitedstate reaction is very convenient for different applications (Demchenko et al. 2013). By switching between them, these dyes demonstrate really dramatic sensitivity of their spectra to polarity and electric fields of their environment (Demchenko and Yesylevskyy 2009; Klymchenko and Demchenko 2002) and also to intermolecular Hbonding (Shynkar et al. 2004), and these effects are much stronger than that observed just with spectral shifts. From the overview presented above, we derive that organic fluorescent dyes are unique in the ability of reporting to the changes of all major types of weak noncovalent intermolecular interactions. The idea visualized schematically in Fig. 5.18 will probably be useful for the development of responsive dyes of new generations. In the state of equilibrium between the reactant and product in the excited state the difference in free energies of these states modulates fluorescence intensities of correspondent bands. If some, even weak, intermolecular interactions of reactant or product molecules change this energy balance, the stronger interacting state (possessing decreased energy) will become more populated, just in accordance with the Boltzmann law. With common solvatochromic dyes, in these cases we observe just the spectral shifts. However, if the two states emit fluorescence and the switching occurs between them, we achieve a strong amplification effect in response—a small relative change in interaction energy between two states changes dramatically the relative intensities of two emissions. Subsequent studies have shown one more remarkable property of 3HC dyes. Observing the spectroscopic changes in protic environments, we started to distinguish and analyze an additional H-N* band. Being characteristic for these environments only, it was attributed to intermolecular hydrogen-bonded form that involves 4-carbonyl group (Shynkar et al. 2004). A new important step was the discovery of strong electrochromism of these dyes (Klymchenko and Demchenko 2002), which opened new dimension in their study and applications. Thus, it was shown that the positions of these emission bands and relative variations of their intensities are highly

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Fig. 5.18 The scheme illustrating the principle of signal amplification in two-band ratiometric fluorescence response (Demchenko 2006). The two excited species (N* and T*) are in dynamic equilibrium. For each of them, the change of intermolecular interactions results in the change of energy separation between ground (N or T) and excited (N* or T*) states. The equilibrium in relative populations of two forms shifts in the direction of the form possessing lower energy, which gives higher fluorescence intensity from a more populated state. According to Boltzmann law, the difference in state populations is an exponential function of the difference in state energies. Therefore, a strongly amplified response is observed in band intensity ratios in comparison with spectral shifts

sensitive to such properties of their microenvironment as polarity, hydrogen bonding and local electric fields. In particular: – In polarity sensing, they exhibit an ultrasensitive two-band ratiometric response to polarity of the environment, up to complete switching between N* to T* bands in emission in a narrow range of variation of this parameter. This range can be adjusted by chemical substitutions (Klymchenko et al. 2003b) covering the range from very low (Ercelen et al. 2002) to very high (Klymchenko et al. 2004) polarities. The origin of this effect, in line with the mechanism described in Fig. 5.18, is a much stronger stabilization of the N* state in polar media, as a result of which this state becomes increasingly populated at equilibrium. The wavelengthratiometric response is observed of such dramatic magnitude, that only small change of polarity is sufficient for total switching between the two bands and a number of dyes with different N* state dipole moments can cover the whole polarity range. – In the sensing of hydrogen bonding potential of the environment (which in biomolecular structures can also be a measure of hydration), a strong response to the presence of proton donor groups is typical for these dyes. In contrast to many other carbonyl-containing fluorescent dyes, in 3HCs no formation or disruption of

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H-bonds occurs in the excited state, so the probe can sense the true concentration of H-bond partners existing in the ground state (Shynkar et al. 2004). Sensitivity to hydrogen bonding can be eliminated by proper chemical substitution that provides sterical screening of this carbonyl group (Klymchenko et al. 2002b). – In electric field sensing, a very strong two-band ratiometric response to the magnitude and direction of the local electric field on molecular scale (the so-called electrochromic modulation of ESIPT reaction) was demonstrated (Klymchenko and Demchenko 2002), and this effect was applied for determination of dipole potential in biomembranes (Klymchenko et al. 2003a). The origin of this response is in electrochromic influence (due to internal Stark effect) on the relative energies of the N* and T* states, as it is shown in Fig. 5.19. By introducing different substituents, the derivatives of these dyes with the ability of vertical insertion and opposite orientations in the membrane were designed (Klymchenko et al. 2003a). They respond to the changes in their intensity ratio of two emission bands in an opposite way in accordance with their inverse orientation. Moreover, a multiparametric analysis based on deconvolution of recorded spectra into individual components showed that these changes are independent from the hydration parameter (M’Baye et al. 2006). In order to avoid the fast internalization of these dyes and/or poor staining of plasma membranes in the living cells, a second generation of probes were introduced (Shynkar et al. 2005).

Fig. 5.19 The effect of covalently attached positively but electronically uncoupled charged groups on the absorption and fluorescence spectra of strongly dipolar 3HC dye. The two bands in fluorescence emission appear as a result of ESIPT reaction. The dye (1), in which the charged group is attached from the side of electron-donor dialkylamino group of dye (2) possesses the absorption spectrum strongly shifted to the blue. Its fluorescence spectrum possesses greatly decreased intensity of the N* band that is also shifted to the blue. The opposite effect is observed for dye (3), in which the positively charged group is attached through a spacer to the opposite side

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In a general sense, the sensitivity to many different types of intermolecular interactions is not an attractive feature in sensing. But in the case of 3HCs, there is a possibility for deriving in the spectral response of a single fluorophore a number of spectroscopic parameters (the wavelengths of absorption and of two emission band maxima together with intensity ratio). Due to this feature, we were able to develop an approach for distinguishing the effects of polarity, electronic polarizability and hydrogen bonding (Klymchenko and Demchenko 2003). The effects of electric field can be evaluated by an analysis of shifts in excitation spectra and by comparing the data obtained for 3HF probes located in different orientations to this field (Klymchenko et al. 2003a). Thus, the molecules of the 3HC family can respond to several, the most important, types of intermolecular interactions. The common response to these interactions, the spectral shift, can be amplified being transformed into the ratio of two band intensities. By chemical modifications, the 3HC dyes can be supplied with a variety of functional units. They were suggested for probing biomembrane structures by locating the dyes at different orientations and depths (Klymchenko et al. 2002a). More sophisticated functional dyes sense variations of dipole potential in membranes of living cells (Shynkar et al. 2005) and are able to detect the early steps of apoptosis (Shynkar et al. 2007). Derivatives of these dyes for covalent labeling of amino and SH-groups were also obtained (Klymchenko et al. 2004), which can be used in various types of protein and peptide biosensors. Their azacrown ether derivatives exhibit dramatic spectral responses to the binding of bivalent cations (Roshal et al. 1998, 1999). Therefore, these reporters can provide a link between chemical sensing and biosensing. Some of their applications will be highlighted in different sections of this book. One can find analogy of this remarkable result—the ability to obtain from a single type of dye a record number of valuable parameters characterizing its environment and of monitoring them with record sensitivity—with the transition from black-andwhite (intensity only) to color (multiple-channel) television. Thus, it is expected that multicolor dyes will occupy their proper position in sensing.

5.3.7 Prospects in Improving the Target-Responsive Sensitivity It is expected that organic dyes will maintain to play the major role in designing the response units of future chemical sensors and biosensors. They offer tremendous possibilities of choice in application to sensor technologies due to their huge number, diversity of their spectroscopic properties, possibilities of variation of these properties by chemical modifications, facile methods of their synthesis and easy spectroscopic control of their properties. They can respond by the changes of all possible fluorescence parameters (positions of wavelength maxima, intensities, anisotropies

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and lifetimes) and allow realizing for that different basic photophysical mechanisms, involving the transfers of electronic charge, proton and excited-state energy. Computer design together with application of rapidly progressing methods of quantum chemistry calculations will aid to improvement of required properties of the dyes. It is also very attractive that the straightforward and facile synthetic routes may lead to direct chemical sensors by coupling the dyes with binders for ions and other small molecules. Their labeling of proteins and nucleic acids and also of polymers and nanoparticles becomes an easy routine procedure. The versatility and practically unlimited possibilities for chemical modifications of organic dyes allow the researcher making the proper choice for any particular application. One has to recognize however, that on a general scale, a scientifically motivated design strategy of fluorescent dyes is still lacking, and their selection is largely empirical. Quantum chemical calculations can predict (and with difficulty) only the excited-state energies, the charge distributions and dipole moments but not the quantum yields or lifetimes. For optimizing them, an empirical rule suggests to choose the most planar molecules without rotating segments and to select among the most efficient analogs. Therefore, the most efficient of presently used approaches is the selection of optimal dye within large libraries of synthetic products.

5.4 Organic Dyes Optimal for Particular Applications 5.4.1 Near-IR Dyes High interest in near-IR absorbing and emitting dyes and their request in different sensing and imaging technologies motivated the author to address them in a separate sub-section. The reason for that is the following. First, the light scattering which often is the problem in fluorescence measurements is strongly reduced in this range (recollect the 1/λ4 Rayleigh scattering law). Second, there are strongly reduced in number potentially contaminating species emitting light in this range. And most important, human and animal tissues are relatively transparent in the near-IR (Volume 2, Chap. 14). This is because the dyes exhibiting the electronic transitions in this range are rare and the light absorbing vibrations of clusters of water still do not provide essential contribution. In our tissues only the porphyrins are strong absorbers of red light, but their intensity decays in the near-IR. As a cutting-edge technology, fluorescence imaging in the near-IR range has attracted tremendous attention. Two windows of relative transparency were identified, NIR-I (650–950 nm) and NIR-II (1000–1700 nm). The second near-infrared window is preferable, and the problem is in finding proper fluorescent dyes (Ding et al. 2019; Wan et al. 2019). However, it is not easy to access this range with artificial dyes. Regarding optical properties, their design and selection is clear. Extending the number of heterocycles or the conjugation length decreases the energy gap between ground and excited states and shifts the

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spectra to longer wavelengths. However, the interaction with the ground-state vibrations in this case becomes stronger, and this results in quenching. Also, the extension of π-electronic structure often reduces chemical stability and photostability. The great request for the dyes absorbing and emitting light in the near-infrared region comes from in vivo imaging technologies and their use as contrast agents in clinical diagnostics of humans (Luo et al. 2011). The near-IR fluorophore approved for this purpose by the Food and Drug Administration (FDA) is indocyanine green (ICG), see Fig. 5.20. This cyanine dye is non-toxic and can be rapidly excreted from the body, and therefore is used for contrasting blood vessels (Reinhart et al. 2016), and in optical monitoring of surgical operations (Rosenthal et al. 2015; Takahashi et al. 2016). Many attempts were made to overcome poor ICG photostability and chemical stability by making chemical substitutions (Su et al. 2015). New generation of long-chain cyanine dyes have been synthesized (Altman et al. 2012; Das et al. 2011; Samanta et al. 2010). Their low water solubility and the tendency to aggregate is overcome by covalent modifications with groups that increase their polarity and also photostability (Samanta et al. 2010; Wiktorowski et al. 2014). Cyanine dyes (see Fig. 5.10) remain central in continuing efforts for improvement of near-IR emitters (Bricks et al. 2015). The cyclization of a chromophore by polymethylene bridges was found to be a promising way to increase their stability. Introduction of one or two, or even three trimethylene bridges in the polymethine chain prevents the dye molecule from conformational transformations, which results in increase of fluorescence quantum yield and in appreciable bathochromic shift of spectra (Tolmachev et al. 1998). As a rule, the near-IR dyes contain both conjugated substituents and different cyclic attachments in the polymethine chain. Such modifications do not change the typical ‘cyanine-like’ shape of the spectral bands, which is manifested by their narrow width and high intensity (Fig. 5.21). Further move to longer wavelengths can be achieved with those of them that form J-aggregated structures (Bricks et al. 2017). Fig. 5.20 Indocyanine green and its absorption (blue) and fluorescence (red) spectra

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Fig. 5.21 Normalized absorption spectra of cyanine dyes with bridged scaffolds leading to strong shifts of their absorption spectra to near-IR region in solution: a λmax = 994 nm, b λmax = 827 nm, c λmax = 804 nm

The recently reported benzothiopyrylium pentamethine cyanines allow operating in the second near-IR window (NIR-II; 1000–1700 nm) that is highly important for biological imaging applications (Wang et al. 2019c). These dyes demonstrate high brightness and superior photostability in aqueous solution and due to these remarkable properties are promising substitutes of ICG. Porphyrins and phthalocyanines and their metal complexes embody some of the most intensively studied dyes because of strong interest in their use in photodynamic cancer therapy (Volume 2, Chap. 17). A series of conjugated porphyrin dimers with intense absorptions ranging from 650 to 800 nm and fluorescence emission from 700 to 800 nm have been demonstrated (Kuimova et al. 2009). High biocompatibility (Samanta et al. 2010) and functionalization potential of novel dyes will allow the sensing of diagnostically important compounds in human tissues (Guo et al. 2014). The squaraines (Fig. 5.12) are the dyes with a resonance stabilized zwitterionic structure (Patsenker et al. 2010b). The central ring is typically appended with donor moieties to afford a donor-acceptor-donor motif. Unsymmetrical squaraines are prospective for inducing their environment sensitivity (Khopkar and Shankarling 2019). The BODIPY (borondipyrromethane) dyes (Fig. 5.8) typically have relatively shorter wavelength emission maxima and smaller extinction coefficients as compared to the cyanines and other near-IR active fluorophores, but by fusing π-excessive aromatic rings to the BODIPY core, significant increases in quantum yield, higher extinction coefficients, improvements in photostability and NIR optical activity could be achieved (Umezawa et al. 2009). Thus, BODIPY-coumarin (Bochkov et al. 2013) and BODIPY-quinoline (Liu et al. 2020) hybrids due to induced charge asymmetry allow achieving together with near-IR emission a strong Stokes shift. Hybrid structures are also designed on rhodamine scaffold (Wang et al. 2019a). In general, a great problem of transferring the responsive properties of the visible dyes to those emitting in the near-IR region (see Sect. 5.3) exists (Chen et al. 2020),

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though some successful solutions were found (Staudinger and Borisov 2015). It is thought to be resolved by making molecular hybrids that with the spectra shifted to IR due to extended π-electronic conjugation will retain some reporting properties of constituting components (Chen et al. 2017). Near-infrared dyes are also in strong demand for photoaccoustic imaging (Wang et al. 2019b), for sensitization of upconverting nanoparticles (Saleh et al. 2019) and in other areas. Great importance of dyes absorbing in the near-IR region is manifested for two-photon microscopy. Such two-photonic dyes allow near-IR excitation and emission in the visible spectral range. The most efficient of them belong to the class of cyanines (Przhonska et al. 2010).

5.4.2 Two-Photonic Dyes Selecting the dye for two-photon spectroscopy or microscopy (discussed in Volume 2, Chap. 15), one has to know that in these excitation conditions some properties are different from those observed at excitation by single photons. Due to different quantum mechanical selection rules, the two-photon and single-photon excitation spectra are not equivalent, and for two photons they are often much broader. The relative molar absorbance of many dyes may exhibit an unexpected variation: high brightness with single photons may transform to low brightness at two-photon excitation. Therefore, a special effort was made for synthesizing organic dyes with especially high two-photonic absorbance (Webster et al. 2008). The major characteristic of two-photonic fluorophores is their two-photonic absorption cross-section measured in Goeppert-Mayer units, (1 GM = 10−50 cm4 s/photon). We can make comparative estimates taking as the reference one of the brightest organic dyes, Rhodamine B. It is characterized by only 210 GM units, and for many dyes, it is much lower. In contrast, the new dyes synthesized with the knowledge of physical two-photonic action, demonstrate the GM values higher by many orders. Thus, for one specially designed polymethine dye, the value of 2280 GM was achieved (Fu et al. 2007), and for fluorene-bridged squaraine dimer, it reached the value 2750 GM (Moreshead et al. 2013), Theoretical analysis suggests that for achieving high GM values, the presence in the same molecule of several sites producing substantial charge transfer in their extended π-conjugated system is needed (Przhonska et al. 2010). Detailed analysis of the linear and nonlinear optical properties of several classes of polymethine dyes, which include symmetrical and asymmetrical combinations of π-conjugated bridges with electron donating (D) or electron accepting (A) terminal groups, such as D– π –D, A– π –A, D– π –A, and a quadrupolar type arrangement of D– π –A– π –D allowed formulating general principle for achieving enhanced two-photonic crosssection. According to this principle, the molecular structure should be formed of the electron-donating or electron-withdrawing parts arranged in symmetric or asymmetric manner with a large amount of π-electrons. In the case when the molecular structure includes two or more fluorophore systems or suitable alignment of the active

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units is reached in an aggregated form, an additional specific effect can be expected due to the intramolecular interactions (Collini 2012). A strong enhancement of the two-photon absorption cross-section of pseudoisocyanine dyes was observed in aqueous solution in the course of their formation of supramolecular J-aggregates (Belfield et al. 2006). This enhancement is attributed to a strong coupling of their molecular transition dipoles. Forming J-aggregates, some styryl dyes were found to be good two-photon emitters for the DNA detection and imaging (Yashchuk et al. 2007). The two-photon dyes have enriched the family of molecular sensors by attachment of molecular recognition units (Kim and Cho 2015; Liu et al. 2017). Two-photon excitation can be used in EET-based technologies (Yuan et al. 2015).

5.4.3 Dyes Demonstrating Phosphorescence and Delayed Fluorescence Phosphorescence is the emission from triplet state. It usually exhibits strong Stokes shifts, long emission lifetimes (up to milliseconds and seconds) and strong temperature-dependent quenching. Some dyes display strong room-temperature phosphorescence and they are used in sensing techniques. Application of phosphorescent dyes as the sensor reporters has important advantages, since a strong Stokes shift allows avoiding the background emission and light-scattering effects, whereas the long lifetimes allow additional possibility of removing fluorescent background by using the time-gated detection (Sect. 3.4). The dyes that display strong room-temperature phosphorescence with the steadystate intensity comparable to that of fluorescence can be used in the λ-ratiometric sensing technologies. Since phosphorescent dyes are, as a rule, also fluorescent and their phosphorescence bands are strongly shifted to longer wavelengths, the ratio of fluorescence to phosphorescence emission intensities can be achieved in a steady-state λ-ratiometric recording as a very precise, convenient and self-calibrating detection technique (Hochreiner et al. 2005). The two emissions can be also easily separated in time domain: fluorescence is a ‘short-lived’ and phosphorescence is a ‘long-lived’ emission. This difference can be used in oxygen sensing in solutions, since oxygen is a collisional quencher of long-lived emission. Among the dyes exhibiting room-temperature phosphorescence are the fluorescein tetra-bromo derivative eosin and tetra-iodo derivative erythrosine (Fig. 5.22). These properties appear due to the presence of heavy atoms of iodine and bromine in their structures that facilitate the intersystem crossing reaction leading to triplet state (Garland and Moore 1979). Much larger number of dyes can become phosphorescent if the dye is protected from quenching by rigid environments. They can be incorporated into porous sol-gel materials or into inorganic or organic polymer matrices, and the optimal detection technique is the emission lifetime (Sanchez-Barragan et al. 2006). The latter can exhibit variation by many orders of magnitude. The heavy atoms

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Fig. 5.22 Phosphorescent dyes eosin (a) and erythrosine (b). Presented in (c) are their delayed emission spectra. The long-wavelength band belongs to phosphorescence and the short-wavelength band is due to the presence of delayed fluorescence

are used to increase the population of phosphorescence state by the so-called ‘Kasha heavy atom effect’. Whereas fluorescein has a quantum yield of 0.9, for eosin it is 0.2 and for erythrosine 0.02. Phosphorescence emission increases correspondingly. It can be further increased by removing oxygen from the medium. Design of novel all-organic dyes with room-temperature phosphorescence is in progress (Chen and Liu 2019). Key important features to achieve are the high probability of intersystem crossing (ISC) to the triplet state and low probability of quenching of this state. The cited paper contains different examples of application of novel phosphorescent dyes. Phosphorescent complexes can be obtained also by incorporating into heterocyclic organic structures such as porphyrins of the noble metal ions platinum, palladium and others (Papkovsky and O’Riordan 2005) and also chelating complexes of ruthenium (Castellano et al. 1998; O’Neal et al. 2004), see Sect. 6.3. Delayed fluorescence is the emission with spectral parameters as those of fluorescence and duration as that of phosphorescence (check the short-wavelength band in Fig. 5.23). It can be obtained by intra- and intermolecular triplet-triplet annihilation and, in rare cases, when the energy gap between the first singlet excited (S1 ) state and the first triplet (T1 ) excited state is small enough for the reverse transition of excitation energy (Liu et al. 2018a). Its application in sensing and imaging technology is still limited. A new discovery of organic dye system that displays long persistent luminescence that was previously known for specially designed inorganic nanocrystals (see Sect. 7.3) opens new horizons (Kabe and Adachi 2017). This type of emission involves the presence of “excitation traps” and can last for hours. It is particularly attractive for long-term biological imaging and monitoring the surgical operations.

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Fig. 5.23 Photochromic electronic energy transfer (EET) (Jares-Erijman and Jovin 2003). The chemical structures depicted correspond to the photochromic dithienylethene in the colored closed form (left) and colorless open form (right). The absorption spectrum of the latter overlaps well with the emission spectrum of the donor; the kernel of the overlap integral (striped) corresponds to the Lucifer Yellow dye as the donor. Ultraviolet light induces the photochromic transition to the closed form (ON), and visible (green) light reverses the process to the open form (OFF)

5.4.4 Organic Photochromic Compounds Molecules that can reversibly change their absorption and emission spectra in response to external stimuli are of central importance to the imaging technologies and development of molecular devices. There are organic photochromic compounds that isomerize reversibly changing color under excitation with an appropriate wavelength of light. Photochromism is a term used for a reversible transformation of a specific molecule between its two isomers induced by two different wavelengths of light (Fukaminato et al. 2018; Raymo 2013; Zhang et al. 2014). The photochromic reaction can be induced by light resulting in the photogenerated closed-ring isomers. During the reversible light-driven process, several physical and chemical properties, such as π-conjugation and absorption spectrum, change dramatically, and fluorescence

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emission can be modulated in ON-OFF manner. Such photochromic compounds operating as photochemical molecular switches have found many applications. The sensing technology benefits from these possibilities. The photoswitching quenching phenomenon provides the light-activated control of the excited states, and, consequently, control over any subsequent energy or electron-transfer processes. The best compounds serving for this purpose are probably spiropyrans. These molecules exist in closed spiro forms absorbing at wavelengths shorter than 400 nm. They undergo a light-driven molecular rearrangement to an open merocyanine form with absorbance at 500–700 nm (Bahr et al. 2001). The very elegant approach was suggested (Giordano et al. 2002) and got the name “photochromic EET ”. The photochromic compounds were used as electronic energy transfer (EET) acceptors having the ability to undergo a reversible transformation between two different structural forms that have different absorption (and in some cases, fluorescence) spectra. Following reversible cycles of photoconversion, in one form they serve as acceptors and in the other form allow emission from the donor. Thus, they offer a possibility of reversible switching the EET effect between “ON” and “OFF” states without any chemical intervention, just by light (Fig. 5.23). In imaging the living cells, the EET switching on and off allows obtaining the necessary control on the distribution of donor molecules and on their fluorescence parameters thus controlling the emission colors (Jares-Erijman and Jovin 2003). The ability to undergo numerous cycles of EET switching is extensively used in super-resolution microscopy (Volume 2, Chap. 15). This allows modulating the number of spatially resolvable emitters located in different positions in image for achieving the nanoscale resolution.

5.4.5 Dyes for Sensing in Protein and Nucleic Acid Science Tryptophan is the natural amino acid that is frequently used as a fluorescent marker and reporter (Demchenko 1986). Excited at 280–290 nm, its emission is observed with the maximum at 330–350 nm. Since in many protein molecules several Trp residues are located in their structures, for obtaining the site-specific information and avoiding spectral heterogeneity, all Trp residues beside one target residue are typically substituted by other groups. Due to environment sensitivity of Trp fluorescence, many interesting results were obtained on protein folding and interactions. Still, because of light absorption and emission in the ultraviolet, Trp is not convenient for sensing applications. Its substitution into more efficient synthetic fluorophore can be provided by both synthetic and biosynthetic means. Proteins possessing reactive –SH groups can be easily modified by covalent attachment of different fluorophore derivatives. An example is the labeling of oligomeric α-crystallin, the molecular chaperone and heat shock protein (Avilov et al. 2005). In this work, the 3HC dye 6-bromomethyl-2-(2-furanyl)-3-hydroxychromone (BMFC) was attached covalently to a sole exposed SH-group in one of subunits of this protein. The same derivative when attached to antigenic peptide allowed detecting its specific

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interaction with antibody (Enander et al. 2008) and being attached to enzyme inhibitor reported on interaction with enzyme elastase (Boudier et al. 2009). The color-switching amino acid analogs have found interesting applications in the studies of interactions of peptides with biological membranes. Substitution by 3-hydroxychromone derivative of two sites in the 26 amino acid sequence of melittin, a well-studied membrane-active peptide, allowed locating these sites with respect to biomembrane based on fluorescence response to local hydration and polarity (Postupalenko et al. 2013). Moreover, using fluorescence microscopy with a polarized light excitation, the preferential orientation of peptide parallel to the membrane surface was validated. The developed amino acid and the proposed methodology will be of interest to study other membrane peptides. Synthetic amino acid analogs containing fluorophore as the side group can be incorporated at desired sites of synthetic peptides. Based on different photophysical mechanisms, they can provide responsive functions (Krueger and Imperiali 2013). Thus, the fluorescent λ-ratiometric analog of natural amino acid tryptophan (Trp) was synthesized and incorporated into the zinc finger domain of the HIV-1 nucleocapsid protein (Shvadchak et al. 2009). This peptide retained in full the Zn2+ binding affinity and also the function of this protein domain. It preserved the folding, the nucleic acid binding and chaperone activity, indicating that the new amino acid can conservatively substitute Trp residues. Due to the exceptional sensitivity of its fluorophore to hydration in highly polar environment, the new λ-ratiometric 3-hydroxychromone derivative in the form of amino acid appears as a promising tool for substituting Trp residues for site-selective investigation of peptide–nucleic acid interactions (Strizhak et al. 2012). A recent example is the use of such fluorescent L-amino acid bearing the 4 -methoxy-3hydroxyflavone fluorophore (M3HFaa) that shows dual emission, as a result of ESIPT reaction (Fig. 5.24). These data demonstrate the two-band switching response demonstrating the interplay of two limiting cases, A30-M3HFaa peptide in aqueous buffer, in which the probe is exposed to water, and when it is incorporated into phospholipid membrane, in which it is hidden in low-polar interior. On binding to SL2 RNA, the probe remains exposed, and the interaction with DNA and TAR RNA demonstrate complete or partial screening in the formed complex. Important information on conformational stability and transitions of native proteins can be derived from the studies of surface exposure and hydration of their Trp residues. The analog of tryptophan, (2,7-aza)tryptophan, (Fig. 5.25) has been recently synthesized and suggested for probing the hydration of particular sites in proteins (Shen et al. 2013). This artificial amino acid can be recognized by biosynthetic machinery as Trp, so when added to culture medium in bacteria it can replace Trp in newly synthesized proteins. Whereas Trp emits fluorescence in the nearUV with polarity-dependent band maxima at ~330–350 nm, (2,7-aza)Trp has two additional fluorescence bands extending to the visible that appear due to watercatalyzed proton transfer reaction. In the presence of water, (2,7-aza)Trp exists in two proton-transfer forms in the ground state, the N(1)-H and N(2)-H isomers, one of which undergoes water-catalyzed excited-state proton transfer (ESPT) reaction, giving a 345-nm emission band and a prominent green isomer 500-nm emission (see

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Fig. 5.24 Environment-sensitive fluorescent color-switching amino acid M3HFaa (Sholokh et al. 2014). a Its structure. b Sequence of the nucleocapsid peptide NC(11-55), where the arrows show the two positions of natural amino acids (A30 and W37) replaced by M3HFaa. c Effect of different oligonucleotide sequences and phospholipid vesicles (LUVs DOPS:PC) on the normalized fluorescence spectra of the labeled NC(11-55) peptide (A30-M3HFaa). Excitation wavelength was 360 nm

Fig. 5.25 The Trp analog (2,7-aza)Trp, its spectra and an example of its application in the studies of protein conformation (Chao et al. 2014; Shen et al. 2013). a The structures of Trp and (2,7aza)Trp. b The spectrum of (2,7-aza)Trp in the absence (left) and presence (right) of water. c The equilibrium between two conformations of RNase T1 detected by hydration of Trp59 site. The existence of N(2)-H isomer in the loop-open form reveals the specific hydrogen bonding between N(2)-H proton and water molecule that bridges N(2)-H and the amide oxygen of Pro60, forming a strong network. In contrast, in loop-close form the water molecules are squeezed out of this site

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Fig. 5.25b). Upon replacing tryptophan, (2,7-aza)Trp successfully recognizes the presence of water in its vicinity by ratiometric changes in relative intensities of these emissions. Essentially, sensing hydration water in proteins occurs without disrupting their native structures (Shen et al. 2013). Using this property, it was found that RNase T1, a small protein composed of 104 amino acid residues, exhibits the structure fluctuating between the two loop-close and loop-open forms differing by exposure to water of a single residue Trp59 (Chao et al. 2014), see Fig. 5.25c. Possessing similar photophysical properties but requiring for proton transfer lining-up of two molecules of water, (2,6-aza)Trp requires for mediating PT the arrangement in chain of two water molecules (Chung et al. 2017). Combination of fluorescence responses of inserted (2,7-aza)Trp and (2,6-aza)Trp residues was suggested for studying the water-associated conformational mobility in the active sites of proteins (Chao et al. 2018). Nitrogen-containing heterocycles which are parts of DNA and RNA, the socalled nucleobases, are non-fluorescent at normal conditions and cannot be used as molecular reporters. To overcome these limitations, many emissive nucleoside analogues have been synthesized and evaluated in the area of molecular sensing (Michel et al. 2020). They found applications in probing the local structure and dynamics of nucleic acids, typing single-nucleotide polymorphism (SNP), investigations of DNA-proteins interactions, etc. (Sinkeldam et al. 2010; Wilson and Kool 2006). Universal nucleoside analogues, which form stable base pairs with all the natural nucleobases, are especially important in the probing of nucleic acids (Liang et al. 2013). Prospective candidates for this role are 3-hydroxychromones due to their extreme sensitivity to local environment (see Fig. 5.17) and a size compatible with that of a base pair, ~10 Å (Kenfack et al. 2012). Featuring exceptional environmental sensitivity by switching between two well-resolved fluorescence bands, they can serve as highly responsive biosensors to site-specifically probe the proximal environment of nucleic acids (Dziuba et al. 2012; Le et al. 2019; Michel et al. 2020). These results are presented in Fig. 5.26. Once incorporated into short DNA sequence, this nucleoside surrogate does not alter the duplex conformation. It is up to 50-fold brighter than 2-aminopurine, the standard fluorescent nucleoside (Dziuba et al. 2012). This strategy of nucleobase replacement was successfully applied to the mechanistic studies of DNA repair involving base excision (Kuznetsova et al. 2014) as well as the HIV-1 replication (Sholokh et al. 2018). The DNA methylation mechanism—implying as a key step, the 5-methylcytosine (5mC) eversion by a chaperon protein UHRF1—was also investigated with this dual-emissive reporter (Kilin et al. 2017). For the study of DNA hydration, another strategy was selected—by incorporating uracil base coupled with 3HC dye (Barthes et al. 2015; Dziuba et al. 2014). The resulting conjugate demonstrated an exquisite sensitivity to hydration that enables to detect and quantify mismatches and helical conformation changes (from B to A-type) via a variation of emission color. These 3HC-based ESIPT probes allowed addressing several bottlenecks encountered with the other sensors currently used for nucleic acid labeling (Kladova et al. 2019). Presently, smart emissive nucleosides with informative responses to their

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Fig. 5.26 Creation and application of fluorescent DNA base analogs and labels based on 3hydroxychromone dyes (Michel et al. 2020). a Internal DNA labeling, e.g. strategy based on the replacement of a nucleobase by a 3HC reporter. b Fluorescence spectra displaying the selective flipping of 5mC (M) by the SRA domain of the UHRF1 chaperone protein. c External DNA labeling, e.g. modification of a uracil base to site specifically incorporate a 3HC reporter in the hydrated major groove. d Application of hydration probing to detect DNA conformational changes and mismatches

environmental disturbances are in urgent need for DNA and RNA research, and the application of λ-ratiometric dyes may offer interesting prospects (Le et al. 2019).

5.5 Visible Fluorescent Proteins We owe to fluorescent proteins the advancement in our understanding the life of the living cell. Being synthesized within the cell, they form the fluorophore spontaneously after their introduction as the gene product, the following synthesis of polypeptide chain and its folding into protein structure. Such spontaneous fluorophore formation within the cell opens great possibilities for the researcher to fuse the genes coding these proteins with that of any desired protein and to let the cell to synthesize hybrids carrying the fluorophore. In this way there appears the possibility of visualizing many cellular processes with spatial and temporal resolution. In this section, after introduction of the basic fluorescent protein and its fluorophore

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structures, the most attention will be directed to the responsive ability of this unique protein family, to enhance it and to reproduce it in model dye systems. The green fluorescent protein (GFP), a naturally fluorescent protein, was derived from jellyfish Aequorea victoria (Tsien 1998). Its mutants and similar forms isolated from other organisms, particularly coral species, possess the absorption and emission spectra covering almost the whole visible range, starting from blue and extending into near-IR (Stepanenko et al. 2011; Tsien 2009; Zimmer 2009). Technology was developed to transfer it to the inside of living cell as genetically engineered fusion products with other proteins that can be incorporated into different biological structures. This opens an easy but efficient way for locating different proteins inside the cell and visualizing the structures formed by them. In fluorescent proteins (FPs), the fluorophore is formed spontaneously but only within the folded protein structure. Denatured FPs are not fluorescent.

5.5.1 Green Fluorescent Protein (GFP) and Its Fluorophore The mechanisms of fluorophore formation coupled with protein folding were established, and a number of its modified forms were obtained (Miyawaki et al. 2005). The fluorescent group in wild-type green fluorescent protein (wtGFP) is formed spontaneously by the cyclization and subsequent oxidation of the protein’s own structure Ser–Tyr–Gly located at positions 65–67 in its amino acid sequence (Fig. 5.27). While the side chain of the first residue participating in this reaction at position 65 can vary, Tyr-66 and Gly-67 are strictly conserved among all natural GFP-like proteins, artificial mutations of Tyr-66 by His, Trp, or Phe are possible, which results in the blue-shifted spectral variants (Chudakov et al. 2010). The role of Tyr-66 and its aromatic substitutes is essential for the fluorophore formation. On the oxidation step, they become the part of delocalized π-electronic system. The fluorophore in GFP is essentially an ‘organic dye’ and it shares the major properties with common synthetic organic dyes described in this chapter. On illumination with light, it is amenable to long-lived transitions between various fluorescent

Fig. 5.27 The mechanism of intramolecular biosynthesis of wtGFP fluorophore. The first step is a nucleophilic attack of the amino group of Gly-67 onto the carbonyl group of Ser-65. Subsequent elimination of water results in the formation of an imidazolidinone ring. On a second step the Cα –Cβ bond of Tyr-66 is oxidized to give a large delocalized π-electronic system p-hydroxy benzylideneimidazolinone

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or non-fluorescent states, resulting in the processes known as photoactivation, photoconversion, or photoswitching (Acharya et al. 2017; Bourgeois and Adam 2012). Their absorption and fluorescent spectra can be modulated in broad ranges by molecular environment. However, the well-arranged local protein environment provides its essential influence. The ‘dye’ is located inside the barrel-like protein structure that is composed of 11 β-strands forming the so-called β-barrel with a diameter of about 30 Å and a length of about 40 Å. The fluorophore occupies the position deeply inside the protein molecule, so that the plane of its location is roughly perpendicular to symmetry-axis of the β-barrel. Its well-determined contacts with surrounding groups of atoms in the protein interior and the absence of contact with the solvent determine its properties (Fig. 5.28). The absorption and excitation spectra of native wtGFP from A. victoria (blue) have two excitation maxima at 395–400 nm and at 470 nm. Excitation at these wavelengths gives rise to the same fluorescence emission spectrum that has a strongly Stokes-shifted peak at 509 nm and a small shoulder at 540 nm (Fig. 5.29). Operation with different mutations has shown that amino acid substitutions in the fluorophore environment allowed obtaining the forms with the blue absorption band only. This made the forms excited by blue (488 nm) light attractive for imaging using blue line of argon ion laser that is used in common instrumentation. The origin of double wtGFP absorption band will be analyzed.

Fig. 5.28 The three-dimensional structure of green fluorescent protein from jellyfish Aequorea victoria in two projections. The sequence of 238 amino acid residues (26.9 kDa) is assembled into 11 antiparallel β strands (shown as yellow ribbons) forming the so-called β-barrel. The fluorophore, located inside the beta-barrel, is shown is space-filling representation. It is formed inside this structure as a part of the central helix and is highly protected from interaction with the solvent

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Fig. 5.29 Chemical structures of the wtGFP (a) and DsRed (b) fluorophores showing the extended π-electronic system of double bonds. c The absorption (blue) and emission (green) spectra of wtGFP and the absorption (orange) and emission (red) spectra of DsRed

5.5.2 Proteins of GFP Family In continuing developments, fluorescent GFP-like proteins were obtained from different sources (Anthozoa, Hydrozoa and Copepoda species), demonstrating the broad evolutionary and spectral diversity of this protein family. Mutagenic studies gave rise to diversified and optimized variants of fluorescent proteins, which have never been encountered in nature (Chudakov et al. 2010; Rodriguez et al. 2017). Many such GFP variants have been obtained by now; see an open database that assembled and arranged FPs according to their different properties (Lambert 2019). Whereas initial discoveries and applications included blue (BFP), cyan (CFP) and yellow (YFP) fluorescent proteins, now the FPs are available in a broad range of different colors. One can select the protein according to the wavelength of light absorption and emission and optimal FRET pair for co-localization studies in live cells. It was found that the fluorescence quantum yield F values and the brightness for these proteins are relatively good in general but depend on the final structure of fluorescent group and mutations around it. Usually, F ranges from 0.17 for BFP to 0.79 for the wild-type of GFP but it is sensitive to pH, temperature, oxygen concentrations and other environment conditions (Giepmans et al. 2006). Very promising in this respect is the red fluorescent protein from Anthrosoa corals (DsRed), mainly due to its strongly red-shifted absorption and fluorescence spectra (Fig. 5.29). A more extended planar π-conjugated system is responsible for the spectral shift to lower energies, in accordance with a common rule. Its absorption spectrum is represented by the band at 559 nm and shoulders at 527 and 485 nm and the fluorescence spectrum is seen as a single band with the maximum at 586 nm. With molar absorption coefficient 57,000 M−1 cm−1 and quantum yield 79%, it competes with GFP and shows superior properties in terms of photostability and independence of spectra on pH in the range 5.0–12.0 (Shrestha and Deo 2006).

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Excited in the range 558–578 nm, DsRed and its mutants display fluorescence with the band maxima located between 583 and 605 nm. This allows their use as efficient FRET acceptors. With these properties, they become valuable as a fluorescent tags in various cell biology applications and of a particular importance as suitable markers for deep-tissue imaging. During the past decade, many advanced red fluorescent proteins, including farred proteins with emission wavelength longer than 620 nm have been developed (Rodriguez et al. 2017; Shcherbakova et al. 2012). Spectral tuning of FPs is then achieved through modifying the extent of π-conjugation within the fluorophore and interactions with surrounding amino acids that can be modulated by subtle changes of protein conformation. Speaking in general terms, the diversity of fluorophore chemical structures, their conformations, protonation states and interactions within the protein molecule determines the wide spectral diversity of natural GFP-like proteins. Why so many fluorescent proteins are needed? First, demonstrating different colors of their fluorescence emission they allow multicolor imaging by labeling different proteins and in different compartments in living cells. Second, within this diversity, the researcher can find easily the optimal donor-acceptor pairs for analyzing the proximity of different proteins and different structures by electronic energy transfer between them (Bajar et al. 2016). And third is the possibility of observing different excited-state reactions carrying information content that are differently presented within a variety of fluorescent proteins.

5.5.3 Excited-State Reactions in Fluorescent Proteins Now we refer to the light absorption spectrum of wtGFP presented in Fig. 5.29. What is the origin of such unusual behavior, the presence of two separated bands? The major band is observed in the near-UV-violet region and the fluorescence in the green range, demonstrating a dramatic Stokes shift of ~5000 cm−1 . It suggests the presence of some reaction occurring in the excited state, one of those described in Sect. 4.1. What is its origin and why excitation at much longer wavelength band 470–480 nm does not show the presence of such reaction? It was explained by the presence of distribution between two ground-state species. The experiments have shown that this distribution appears due to the presence of the neutral and anionic forms in dynamic equilibrium and the neutral state has to dissociate before the emission. It was found that the donor in proton transfer reaction is the phenol moiety of the fluorophore and the acceptor is the acidic residue Glu-222 within the protein structure. Interestingly, proton donor group and Glu-222 do not contact directly; they are connected via proton relay formed by other protein groups, involving Ser-205. The reaction proceeds in the absence of any other proton acceptor (the protein interior is screened from water). This proton loss is ultrafast and fully reversible. Excitation at the long-wavelength band results in green emission directly from the initially charged form. The amplitude of the two excitation peaks changes inversely being dependent on the equilibrium between the neutral and the anionic forms.

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This equilibrium can be modified through environmental or structural perturbations (Meech 2009). Mutations were performed to change the proton-acceptor potential of Glu-222 and thus shift the equilibrium to populate the proton-dissociated form (Campbell et al. 2018). In a dense protein interior, the fluorophore part derived from Tyr-66, as well as Glu-222, contacts with several amino acid residues, and their mutation influences the equilibrium. The work on selecting proper mutants was successful, and as a result, the GFP mutant form was obtained with the absence of proton transfer dynamics and with the excitation/emission wavelengths similar to fluorescein. They became convenient for live-cell studies.

5.5.4 Labeling and Sensing Applications of Fluorescent Proteins Due to the ability to be co-expressed as fusion proteins with many proteins that are naturally synthesized and functioning inside the living cells, GFP and its variants serve as unique tools in molecular biology and cell imaging. Presently, they are the only fully genetically encoded fluorescent probes available to a broad range of researchers. The benefits of using such markers and sensors are obvious because they obviate the issues of intracellular delivering and targeting of synthetic fluorophores. Their numerous applications include biological labeling to track and quantify individual or multiple protein species, probing to monitor protein–protein interactions, sensing to describe intracellular events and detecting different targets. The detection techniques described in Chap. 3 are efficiently used both in spectroscopy and microscopy formats. Fluorescent proteins can be applied in a large variety of studies related to various aspects of living systems, and the range of these applications is continuously expanding (Chudakov et al. 2010; Rodriguez et al. 2017). In addition to bringing valuable information on the location and dynamics of labeled proteins, their interactions and incorporation into organelles, FPs can perform the work of subcellular sensors. The FRET-based technologies are actively used for that, including the acceptor photobleaching technique (Dinant et al. 2008). The GFP mutants and circularly permuted GFP variants have been used to develop fluorescent probes that sense physiological signals such as membrane potential and concentrations of free calcium (Miyawaki et al. 2005). An important step towards the improvement of fluorescent proteins and extending the range of their applications was obtaining the species that become fluorescent (photoactivated) or change the colors of emission (photoswitchable) upon illumination at specific wavelengths (Acharya et al. 2017). Photoactivation approach can be realized with these proteins since they can isomerize or change interaction with surrounding under intensive irradiation by light. Their fluorescence can be controlled within living cells by light of specific wavelengths, which is especially needed for superresolution fluorescence microscopy.

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Intensity sensing (Sect. 3.2) is a simple approach but it is not attractive due to response depending on fluorophore concentration. In the present case of cellular studies, it depends on the protein expression level that is hard to measure. If a relative signal level is sufficient or if the qualitative cell image demonstrating the distribution of targeted protein is the result to be obtained then the fluorescent protein can be nonresponsive in a sense explained in Sect. 5.2. In required cases the reversible switching between emissive and non-emissive states can be activated by light. The quenching effect can be realized in protonation and cis-trans isomerization reactions (Bourgeois and Adam 2012). The split-protein systems can be used for detecting ions (Lee et al. 2019). In this case the ion coordination approaches two parts of the protein generating fluorescence enhancement. Wavelength-ratiometric sensing (Sect. 3.6) is a very efficient sensing technique since it allows eliminating the dependence of output signal of sensor concentration. In intracellular sensing with FPs, this fact is especially important since it means the independence of the level of probe expression. Different FPs demonstrate dual fluorescence (Chatterjee et al. 2020), in addition to dual excitation, and its use in sensor design would demonstrate the skill of researcher. Fluorescent proteins of different colors can be used to construct the FRET pairs. Classical is the design of Ca++ sensor based on FRET between GFP and YFP with Ca++ -binding protein calmodulin fused between them. From scheme on top of Fig. 5.30 the reader will understand why such constructions are called “chameleontype indicators”. Chameleon-type indicators produce a λ-ratiometric fluorescent

Fig. 5.30 Fluorescence properties of sensor for Ca++ ions made on the basis of calmodulin with appended GFP and YFP (Truong et al. 2001). a Emission spectrum in vitro acquired at an excitation wavelength of 432 nm for zero (1 mM EGTA) and saturating Ca++ concentrations (5 mM CaCl2 ). b The Ca++ titration curve acquired using Ca++ /EGTA and Ca++ /EGTA-OH systems below 10−5 M free Ca++ in a 50 mM HEPES/KOH, pH 7.4, and 100 mM KCl buffer. The changes in emission ratio (530–480 nm) were normalized to the effects of full Ca++ saturation

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signal as a result of Ca++ -dependent changes in the efficiency of FRET from a blueshifted donor to a red-shifted acceptor. Without binding these ions, calmodulin is unfolded keeping the proteins apart, but on binding four Ca++ ions it folds resulting in their close location. As a result, the green GFP donor emission is switched to yellow emission of acceptor YFP (Miyawaki et al. 1997), see Fig. 5.30. Lifetime-based sensing has been realized using fluorescent proteins as FRET donors and acceptors in fluorescence lifetime imaging microscopy (FLIM) (Mérola et al. 2014). Its primary advantages are the independence of protein concentration and little or no sensitivity to cellular parameters. FLIM, however, requires expensive instrumentation that is not yet widely available. The response by dynamic fluorescence quenching of thiol redox-sensitive sensors can be followed by time-resolved fluorescence microscopy (Li et al. 2019). The fact is that all FPs are so constructed that the fluorophore is located in a dense protein surrounding and the direct sensing based on modulation of its emission is difficult to realize. The good point is that if such sensor is created, it is screened to more extent from non-specific perturbations, which gives essential advantages to the researcher. A good example is the design of redox sensor based on GFP (roGFP) that was engineered by introduction of two Cys residues into β-barrel protein structure. They are located in proximity to the fluorophore and are readily capable of forming a disulfide bond, leading to small structural changes in protein that influence the fluorescence. This allows a real-time read out of the redox state of the fluorescent protein thiol-disulfide pair. This sensor exists in two states, with reduced thiols and with their oxidation to disulfide, and this switch produces informative signal (Hanson et al. 2004). They allowed combining the real-time monitoring of thiol redox dynamics with precise genetic targeting to any specific subcellular location. Nowadays, the genetically encoded sensors based on FPs are great in number (Sanford and Palmer 2017). In addition to sensing metal cations, pH and redox states, they can trace different metabolites, respond to biomembrane voltage and follow cell signaling (Chudakov et al. 2010; Newman et al. 2011).

5.5.5 Other Proteins with Visible Fluorescence Emission In addition to GFP and its relatives, there are other proteins that contain strongly fluorescent pigments. They can be isolated from species containing photosynthetic systems. Those are algae phycobiliproteins whose function is to absorb energy of light and transfer it to chlorophyll. They possess absorption maxima at 545–650 nm with the molar absorbance as large as 2.4 × 106 M−1 cm−1 . Their Stokes shift is not very large, 10–25 nm, but quantum yield may reach 85%. Such a high quantum yield is achieved because the protein matrix provides not only the location sites for a number of fluorescent pigments but also avoiding their contact that could result in self-quenching. Due to their high brightness and relatively long-wavelength emission, these proteins have found important applications primarily in flow cytometry, where they can be combined with different dyes for multicolor labeling of cells.

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Meantime, at present it is not possible to synthesize them in fully assembled fluorescent forms inside the living cells, as it is done with GFP-type proteins. This limits their application to in vitro testing. Due to their high brightness and long-wavelength emission, phycobiliproteins and, in particular, B-phycoerythrin, are used in FRET-based immunoassays, in which they make complexes with antibodies (Petrou et al. 2002). They are also useful as acceptors in sensing technologies based on FRET. Their major disadvantage in these applications is their large molecular size (1–1.5 times the molecular mass of IgG), which results in their slow diffusion leading to long incubation time. In this application, the nanoparticles are expected to be more prospective, regarding availability, stability and price. The search for fluorescent proteins among animal sources has led to an important discovery of a protein that has no intrinsic fluorescence but shows bright green emission in mammalian cells and biological mixtures even under anaerobic conditions. It was found that it binds with high affinity the ligand that also does not fluoresce. But if they make the complex, a strong fluorescence appears just as a result of molecular recognition without covalent bond formation or participation of additional reactant (Kumagai et al. 2013). The protein UnaG isolated from Japanese eel belongs to the fatty-acid-binding protein family and the ligand is bilirubin, a membrane-permeable heme metabolite and clinical health biomarker related to liver disease. This observation enabled the establishment of a human bilirubin assay with clinical significance. Based on UnaG mutant, the thiol redox-sensitive sensor was developed for cellular studies (Li et al. 2019). UnaG may serve as the prototype for a versatile class of ligand-activated fluorescent proteins, with applications in research, medicine, and bioengineering.

5.5.6 Finding Analogs of Fluorescent Protein Fluorophores Success in discovery, improvement and application of fluorescent proteins has stimulated focused research directed on finding ‘biology-inspired’ synthetic dyes. In Sect. 5.1.3, we discussed different trends in discovery of new dye families, such as targeted modifications within existing fluorophores, making hybrids between them and combinatorial approach exploring large libraries. Here, the nature suggests us to follow its unique achievements. Fluorescent proteins have shown that spontaneous reactions between amino acid residues in a peptide chain with fluorophore formation are possible. This justified the search for simpler peptide sequences that can give rise to self-assembling and self-catalyzing reactions leading to formation of fluorescent species. The focused search within constructed ~500,000-member solid-phase combinatorial library of Trp-containing Ala-flanked peptide hexamers led to successful findings that several non-fluorescent peptides selected from the library after UV irradiation and oxidation in the air attain intensive visible fluorescence (Juskowiak et al. 2004). A number of such analogs of GFP fluorophore have been synthesized (Ivashkin et al. 2009).

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It should be noted, however, that the photophysical properties of synthetic dyes in solutions are strikingly different from the corresponding structures within the parent fluorescent proteins. The units corresponding to protein fluorophores do not fluoresce in solutions (Meech 2009; Tsien 1998). The other trend in this line is to start from GFP fluorophore, to learn why it is not fluorescent in solution and to make the analogs, in which this problem can be removed. Thus, the search was focused on fluorophores that exhibit the proton transfer reaction (see Sect. 4.1), in which fluorescence is not quenched in liquid media. The dye o-HBDI (Fig. 5.31a) synthesized by Pi-Tai Chou group (Chen et al. 2007) differs from GFP fluorophore p-HBDI by the transfer of functional OH-group from para- to ortho-position. This allowed avoiding internal dye flexibility; the proton transfer became intramolecular, independent from proton acceptor in the surrounding. Intramolecularly locked structures were reported as the results of further developments. Thus, radiationless decay due to flexible segments is prevented by attaching the BF2 entity (Wu and Burgess 2008), see Fig. 5.31a. In further attempts to modify the GFP core, crease of photoacidity together with conformational locking the backbone and multiple fluorination of the phenolic ring

Fig. 5.31 Trends in design of synthetic analogs of fluorescent protein fluorophores. Parent fluorophore of GFP, p-HBDI, is on top. a The dye intramolecularly locked by H-bonding (Chen et al. 2007), the structure locked with the BODIPY-type BF2 moiety (Wu and Burgess 2008) and the dye with free ionizable OH-group, the BF2 entity and polyfluorination at the R sites (Chen et al. 2019). b A series of fluorescent interlocked compounds developed by Pi-Tai Chou group (Hsu et al. 2014). c The structures of core fluorophores of red fluorescent proteins and of their synthetic analogs (Feng et al. 2017). R is the site of attachment of aromatic extensions. The structure becomes rigid on incorporation to DNA G-quadruplexes, and fluorescence is enhanced

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allowed achieving bright and strongly Stokes-shifted emission (Chen et al. 2019). The planar and locked derivatives presented in Fig. 5.31 demonstrate high fluorescence quantum yield and for p-LHBDI and H2BDI the emission of anionic forms is observed in water (Hsu et al. 2014). Dual (cyan and red) emission of sterically locked derivatives was also observed, which depended on the conformation of appended phenyl ring (Chatterjee et al. 2020). Based on the fluorophores of red fluorescent proteins (Fig. 5.31c), a series of redemitting fluorophores were synthesized (Feng et al. 2017). These dyes were fluorinated and contained aromatic attachments. They were not internally locked since the aim was different, to provide informative signal on restriction of internal mobility. They did not demonstrate fluorescence in solutions but became bright emitters on encapsulation into canonical DNA G-quadruplexes. Such binding greatly restricts radiationless deactivation, and they start to demonstrate bright Stokes-shifted fluorescence spanning red and far-red spectral regions (583–668 nm). It was reported on their excellent anti-photobleaching properties, and on efficient two-photon fluorescence. The same principle of fluorescence enhancement by suppressing molecular twisting was applied in dye design for detecting aggregated protein forms (Liu et al. 2018b). Thus, simulating the structures and photophysical properties of the fluorophores in fluorescent proteins has brought a lot of ideas and technical solutions. Just the suppression of free rotation around an aryl-alkene bond that is restricted in β-barrel protein environment has increased the fluorescence yield of synthetic dyes when they are studied in solutions. The stability could be increased by insertion of BF2 group and fluorination. The desired modulation of proton-donation acidity can be achieved with the generation of dual emission. The redder and brighter fluorophores were obtained. All that will inspire chemical synthesis methods and advance a broad range of sensing and imaging applications.

5.5.7 Prospects An exciting new development in fluorescent imaging and sensing has appeared with the introduction of naturally fluorescent proteins. The interest towards these proteins is great, especially to those that can be fused with other proteins on a genetic level producing hybrids that can be synthesized inside the living cells. One of already traditional applications is the co-localization of labeled structures, which can involve split fragments providing fluorescent signal on their association (Avilov and Aleksandrova 2018). These hybrids are unique intracellular markers and tags. The other popular application is monitoring the expression of fluorescent protein, so that the whole cell serves as a sensor for monitoring its life conditions, e.g. checking for toxic pollutants. With the increase of information content, the single cells with incorporated smart fluorescence proteins will become ideal whole-cell biosensors (Gui et al. 2017).

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Different design strategies for biosensing and bioimaging have been reviewed recently (Deng et al. 2019). They highlight the new fluorescent signal-switching mechanisms and the target response of the engineered FPs, which extends the range of applications. Meantime, their application will not probably go far beyond intracellular research because of the size of the proteins as sensors and the cost of their production. In contrast, the organic synthetic products inspired by fluorescent proteins may have a future life with very broad scope of applications.

5.6 Sensing and Thinking. The Search for Novel Dyes and for New Mechanisms of Their Response Design and optimization of fluorescence reporter units remains a key determinant for the future success of sensing technologies. Presently, organic dyes dominate as fluorescence reporters. Their advantage is a small size and extreme diversity of spectroscopic properties. Many synthetic procedures exist for modulating these properties and for incorporation of these dyes into different sensor constructs. Why these dyes are so valuable? Because they possess unique features, responding to the changes of intermolecular interactions with the environment by changes of parameters of their emission. This allows them to participate directly in molecular sensing events. However, with each step of technology development, more flexibility is required from these dyes together with increased brightness and photostability, and often the traditional dyes are simply unable to meet the necessary demands. Discovery of fluorescent proteins was a great stimulus to synthetic chemists. It demonstrated that new unexpected dye families can be found in organic world. Other important findings of recent time are the organic dyes that can be efficiently incorporated into protein and nucleic acid structures. It opened new avenues in designing new specific and efficient biosensors. These achievements encourage researchers to find application of their creativity and enthusiasm in this fascinating field. In order to check the acquired knowledge reading this chapter, the reader is asked to respond to following questions: 1.

2.

3.

List the most important properties of fluorescence reporter dyes. In what directions they should be optimized for application in sensor technologies described in Chap. 3? The dye brightness and quantum yield are connected with fluorescence intensity that is measured in experiment via unknown factor of proportionality. What are the possibilities to evaluate them? Working with very small amounts of dye, it is frequently needed to determine its concentration from absorbance and evaluate absorbance from concentration. Estimate the dye absorbance in concentrations 10−5 and 10−6 M. Estimate the dye concentrations for the measured absorbances 0.5 and 0.05. Assume that the dye molar absorbance is 40,000 M−1 cm−1 and the measurements are made in a cell with 1 cm path length.

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4.

What is photobleaching? On what physical and chemical factors does it depend and what are the possibilities to reduce it? Can this unpleasant property find a useful application? 5. Let the reporter dye be deposited onto the surface of sensor molecule and on sensor-target interaction let it be located at their contact interface. Specify all possible effects that could be used to provide its quenching/enhancement response. 6. Explain the mechanisms by which the fluorescence spectra may shift on the wavelength scale. Suggest several examples of application of this effect in sensing. 7. What are the basic requirements for a dye to be sensitive to polarity? To hydrogen bonding? To electric fields? 8. In view, that the optical cross-section (that determines molar absorbance) cannot be larger than the fluorophore geometrical cross-section and that the quantum yield cannot be higher than 100%, evaluate the possibilities for improvement of presently used organic dyes. 9. How the fluorophores of GFP-type fluorescence proteins are synthesized? What is the advantage of their use in cellular studies? 10. The photophysical and photochemical properties of fluorescent proteins are largely similar to those of some synthetic dyes. What are the problems in making their synthetic analogs and what could be the routes to overcome them?

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Webster S, Fu J, Padilha LA, Przhonska OV, Hagan DJ et al (2008) Comparison of nonlinear absorption in three similar dyes: polymethine, squaraine and tetraone. Chem Phys 348:143–151 Whitaker JE, Haugland RP, Prendergast FG (1991) Spectral and photophysical studies of benzo[c]xanthene dyes: dual emission pH sensors. Anal Biochem 194:330–344 Widengren J, Chmyrov A, Eggeling C, Lofdahl PA, Seidel CAM (2007) Strategies to improve photostabilities in ultrasensitive fluorescence spectroscopy. J Phys Chem A 111:429–440 Wiktorowski S, Rosazza C, Winterhalder MJ, Daltrozzo E, Zumbusch A (2014) Water-soluble pyrrolopyrrole cyanine (PPCy) NIR fluorophores. Chem Commun 50:4755–4758 Willets KA, Wilson AJ, Sundaresan V, Joshi PB (2017) Super-resolution imaging and plasmonics. Chem Rev 117:7538–7582 Wilson JN, Kool ET (2006) Fluorescent DNA base replacements: reporters and sensors for biological systems. Org Biomol Chem 4:4265–4274 Wittmershaus BP, Skibicki JJ, McLafferty JB, Zhang Y-Z, Swan S (2001) Spectral properties of single BODIPY dyes in polystyrene microspheres and in solutions. J Fluoresc 11:119–128 Wu L, Burgess K (2008) Syntheses of highly fluorescent GFP-chromophore analogues. J Am Chem Soc 130:4089–4096 Wuskell JP, Boudreau D, Wei MD, Jin L, Engl R et al (2006) Synthesis, spectra, delivery and potentiometric responses of new styryl dyes with extended spectral ranges. J Neurosci Methods 151:200–215 Wysocki LM, Lavis LD (2011) Advances in the chemistry of small molecule fluorescent probes. Curr Opin Chem Biol 15:752–759 Xia G, Wang H (2017) Squaraine dyes: the hierarchical synthesis and its application in optical detection. J Photochem Photobiol C Photochem Rev 31:84–113 Yang CJ, Jockusch S, Vicens M, Turro NJ, Tan W (2005) Light-switching excimer probes for rapid protein monitoring in complex biological fluids. Proc Natl Acad Sci USA 102:17278–17283 Yang L, Liu Y, Ma C, Liu W, Li Y, Li L (2015) Naphthalene-fused BODIPY with large Stokes shift as saturated-red fluorescent dye for living cell imaging. Dyes Pigm 122:1–5 Yashchuk VM, Gusak VV, Drnytruk IM, Prokopets VM, Kudrya VY et al (2007) Two-photon excited luminescent styryl dyes as probes for the DNA detection and imaging. Photostability and phototoxic influence on DNA. Mol Cryst Liq Cryst 467:325–338 Yau CS, Pascu SI, Odom SA, Warren JE, Klotz EJ et al (2008) Stabilisation of a heptamethine cyanine dye by rotaxane encapsulation. Chem Commun 2897–2899 Yesylevskyy SO, Klymchenko AS, Demchenko AP (2005) Semi-empirical study of two-color fluorescent dyes based on 3-hydroxychromone. J Mol Struct Theochem 755:229–239 Yu C, Jiao L, Li T, Wu Q, Miao W et al (2015) Fusion and planarization of bisBODIPY: a new family of photostable near infrared dyes. Chem Commun 51:16852–16855 Yuan L, Jin F, Zeng Z, Liu C, Luo S, Wu J (2015) Engineering a FRET strategy to achieve a ratiometric two-photon fluorescence response with a large emission shift and its application to fluorescence imaging. Chem Sci 6:2360–2365 Yushchenko DA, Shvadchak VV, Bilokin MD, Klymchenko AS, Duportail G et al (2006a) Modulation of dual fluorescence in a 3-hydroxyquinolone dye by perturbation of its intramolecular proton transfer with solvent polarity and basicity. Photochem Photobiol Sci 5:1038–1044 Yushchenko DA, Shvadchak VV, Klymchenko AS, Duportail G, Mély Y, Pivovarenko VG (2006b) 2-Aryl-3-hydroxyquinolones, a new class of dyes with solvent dependent dual emission due to excited state intramolecular proton transfer. New J Chem 30:774–781 Zatsikha YV, Yakubovskyi VP, Shandura MP, Kovtun YP (2013) Boradipyrromethenecyanines on the base of a BODIPY nucleus annelated with a pyridone ring: a new approach to long-wavelength dual fluorescent probe design. RSC Adv 3:24193–24201 Zatsikha YV, Yakubovskyi VP, Shandura MP, Kovtun YP (2015) Functionalized bispyridoneannelated BODIPY—bright long-wavelength fluorophores. Dyes Pigm 114:215–221 Zhang J, Wang J, Tian H (2014) Taking orders from light: progress in photochromic bio-materials. Mater Horiz 1:169–184

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Chapter 6

Small Luminescent Associates Based on Inorganic Atoms and Ions

In this chapter, we present new materials for sensing and imaging that can be viewed together due to the central role in their function of inorganic atoms and ions. They represent three different mechanisms of generation of light emission. Noble metal clusters composed of only several atoms exhibit molecular-type fluorescent emission that is very similar to that of organic dyes. It is characterized by smooth emission spectra, short lifetimes, high polarization of emission and brightness. In contrast, long lifetimes in the millisecond time range are characteristic of complexes formed of transition metals and noble metal ions of platinum group, and their emission is typical phosphorescence. The lanthanides Eu and Tb demonstrate luminescence spectra that are presented by series of very sharp emission bands demonstrating very long lifetimes. Their metal-centered luminescence is sensitized by energy transfer from the ligand. In all these cases the organic groups of atoms surrounding the metal species play their important but different role. Being of much smaller size than semiconductor quantum dots or upconverting nanocrystals and exhibiting such diverse properties, these materials demonstrate a broad range of applications that will be overviewed. The final section “Sensing and thinking” will be accompanied by questions and problems. Pigments of inorganic origin were known for many centuries. They were used as bright and very stable paints decorating the human life. Their photoinduced light emission was usually unnoticed until the scientists started to look for new light emissive materials. These materials were found, and now they demonstrate increasing popularity for developing new sensing and imaging technologies. In this Chapter such materials that can efficiently operate on atomic-molecular level are discussed. We start the discussion from the few-atom clusters of noble metals that represent new and very efficient family of fluorescent labels and probes. Noble metal clusters composed of only several atoms exhibit molecular-type fluorescent emission that is very similar to that of organic dyes. It is characterized by smooth emission spectra, short lifetimes, high polarization of emission and brightness. They exhibit excellent photophysical properties particularly for biological imaging such as high photostability, long fluorescence lifetime, strong Stokes shift and large two-photon © Springer Nature Switzerland AG 2020 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-030-60155-3_6

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a

b

Ag0 Ag0

c

Rb3+

La3+ Fig. 6.1 The small composite structures based on inorganic atoms and ions that generate luminescence on light excitation. a The few-atom clusters of Ag, Au and Cu that generate the ligand-to-metal charge transfer (LMCT) fluorescence. The cluster is stabilized by –SH, –COOH, –OH, –NH2 and = NH groups belonging to small molecules or polymer chains. b Complexes of ions of transition metals Ru, Os and Re stabilized by organic ligands that demonstrate metal to ligand charge transfer (MLCT) that generates phosphorescence. In both cases the dipole moment can be observed in the excited state (shown as arrows). c The rare earth (lanthanide) ions stabilized by organic ligand demonstrating luminescence sensitized by energy transfer from the ligand

absorbance. Furthermore, their small size, the easy preparation in aqueous solution, the ability to functionalize them, and their biocompatibility make them very promising for future materials and devices. In sensing technologies there is a strong need to overcome limitations in lifetime values and to extend the time ranges to microseconds and milliseconds. Longer lifetimes should allow observing molecular motions of large molecules and particles and using simpler instrumentation for lifetime analysis. They offer a simple way to eliminate the light-scattering effects and short-living background fluorescence, thus increasing significantly the sensitivity and precision of assays. Ions of transition metals when decorated by organic heterocyclic structures can attain a very attractive feature—they become phosphorescent. Similar properties demonstrate the noble metal ions of platinum group when they are incorporated into porphyrin-like structures. Their long lifetimes in the millisecond time range make them ideal oxygen sensors. Demonstrating high polarization of their emission, they allow extending the assays based on response of fluorescence anisotropy to larger molecular units. Long lifetimes can also be achieved with coordination complexes of lanthanide ions. The lanthanides Eu and Tb demonstrate luminescence spectra that are presented by series of very sharp emission bands showing very long lifetimes. Their metalcentered luminescence is sensitized by energy transfer from the ligand that operates as antenna collecting the absorbed light and thus increasing the brightness. Thus, in this Chapter the reader will become acquainted with new materials for sensing and imaging that can be viewed together due to the central role in their function of inorganic atoms and ions. They represent three different mechanisms of generation of light emission, and all of them are of value. They are schematically presented in Fig. 6.1. The organic groups of atoms surrounding the metal species allow not only realizing important photophysical mechanisms but also increasing

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the stability, solubility in different solvents, including water, and also biocompatibility. Being of much smaller size than semiconductor quantum dots or upconverting nanocrystals and exhibiting such diverse properties these materials demonstrate a broad range of applications that will be overviewed. The reader is initiated to think on a new family of materials in the context of luminescence imaging and other applications.

6.1 Fluorescent Few-Atom Clusters of Noble Metals Fluorescent noble metal clusters containing only several atoms (often called quantum clusters) have attracted a lot of attention as a new class of fluorophores (Chakraborty and Pradeep 2017; Jin et al. 2016). They demonstrate unique electronic structures and, subsequently, unusual physical and chemical properties. Like organic dyes, they possess discrete electronic states, and the electronic transitions between these states result in strong visible fluorescence. In view of their very small size comparable with that of organic dyes, of their biocompatibility and the absence of photobleaching, these clusters represent an attractive alternative to other types of molecular and nanoscale fluorophores in labeling, sensing and, especially, in imaging (Chen et al. 2015; Zhao et al. 2019).

6.1.1 The Cluster Structures and Their Stability Up to now, significant efforts have been dedicated to the synthesis, studies of properties and application studies of gold (Muhammed and Pradeep 2010) and silver (Choi et al. 2012a; Diez and Ras 2011) clusters. Recently, a growing number of studies on copper clusters have also been reported (Hu et al. 2016; Shahsavari et al. 2019). These clusters can be easily obtained by chemical or photochemical reduction of ions in solutions. The application of strong chemical reductant, such as NaBH4 , can be substituted by treatment with UV or visible light, by electrochemical reduction or by other means. The problem is only to stabilize them by choosing appropriate scaffolds. In the case of silver, reduction (Ag+ to Ag0 ) is coupled directly with cluster formation. Organic scaffolds are commonly used for maintaining the cluster stability and suppressing further increase in their size. The clusters are recognized by characteristic excitation and emission bands in the visible range of spectrum demonstrating behavior similar to that of organic dyes (Diez et al. 2012). The results of recent efforts for implementation of silver clusters (Choi et al. 2012a; Diez and Ras 2011) can be summarized as follows (see Table 6.1): (a) The clusters of well-defined size and composition can be synthesized in both aqueous (Wu et al. 2009) and nonaqueous (Diez et al. 2012) solvents. During

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Table 6.1 The properties of few-atom silver clusters Parameter

Property

Size

< 1 nm but need stabilizing template

Absorption spectra

Commonly corrupted by light-scattering. No vibronic structure observed

Molar absorbance, ε

Of the order of 105 M−1 cm−1

Excitation spectra

Resolved, broad (~ 60 nm half-width) and symmetric

Emission spectra

Broad (~ 70 nm half-width), symmetric, inhomogeneously broad

Stokes shifts

~100 nm, low sensitive to intermolecular interactions

Fluorescence quantum yield, 

Can be relatively high, 20-30%

Excited-state lifetime, τF

Insufficiently studied. Can be between 10 and 200 ns.

Two-photonic cross-section, α

>10 000 GM

Fundamental emission anisotropy, r 0

Unexpectedly high, ~ 0.35

Photostability

High

Availability of chemical modifications

Depends on stabilizing template

(b)

(c)

(d)

(e)

the process of reduction and cluster formation the number of clusters increases without affecting the type of emitters and the shape and position of emission spectra. Their excitation and emission spectra in the visible range of spectrum are characteristic. They may not depend on the method and duration of treatment but strongly depend on the scaffold. When one increases the time of photoreduction treatment, only the intensity commonly increases, which indicates the increase of production of the same clusters but not the appearance of different ones. The excitation and emission spectra are smooth and well separated (Stokes shifts are strong) resembling the features of charge-transfer organic dyes (see Sect. 5.3). Their stability is variable and strongly dependent on the support. With synthetic polymers (Diez et al. 2009), peptides and proteins (Yu et al. 2007) and DNA fragments (Vosch et al. 2007) sufficient stability for many applications has been achieved. The factors determining their stability and its dependence on scaffold are still not well understood. They exhibit a remarkable photostability, in contrast to organic dyes that are destroyed after 105 –107 excitation-decay cycles (see Sect. 5.1.2).

In many cases the geometrical shapes of these clusters are not well known at present, but it is clear that interaction with the support atoms and their groups is a strong determinant for their formation. This interaction determines the cluster size and stability and, finally, the parameters of fluorescence emission. There is a difference in formation of gold and silver clusters. The gold clusters are usually larger in size incorporating several tens of atoms. To date, several atomically precise Aun clusters with thiolate ligands or stabilized by protein templates (Ghosh

6.1 Fluorescent Few-Atom Clusters of Noble Metals

241

et al. 2014) have been synthesized, and their electronic and optical properties studied. In contrast, silver clusters can be formed of only 2–3 atoms. As a rule, the fluorescence quantum yield in gold clusters is much lower than that in the clusters of silver. This is probably because the silver species are commonly obtained in smaller size and thus can occupy a smaller density of states.

6.1.2 The Mechanisms of Light Absorption and Emission in Noble Metal Clusters In bulk metal materials the electrons are free to move. However, when the size in metallic particles is decreased to become smaller than the free path length of an electron (~20–30 nm for silver and gold) the electron motions become restricted and the localized plasmonic effects are observed (Fig. 6.2). They are responsible for strong light absorption and scattering. Due to the generation of strong electromagnetic fields that appear at atomic distances in their surrounding there appear dramatic plasmonic effects (discussed in Chap. 13). Due to these effects, an enhancement in the spectra of Raman scattering and also in fluorescence emission of different closely located fluorophores is observed.

Atomic resonance absorption

Plasmonic effects Mie scattering Metallic properties Quasi-continuous electron energy states

Fig. 6.2 The effect of size on metal materials. Whereas bulk metals and metal nanoparticles have a continuous energy profiles, the limited number of atoms in metal clusters results in discrete energy levels. The electronic transitions between these levels on the absorption of light quanta result in the appearance of molecular-type fluorescence excitation and emission spectra

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If we further decrease the particle size down to several atoms, plasmonic properties disappear. Being too small for forming the continuous density of states that are necessary for generating plasmons, the few-atom clusters start to behave like molecules (Choi et al. 2012a; Diez and Ras 2011). This transition is very sharp and it is reflected not only by enhancement of fluorescence but also by the change of other properties, such as two-photon absorption (Jin et al. 2016). They start to possess discrete electronic states and the electronic transitions between these states. With these new properties they bridge the gap between single atoms and nanoparticles. The number of atoms in these clusters varies between two and about one hundred. The ‘magic size’ clusters that conform to some symmetry rules were found. For gold clusters the sizes Au5 , Au8 , Au13 , Au23 and Au31 were described (Jin et al. 2016; Zheng et al. 2007). The presence of magic compositions has been confirmed also for the silver clusters (Chakraborty and Pradeep 2017).

6.1.3 Spectroscopic Properties It is interesting that the absorption spectra of metal clusters in solutions demonstrate continuous density of states extending to the UV range, but the excitation and emission spectra are smooth, rather symmetric and behaving like that of organic dyes. Fluorescence emission is commonly represented by single emission bands that for different clusters may be observed in the whole visible range, extending to near-IR. The spectral positions of these bands depend on two major variables—the nature of scaffold and the cluster size. The general tendency observed for silver (Choi et al. 2012b) and gold (Zheng et al. 2007) clusters is that with the increase of cluster size the excitation and emission bands shift to longer wavelengths. The excited state lifetimes are of the order of 1-10 ns, so we can attribute their emission to fluorescence. In comparison with gold clusters, the silver clusters are usually smaller and brighter; the positions of their spectral bands do not depend so clearly on the cluster size because of stronger dependence on supporting scaffold. Their excitation and emission spectra are usually broad, symmetric and do not possess any fine structure. When formed on polymeric scaffolds they display mirror symmetry between excitation and emission spectra. Remarkable is their intra-band spectral heterogeneity with the retention of such symmetry (Fig. 6.3). which can be explained by distribution in perturbing action of the scaffold and surrounding media (Diez et al. 2013). Heterogeneity arising due to the presence of clusters of different sizes generates new bands, which can provide sharp distinction arising from the presence in the system of particles of different size. Notably, the strong Stokes shifts (~2,000 cm−1 ) are characteristic for practically all silver and gold clusters of several atoms studied in solutions so far. Careful inspection of result presented in Fig. 6.2 allows deriving that the Stokes shift is the same when scanning is provided within the contours of three observed bands and also when the results are compared between the bands. Such behavior can be explained by the involvement of the ligand-to-metal charge transfer (LMCT) states (Díez et al.

6.1 Fluorescent Few-Atom Clusters of Noble Metals

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Fig. 6.3 The heterogeneities in excitation and emission spectra of clusters produced in water by photochemical reduction of silver salt on amphiphilic polystyrene-block-poly(methacrylic acid) block copolymer (Díez et al. 2013). a Fluorescence emission spectra as a function of excitation wavelength and b excitation spectra as a function of emission wavelength. c Representation of the positions of fluorescence emission spectra (lmax em ) as a function of excitation wavelength (lex ) and d the positions of excitation spectra (lmax ex ) as a function of emission wavelength (lem ) in the visible range derived from the data of a and b respectively

2013). Here the electron-rich groups of ligands serve as the electron donors (D) and the neutral atomic cluster as the electron acceptor (A), so that the LMCT states are formed in both ground and excited states. In the present case (Fig. 6.3), three spectroscopically distinguishable species are observed. They probably differ by the number of silver atoms so that its increase shifts the spectra to longer wavelengths. Both the ground-state and excited-state energies of these complexes are distributed on their interaction with the environment forming the inhomogeneous contour in spectra and allowing the site-selectivity by small gradual shifts of excitation or emission energy. The strong Stokes-shifted fluorescence emission is commonly observed with gold clusters that can be variable and dependent on cluster size (Zheng et al. 2007). Probably, the record value was recently obtained for the clusters synthesized and protected in ovalbumin environment (Rajamanikandan and Ilanchelian 2018). Possessing excitation maximum in the violet, at 390 nm, these clusters emit red light demonstrating band maximum at 650 nm. The mechanism of LMCT can explain that. Unexpectedly, a high level of anisotropy of fluorescence emission was detected for the silver clusters made on polymer support, reaching the values r = 0.35–0.37 (Diez et al. 2013). Polarized emission was also found for gold clusters incorporated into protein molecules (Raut et al. 2013). This witnesses not only for the absence of rotational mobility of the clusters during fluorescence lifetime but also suggests the presence of nonzero excited-state dipole moment in the excited state (check Sect. 3.3). Since the cluster is neutral, this fact can be due to its strong interaction with electron donor group of the ligand (presumably, of the carbonyl group of the polymeric support) forming the LMCT complexes.

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The silver clusters can be obtained and studied in nonaqueous media (Diez et al. 2012). They always show high almost solvent-independent Stokes shifts and spectral inhomogeneous broadening observed in excitation and emission. Together with interesting facts, such as the presence of a second excitation band in the UV region and the drop of fluorescence polarization when fluorescence is excited at this band, this supports the LMCT origin of their emission (Diez et al. 2013). The two-photon absorption (TPA) is the simultaneous absorption of two photons of identical or different energies in order to excite a molecule by adding these photon energies (Chap. 16). Thus, with the near-IR light pulse, one can excite the state leading to emission in the visible range. This nonlinear effect allowed obtaining very sharp images in microscopy and because of that it is a very valuable feature of any fluorophore. The two-photon cross-section expressed in Göpert-Mayer (GM) units, 10−50 cm4 s, is the measure of fluorophore ability to absorb two photons. Surprisingly, very high values were found for metal clusters. Different research groups reported for silver clusters the values of the order of 100,000 GM (Patel et al. 2008) and quite similar data were obtained for gold clusters (Polavarapu et al. 2011). The reader may compare these values with the data obtained for organic dyes (Sect. 5.4.2) and see that the difference is by 3-5 orders of magnitude! Such superiority over organic fluorophores can be explained by unique strongly asymmetric nature of the ground and excited states that have to be considered together with the ligands as integrated systems.

6.1.4 Formation, Stabilization and Protection of Metal Clusters The stable existence of noble metal clusters cannot be observed without interaction with the ligands forming their surrounding that can be rather strong. Protection preventing the cluster aggregation can be achieved by small molecules serving as electron donors in LMCT states. Based on numerous existing data, it can be concluded that –SH, –COOH, –OH, –NH2 and = NH are the main groups that interact with Ag atoms to stabilize the clusters. Amino acids and glutathione contain these functional groups and they have been frequently used to synthesize the clusters. The ratio of efficient functional groups of stabilizer to Ag atoms is important but their configuration should play a dominant role when small molecules are used as stabilizers. The chirooptical properties can be demonstrated for the clusters possessing no inherent chirality when they are synthesized using different combinations of chiral thiol ligands (Cathcart and Kitaev 2010). Chiral electronic transitions in fluorescent silver clusters stabilized by DNA were also described (Swasey et al. 2014). Stabilization and protection is important for Au clusters, but for Ag clusters the role of stabilizing groups is crucial due to higher reactivity of these atoms and the fact that they are easier oxidized.

6.1 Fluorescent Few-Atom Clusters of Noble Metals

245

Protection by synthetic polymers can be achieved in many ways applying macromolecules of different chain length and also dendritic and block-copolymer structures. The amphiphilic polystyrene-block-poly(methacrylic acid) block copolymers can be universally used as their support medium (Diez et al. 2012). Strongly luminescent silver nanoclusters with tunable emission can be directly synthesized in organic polar and apolar solvents. When studied in solvents of different polarities they demonstrate a remarkable similarity in spectroscopic properties between these clusters and the charge-transfer organic dyes. The water-soluble red and near-IR-emitting silver clusters can be easily encapsulated into DNA molecules. They were shown to exhibit extremely bright and photostable emission on the single-molecule and bulk levels. DNA is an effective template for the preparation of fluorescent Ag clusters due to the presence of both carbonyl oxygen and the doubly bonded ring nitrogen in heterocyclic bases that also can participate in interaction with the clusters. The affinities and fluorescence spectra of studied clusters are highly DNA-sequence-dependent owing to cytosine (C) bases that exhibit strong affinity for silver ions (Choi et al. 2012b). The affinity is much stronger of single-stranded than of double-stranded DNA. Moreover, as it was shown in the analysis of 12-mer DNA strands of different composition, the nature of Ag clusters and their fluorescence are strongly variable (Richards et al. 2008). The DNA sequence and its conformation determines the well-defined silver clusters sizes, which results in distinct emission properties ranging from visible to near-IR (Wang et al. 2016). Protection and co-synthetic incorporation of clusters into protein molecules (Chevrier et al. 2018; Hu et al. 2015; Rajamanikandan and Ilanchelian 2018) can be made in amazingly simple way. Upon addition of Au ions to the aqueous solution of bovine serum albumin (BSA) solution the protein molecules sequestered these ions and entrapped them. Then pH is raised to 12, which makes it denatured and allows the ions assemble in protein interior and are reduced spontaneously. Then pH is decreased, protein re-folds and we have bright clusters with red emission within the protein interior (Xie et al. 2009). Importantly, such gold clusters are in a rigidified state within the protein scaffold, offering an explanation for their highly luminescent character (Chevrier et al. 2018). Highly fluorescent gold clusters have also been synthesized by similar approach in basic aqueous solution using lysozyme as a reducing and stabilizing agent (Wei et al. 2010). It is thought that the proteins tyrosine residues could participate in the reduction of Au ions to neutral atoms before their assembly. Organic dyes can serve not only as sensitizers in reactions of photoinduced reduction of metal ions but also as molecular supports and protection factors for the formed clusters. This allows obtaining novel fluorescent nanoscale materials of precise stoichiometry in a simple one-step process just by UV light illumination. The results of realization of this idea are presented in Fig. 6.4. A mixed solution of silver salt with organic dye Thioflavin T after UV light treatment changes the light absorption and emission properties of Thioflavin T into novel properties of the cluster. In comparison to metal clusters formed on polymeric or DNA carriers, these fluorescent metal-dye composites are the structures very small in size and with well-defined stoichiometry. Based on mass spectrometry data it was suggested that they consist

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6 Small Luminescent Associates Based on Inorganic Atoms and Ions

a

Fluorescence intensity

b

DMF

Fig. 6.4 The photosensitized reduction of Ag+ ions and stabilization of Ag+n clusters by Thiophlavine T (ThioT). a Illustration of formation of ThioT2 -Ag02 composite. b Fluorescence spectra of these clusters in different solvents (Kanyuk and Demchenko 2013)

of two silver atoms and two dye molecules. The most stable composite structures are obtained in 2-propanol, though sufficient stability can be achieved in other solvents. Their excitation and emission spectra possess the maxima at 340 and 450 nm correspondingly and are solvent-independent (see Fig. 6.4b). They demonstrate dramatic Stokes shift, so that the overlap between excitation and emission bands is practically absent. It can be expected that such new fluorescent nanoscale materials will find many applications in biosensing and biological imaging technologies. Thus, organic molecules of different size and organization play an essential role by forming the sites for cluster formation, catalyzing the ion reduction, being a shell protecting from oxidation and, probably, forming the metal-ligand charge transfer states.

6.1.5 Applications in Sensing and Imaging Generation of reporting signal as the change of fluorescence intensity (see Sect. 3.2) is the most common application of Ag and Au clusters in sensing technologies.

6.1 Fluorescent Few-Atom Clusters of Noble Metals

247

Quenching of silver cluster fluorescence is suitable to detect the presence of small analyte molecules, such as cysteine and metal cations, which is possible with a low detection limit and high selectivity. Using different DNA templates, it was possible to quantitatively detect bivalent cations. Interestingly, such sensors could show selective response for Hg2+ in contrast to Cu2+ (Guo et al. 2009), but also the selectivity for Cu2+ , not for Hg2+ (Lan et al. 2010). At first sight, this may seem a discrepancy, but it is rather a demonstration of an essential role of template that was different in these cases. In fact, the easiest and the most spectacular are the determinations of mercury ions that form strong nonemissive complexes with noble metal clusters (Yuan et al. 2012). The significant quenching by cysteine in solutions of silver clusters incorporated into polymer scaffold was used for sensing this amino acid on the background of almost zero response by other amino acids (Shang and Dong 2009). In all these cases the quenching indicates that there is a strong interaction between the clusters and cysteine molecules and the quenching occurs due to the excited electron transfer reaction (see Sect. 4.1). In view that the wavelength-shifting response from a typical cluster is small (Diez et al. 2009; Qu et al. 2013) and not always observable, for λ-ratiometric reporting (see Sect. 3.6) the excitation energy transfer (EET) between donor and acceptor is used. Thus, in aptamer devised for the detection of T-2 toxin, the Ag clusters acted as energy donors, whereas the probe employed as fluorescent acceptor was used as a target-dependent quencher (Khan et al. 2018). A broad range of applications of noble metal clusters was a subject of many reviews (Zhang and Wang 2014). Several the most popular of them can be outlined. In addition to metal ion sensing in aqueous media, sensing the small molecules, such as ATP, using the aptamer sensor configuration can be achieved (Zhang et al. 2013), Thus the few-atom clusters of noble metals represent new family of fluorescent labels and probes. These clusters demonstrate excellent photophysical properties. They are among the strongest emitters, possessing high quantum yields, high molar absorbance, nanosecond lifetimes, and still retaining small sizes of core units. Being electron dense they fit equally well for the electron microscopy imaging. Biological imaging has to benefit from their excellent photostability, lack of blinking, strong Stokes shifts and large two-photon absorbance. Furthermore, their easy preparation in aqueous solution, the ability to functionalize them, and their biocompatibility make them very promising for future materials and devices. Among already demonstrated but by now poorly explored possibilities are the mixed Ag-Au clusters, the clusters composed of neutral atoms together with the corresponding ions, decoration with EET donor ligands (Cantelli et al. 2017). Proper selection of supporting materials is the critical issue for different technologies that use them.

6.2 Metal Complexes that Exhibit Phosphorescence In contrast to metal clusters discussed above, phosphorescence is the emission of light quanta from the triplet state. In order to emit phosphorescence, the fluorophore has to be excited to the singlet state, then the formally forbidden intersystem crossing

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6 Small Luminescent Associates Based on Inorganic Atoms and Ions

(ISC) should occur to the triplet state generating the emission. The probability of singlet-triplet transition depends on spin-orbit coupling. In phosphorescent organic dyes it can be enhanced by heavy atoms that can be the part of dye structure (see Sect. 5.4). Below, we will see that some metal ions in organic environment can play a central role generating phosphorescence. Like that of organic dyes, this emission is characteristic by strong Stokes shifts, long lifetimes, ability to be quenched by temperature and oxygen.

6.2.1 Metal-to-Ligand Charge Transfer (MLCT) Phosphorescence The long-living emission of transition metal complexes is due to their ability for intersystem crossing to the triplet state and to emit phosphorescence. The emission properties of complexes of ruthenium (Ru), osmium (Os) and rhenium (Re) ions with organic ligands, such as pyridine groups, are the most frequently studied and used. They exhibit the metal-to-ligand charge transfer (MLCT) phosphorescence (Balzani et al. 2000). The absorption and emission spectra of these complexes are broad and structureless, with strong Stokes shifts; this emission is highly polarized (Balzani et al. 2000). The structure of one of such complexes of Ru3+ with ligands, tris-(2,2’bipyridine) chelate, Ru(bpy)2+ 3 , and its absorption and emission spectra (Piszczek 2006) are presented in Fig. 6.5.

Emission intensity

b

Absorption

a

Ru(bpy)2mcbpy

400

500

600

700

Wavelength, nm

Fig. 6.5 An example of chelating complex incorporating ruthenium ions and containing a reactive group for covalent binding. a The structure of Ru bis(2,2’-bipyridine)-4’-methyl-4carboxybipyridine N-succinimidyl ester, (Ru(bpy)2 mcbpy). b Normalized absorption and emission spectra of (Ru(bpy)2 dcbpy) in aqueous buffer, pH 7.4 and when it is conjugated to protein IgG (slightly shifted to the red)

6.2 Metal Complexes that Exhibit Phosphorescence

249

We can see that despite visual similarity in formation of ion-heterocyclic chelates, the origin of such emission is quite different from that of lanthanide chelating complexes that will be discussed below. Since it is the phosphorescence in MLCT complexes, their absorption and phosphorescence emission spectra are similar to that of typical organic dyes. The absorption spectra are broad. For Ru2+ complexes they are located around 470 nm with the width of ~100 nm. Similarly broad are the emission spectra that are observed at 610-670 nm, demonstrating a large Stokes shift. The molar absorbance of these complexes is on the level of ~ 10,000–30,000 M−1 cm−1 , which is sufficient for different applications in sensing. Since phosphorescent emission occurs from the triplet state, its duration is long and can be observed on a scale of several microseconds. Remarkable, this emission is highly sensitive to temperature and concentrations of oxygen in the medium, as it should be for phosphorescence. Long lifetime of ruthenium chelate complexes allow using them as reference emitters in the two-channel λ-ratiometric sensors based on the principle of intensity sensing with the reference (Sect. 3.2). This principle was realized in the design of double labeled glucose sensor (Shav-Tal et al. 2004) and in glutamine sensor (Lakowicz et al. 1999) based on bacterial periplasmic glucose and glutamine binding proteins, correspondingly. In these sensors the organic environment-sensitive dye Acrylodan was the responsive dye reacting to the target binding by quenching its emission. Ruthenium bis-(2,2’-bipyridyl)-1,10-phenanthroline-9-isothiocyanate served as a nonresponsive long-lived reference. This allowed recording the ratio of emission intensities of Acrylodan dye and ruthenium (at 515 and 610 nm) as a reporting signal, which allows increasing substantially the accuracy of sensor operation. The other important application of metal chelate complexes is in fluorescence anisotropy sensors and polarization immunoassays. Since these complexes exhibit large fundamental anisotropy values, this allows examination of rotational dynamics on the time scale of microseconds. The problem here (as discussed in Sect. 3.3) is to develop luminescence probes with lifetimes that are comparable to rotational correlation times of the antibody, the antigen, and the bioconjugates that they form. Ruthenium or rhodium chelate complexes with lifetimes 1000-fold longer than of the common dyes allow achieving this goal.

6.2.2 Phosphorescent Metal-Chelating Porphyrins The porphyrins are among organic heterocyclic compounds that can incorporate the transition metal ions with the formation of phosphorescent complexes. They possess a tetrapyrrolic structure and may contain different side substituents. These compounds can be widely found in nature forming various heme derivatives and chlorophylls. Many of these structures can be now produced by chemical synthesis. Interesting for sensor applications are metalloporphyrin complexes of platinum (Pt) and palladium

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Absorption

Luminescence intensity

(Pd). They display narrow and intensive absorption bands at 360–400 nm with molar absorbance 1–5 × 105 M−1 cm−1 (Soret band) and also the absorbance in 500550 region (Q-bands). In this green part of the spectrum their absorbance is much weaker, on the level of 20,000 M−1 cm−1 . The red (600–750 nm) phosphorescence emission exhibits lifetimes in the range from 0.01 to 1 ms (Papkovsky and Ponomarev 2001). These features make luminophores useful as probes for a number of analytical applications, particularly of those employing the time-resolved detection (Papkovsky and O’Riordan 2005). A number of analytical systems have been developed that are based on the use of phosphorescent porphyrin probes (Papkovsky et al. 2000). Among them are the fiberoptic phosphorescence lifetime-based oxygen sensors constructed on the basis of hydrophobic platinum-porphyrins and also the biosensors for metabolites (glucose; lactate) based on the detection of oxygen consumption by glucose oxidase. Watersoluble platinum- and palladium-porphyrins can also be used as the labels for ultrasensitive time-resolved phosphorescence immunoassays. There is a demand for sensing oxygen in biological systems within the near-IR optical window, which can be satisfied with the extension of porphyrin π-electronic system. Thus, phosphorescent palladium(II) and platinum(II) complexes of tetrabenzoporphyrins and tetranaphthoporphyrins were suggested. They show more favorable spectral properties and are excitable at 600–640 and 690 − 720 nm, respectively. They possess moderate to high brightness and relatively long phosphorescence decay times of 8–400 μs. On the step to further improvement their analogs azatetrabenzoporphyrins were synthesized and their improved photostability was demonstrated (Borisov et al. 2010). Due to very sharp absorption spectrum that allows providing excitation by red diode laser and sharp near-IR emission spectrum (Fig. 6.6) they can be efficient oxygen sensors in vivo.

Wavelength, nm

Fig. 6.6 Absorption and luminescence spectra of phosphorescent platinum(II) complex with azatetrabenzoporphyrin dye absorbing red and emitting near-IR light (Borisov et al 2010)

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The techniques of labeling proteins and nucleic acids with porphyrins as the probes have been developed (O’Riordan et al. 2001). They can be used for labeling oligonucleotides that are applied in hybridization assays. Upon hybridization with complementary sequences bearing common organic dyes, the metal chelate probes display a strong quenching due to close proximity effects, which allows establishing separation-free hybridization assays. Their attractive field of application is in timeresolved luminescence microscopy (Soini et al. 2002). Summing up, due to their long lifetimes, the emitting complexes of transition and noble metals can respond to rather slow motions of molecules and nanoparticles. This can realize the mechanism of reporting on the binding of targets of relatively large size. In sensor operation, different quenching effects can be used as the mechanism of on/off switching on the long time scale.

6.2.3 Upconversion by Triplet-Triplet Annihilation Phosphorescent materials are able to perform the upconversion process. The physical term ‘upconversion’ means the excitation from already populated excited state to a higher energy state, from which the emission can proceed with the energy higher than the energy of excitation light. Special materials are needed for that, in which the emissive states are reached by two quanta: first quantum excites to a long-living state, in which the electrons can wait for the second quantum, and the second quantum allows reaching the state, from which the emission occurs. In Sect. 7.5 we discuss the upconverting properties of nanocrystals made of lanthanide ions. They form intermediate states, on which the electrons of intermediate energies can stay waiting for second photon to come. With phosphorescent chelated ions a different effect can be realized. The triplet-state energy can be transferred from one luminophore to another (triplet-triplet excitation energy transfer, TTET), and if the latter is already excited, the energy can be doubled and become sufficient to excite luminophore with higher excited-state energy. Then the intersystem crossing can occur in reverse direction but populating the electronic state with higher energy. In this way, it is possible to emit light in the form of fluorescence of energy higher than the energy of excitation quanta. This process is illustrated in Fig. 6.7. Polycyclic complexes of platinum and palladium in the form of metal-porphyrins are commonly used as sensitizers as well as phosphorescent complexes of ruthenium and rhenium (Zhou et al. 2015). Regarding annihilators, polycyclic aromatic hydrocarbons can better serve to this purpose. Such systems can be assembled into nanoparticles. Compared to lanthanide-based upconverters (Sect. 7.5) they can be excited at lower photon power, exhibit higher brightness and broader variability of excitation and emission wavelengths. The water-soluble nanoparticles containing TTA active components incorporated into silica matrix were successfully applied in imaging (Liu et al. 2012). In this way, blue emission was observed with near-IR excitation that penetrates deeply into living tissues. Two-color imaging of cancer cells in vivo was achieved with simultaneously

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6 Small Luminescent Associates Based on Inorganic Atoms and Ions 3T 1

ISC 1S

1

1

0

Phosphorescence

Fluorescence

Absorption 1S

TTET

1S

Donor

0

Phosphorescen ce

3T 1

3T 1

Phosphorescen ce

1S

Fluorescence

ISC

Acceptor

Fig. 6.7 The energy level diagram of the upconversion process based on triplet − triplet annihilation (TTA) between the triplet states (3 T1 ) of the donor (sensitizer) and acceptor (annihilator). The donor is excited to its singlet excited state 1 S1 by the long-wavelength excitation, and the excitation is followed by intersystem crossing (ISC) to achieve the triplet state 3 T1 . The donor then transfers its triplet energy to the acceptor (triplet − triplet excitation energy transfer, TTET). This process is repeated to produce a large number of annihilator long-lived triplets, thus enabling the TTA process to take place efficiently. Finally, the high-energy triplet excited state of the acceptor is achieved. After ISC to singlet state it emits light in the form of fluorescence but with long lifetime as that of phosphorescence. The dotted downward arrows represent virtual emissive and nonradiative transitions

excited upconverting composites (Kwon et al. 2016). The weak point of this approach is the insufficiently weak photostability associated with the presence of oxygen that may be not only a quencher; it may generate singlet oxygen producing photochemical damage. Incorporation into nanoparticles is able to produce protective effect (Huang et al. 2019; Wang et al. 2020).

6.3 Lanthanides, Their Complexes and Conjugates The lanthanides that are also known as rare earth elements (general chemical symbol Ln) usually exist as trivalent cations and only in this oxidation state they demonstrate appreciable light emission. Strictly speaking, their emission is not a fluorescence (it does not proceed from singlet-singlet transitions) and therefore a broader term ‘luminescence’ has to be applied in this case. Only four of lanthanide ions can emit light of detectable intensity in the visible region: terbium (Tb+3 ), europium (Eu+3 ), samarium(Sm+3 ) and dysprosium (Dy+3 ), and only the first two exhibit a relatively high emission intensity (Armelao et al. 2010).

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6.3.1 Photophysics of Lanthanide Chelates The light emission of the trivalent lanthanide ions comes from the formally forbidden transitions of f electrons (inner-shell 4f -4f transitions). Their valence electrons in the 4f orbitals are shielded by the filled 5 s2 and 5p6 subshells. This results in weak interactions of the valence electrons with the environment and minimal electron-phonon coupling interactions. Because of this, the trivalent lanthanides exhibit sharp and narrow 4f–4f transition peaks and their luminescence spectra are largely unaffected by the external environment. Moreover, they can be incorporated into a wide variety of inorganic matrices, such as upconverting nanoparticles discussed in Sect. 7.5, still exhibiting their characteristic electronic properties. Since the electronic transitions in lanthanide chelates are partially forbidden, the lifetimes of their excited states are long, extending to μs-ms range, which allows the use of simple and convenient version of time-resolved detection. Because of the fact that the emission of lanthanide ions is almost insensitive to intermolecular interactions, there is no observed quenching of their long-lifetime emission on collisions with molecular oxygen (Bunzli and Piguet 2005a). The major problem with direct excitation of these ions is in their very low molar absorbance (ε ~ 1 M−1 cm−1 ). Therefore for overcoming this drawback they are incorporated into chelating complexes formed by light-absorbing organic heterocycles that play the role of antennas (Fig. 6.8). Being primarily excited, the latter transfer the excitation energy to lanthanide ions, which become the sources of a much more efficient emission than on direct excitation (Wang et al. 2006). This allows increasing their molar absorbance to the values above 10 000 M−1 cm−1 , which is sufficient for many applications in fluorescence sensing (Hagan and Zuchner 2011). Synthetic strategies for incorporation of lanthanide ions into molecular complexes are well developed. They use pre-organized macrocycles (such as cryptands and crowns), pre-disposed macrocycles (such as substituted cyclens), podands. Different

Fig. 6.8 Typical structures of complexes combining chelating and antenna functions with europium and terbium ions. For cryptates (left) and terpyridine (center), the chelate and antenna are logically the same entity. The complex may be composed of a chelating group and an appended antenna (right)

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structures formed by self-assembly process from complex heterocycles such as substituted benzimidazole pyridines (Bunzli 2006; Hagan and Zuchner 2011) can be also efficiently used. Modified calixarenes were also suggested as ligands (Oueslati et al. 2006). This allows realizing many possibilities for modification, functionalization and incorporation into desired systems. In addition, all these complexes should be able to play their major role: to minimize the vibration-induced quenching and protect the ion from interaction with the solvent. The primary role of chelating group is to provide indirect excitation of lanthanides via antenna effect. It is realized in three steps (Bunzli and Piguet 2005b). First, light is absorbed by aromatic ligands surrounding the ion, leading to its singlet excited state. Then an intersystem crossing (ISC) occurs to their triplet (long-lifetime, phosphorescent) state, from which the energy is transferred to emitting lanthanide ion. Figure 6.9 modified from (Hagan and Zuchner 2011) shows sequential steps of this process. The ligand singlet excited state may also transfer its energy, but due to short lifetime of this state, such process is not efficient. As a result, luminescence of lanthanide-chelator complexes attains long lifetimes (τ) that may extend from ~100 ns to milliseconds. Such property is their primarily attraction point that offers many advantages in sensing. These complexes allow eliminating the background fluorescence and light-scattering by time-resolved detection, or even by technically simple time-gated detection collecting the emission after microsecond time delay (see Fig. 3.11). With τ values in the μs-ms time range the

ISC

EET

Fig. 6.9 Schematic representations of excited-state transformations for a typical europium chelate. The light quanta are absorbed by aromatic ligands (antenna) and after intersystem crossing (ISC) from singlet (S1 ) to triplet (T1 ) level the triplet-state energy is transferred by excitation energy transfer (EET) mechanism to chelated lanthanide ion, which emits light via several transitions

6.3 Lanthanides, Their Complexes and Conjugates

255

lanthanide emission can be easily distinguished from that of conventional fluorescent dyes with τF ~ 1–5 ns, and this is advantageous for multiplex sensing.

6.3.2 Luminescence Spectra of Lanthanides Lanthanide chelates exhibit broad excitation spectra (owing to their organic ligands) and several very characteristic luminescence emission bands of the lanthanide ions (Pandya et al. 2006). They are composed of several discrete sharply spiked bands (less than 10 nm in width). Being excited in the near-UV, they exhibit very strong Stokes shifts. Thus, for terbium chelates with excitation at about 340 nm, a series of emission bands is observed, starting from about 500 nm and extending to longer wavelengths giving, predominantly green light emission (Fig. 6.10). Regarding chelates of europium ions, multiple peaks of their emission are located in the orange-red spectral region (Fig. 6.11). Here also, the strong spectral separation between excitation and emission bands is the second, beside the long lifetime, attractive property of lanthanide ion complexes (Pandya et al. 2006). Luminescence quantum yields of lanthanide probes can be as high as 0.1-0.4, but often they are much lower, ~0.05. Its severe quenching by water can be observed (Bunzli and Piguet 2005b). In contrast to organic dyes (in which the quenching usually occurs due to electron transfer to traps formed in bulk water) the quenching here is due to coordinating water molecules. The latter, interacting with inner coordinating sphere, dissipate energy via O–H vibrations. Thus, a special effort has been made for achieving the high emissive properties of these complexes in water (Charbonniere et al. 2007) by constructing the saturated co-ordination sphere around the

Wavelength (nm)

Wavelength (nm)

Fig. 6.10 Emission spectra for selected terbium complexes. The left part shows residual ligand fluorescence from the aza-thiaxanthone chromophore

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Eu thiaxanthone

Fig. 6.11 Absorption and total emission spectra (λex = 384 nm) for the aqua–europium complex, highlighting the short-lived ligand-based fluorescence (left) and the long-lived europium luminescence(right). The latter is observed uniquely when a 100 μs time delay is used

ion. Finally, this allowed realizing different simple and sensitive bioassays (Yuan and Wang 2006). On a general scale, severe quenching by water molecules is a weak point in application of these luminophores. This requires their shielding from water but also allows constructing different types of molecular sensors exploring this property (Armelao et al. 2010). The properties of lanthanide chelating complexes are summarized in Table 6.2.

6.3.3 Lanthanide Chelates as Labels and Reference Emitters Lanthanide coordination compounds are very efficient labels. This is not only because of their intrinsic properties, such as high brightness and many possibilities of their covalent and non-covalent attachments, but also because they do not exhibit concentration quenching and therefore can be used for loading macromolecules and nanoparticles in very high concentrations (Kokko et al. 2007). Lanthanide complexes are ideal as the reference dyes in fluorescence sensors based on two-wavelength ratiometric recording (see Sect. 7.4). Their broad excitation spectra allow exciting them by light of the same wavelength as the reporting dyes and providing stable signal that, being presented by series of sharp bands, can be well separated from that of the reporter. The response of this reference should

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Table 6.2 The properties of lanthanide ion chelating complexes Parameter

Property

Size

~1-2 nm

Absorption spectra

The broad band in the range 337-420 nm, depending on the ligand

Molar absorbance, ε

Very small (~ 1 M−1 cm−1 ) upon direct excitation. Highly increased up to ~ 10 000 M−1 cm−1 in chelating complex

Emission spectra

Series of strongly separated, narrow (~ 10 nm half-width) bands

Tunability of emission spectra

Possible only in a very limited range (several nm)

Stokes shifts

Large, 100-200 nm

Excited-state lifetime, τF

Very long, up to ~ 1 ms, normally insensitive to oxygen quenching; sensitivity may appear due to quenching of ligand emission

Photoluminescence quantum yield, Φ

Variable, from ~ 0.05 to ~ 0.4; dramatic quenching by coordinated water

Fundamental anisotropy, r 0

Zero

Photostability

Relatively high but not ideal

Chemical stability

Often low, due to low binding energy in chelating complex. Can be increased by selecting proper ligands

Availability of chemical modification/functionalization

High, depending upon the ligand

Cell toxicity

Definitive data are absent

be independent of target binding and of any external perturbation. It is easily distinguishable from reporter signal by emission wavelength or lifetime. Low dependence of emission spectra on the temperature and on the presence of quenchers makes lanthanide chelates very attractive for this purpose. Lanthanide probes are very important tools in cellular imaging that are beneficial with the use of lifetime imaging techniques (Heffern et al. 2013). The other attractive application is the time-resolved immunoassays (Wang et al. 2013). In a latter case their composites with quantum dots were suggested to be used as efficient FRET systems, in which they serve as excitation energy donors providing the property of long lifetime emission to the acceptor (Cywincki et al. 2014; Hildebrandt et al. 2014). For more detail about this mechanism and its applications see Sect. 10.3.

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6.3.4 Lanthanide-Based Heterogeneous and Homogeneous Immunoassays Formation-dissociation of lanthanide complexes result in dramatic (by orders of magnitude) change in their luminescence emission intensity and lifetime by onoff switching of the antenna effect, so the idea of application of this property in sensing is straightforward. Moreover, the chelating complexes can be both strong and weak (easily dissociating), and this fact suggests very elegant ideas in applications, particularly in immunoassays. In Dissociation-Enhanced Lanthanide Fluorometric Immunoassay (DELFIA) and related technologies (Hemmila and Laitala 2005) the non-luminescent lanthanide Eu3+ and Tb3+ chelates are used as labels for antibodies. The complexes are formed in such a way that metal ions are loosely bound within them and can dissociate into the medium. After performing immunoassay, in which the antibodies are bound by the antigens, the lanthanide ions dissociate from their ligands. On the next step, they are transferred to development solution, where they form new strongly emitting complexes with the high-affinity chelators that are present in this solution. These complexes can be easily detected and quantified (Fig. 6.12). The desired analytical signal can be obtained by time-resolved detection. This technique and its versions are very popular both in research and in clinical analysis because of high sensitivity and reproducibility. The comparative studies of several sensing technologies for detecting cAMP have shown that DELFIA has the highest sensitivity (Gabriel et al. 2003). Already covering the whole picomolar range, it can be further enhanced dramatically by assembling the lanthanide chelates into nanoparticles (Zhou et al. 2014), by application of detergent micelles and by other means. We cannot forget, meantime, that this and related assays are the techniques based on the concept of heterogeneous assays that have the disadvantage of necessary separation and reagent addition steps (see Sect. 1.1). In these assays the elimination of unreacted labeled antibodies is required before reading the luminescence signal, which implies several washing phases. Simplification of these techniques by transforming them to homogeneous assay format is a great challenge. Homogeneous assays are based on direct modulation of the label luminescence that can be performed in solutions. The antigen of interest is coupled to two antibodies, one decorated with a lanthanide label and the other one with an organic dye serving as EET acceptor. The latter emits at a wavelength distinct from the lanthanide donor, which allows generating the λ-ratiometric signal. Measurement in time-resolved mode allows one to eliminate the fast decaying luminescence signals coming from the background and detect excitation of organic conjugate that is not bound to the antigen (Bünzli 2010).

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Fig. 6.12 Principle of heterogeneous luminescent immunoassay realized with specific antibodies (Ab). Step 1 is the fixation of the analyte; Step 2 is the immunochemical reaction with the labeled antibody of LnIII followed by complexation and insertion into micelles (step 3). From (Bünzli 2010)

6.3.5 Switchable Lanthanide Chelates in Sensing For producing direct sensing, one can choose among many possibilities to incorporate recognizing and transducing units (Fig. 6.13). Because the primary light absorption sites in lanthanide complexes are the chelating aromatic antenna groups (sensitizers) and emission properties are determined by lanthanide ion, there are basically two application sites for inducing the changes in emission intensity and lifetime that could be used to produce sensing response (Pandya et al. 2006): (a) Perturbation of sensitizers. The reporting signal can be generated by the influence on two (singlet and triplet) excited states of sensitizer groups. Photoinduced electron transfer (PET) can be used as a common mechanism of excited-state quenching, in which an electron is transferred from an electron donor moiety to an acceptor site (see Sect. 4.1). With lanthanide complexes demonstrating PET reaction, the electron donor group can be covalently attached to chelating heterocycle, so that coupling-decoupling of this quencher can provide the response in intensity of emission (Terai et al. 2006).

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a

EMISSION

Ln3+ b

NO EMISSION

T

NO EMISSION

Ln3+

Ln3+ EMISSION

T

Ln3+

Fig. 6.13 Illustrations of the mechanisms in the background of modulation of lanthanide emission on a reversible binding of the target (T): a Influence of tatget on the excited-state transitions of antenna via PET mechanism leading to suppression of energy transfer to lanthanide ion. b Direct influence on luminescence of lanthanide by screening the access of lanthanide ion to water molecules

(b) Perturbation of the lanthanide ion. This modulation of emission can be provided via coupling/decoupling with the sensor operation that modulates the access of La ion to water molecules, in view that they are extremely potent quenchers (Bunzli and Piguet 2005b). When these quenching water molecules are displaced, for instance, by reversible binding of anion, high emission intensity is restored with the correspondent increase of lifetime. Better protection from interaction with water can be achieved on incorporation of the whole complex into detergent micelles and into different nanostructures. This offers new possibilities to impose the sensor response. There are many molecular constructions reported on functionalization of Ln chelates for different sensing and imaging purposes (Wang et al. 2014; Zhang et al. 2018). The examples presented below (Fig. 6.14) can be the illustrations of that. In the first case (Thibon and Pierre 2009) the smart potassium sensor is made in such a way that antenna and Tb3+ site are separated by azacrown ether. In the absence of K+ ion, this chelate displayed weak luminescence because of low EET efficiency due to a long distance between the Tb3+ site and antenna. Binding of K+ ion by the azacrown ether favored a cation—π interaction with the aryl ether, leading to a close proximity between the Tb3+ site and the antenna. This results in significant luminescence enhancement. The other example represents a similar construction with a difference that the K+ binding provides perturbation of electronic energy levels of antenna (Weitz and Pierre 2011). As a result, the energy exciting the Eu3+ site

6.3 Lanthanides, Their Complexes and Conjugates

a

K+

261

b

EET

K+

ICT

EET

Fig. 6.14 Examples of application of smart lanthanide-based fluorescence probes for ion sensing. a The Ln site and antenna are separated by azacrown ether coordinating potassium ion. On K+ binding the conformational change occurs so that antenna group approaches closer the Ln site enhancing EET and thus increasing the Ln emission providing the sensor response. b On K+ binding the charge perturbation of electronic state of antenna occurs, so that the luminescence excitation spectrum changes generating the ratiometric response

can be changed by modulation of excitation wavelength. In this way, the ratiometric response can be obtained on ion binding. The lanthanide complexes with ionizable groups incorporated into antenna may respond to the change of pH not only by variation of emission intensity but also by redistribution of intensity between emission bands. This opens the pathway for constructing the probes that are valuable for luminescent ratiometric pH imaging (Shaner et al. 2004; Woods and Sherry 2003). In fact, any perturbation of the ligand by inducing the excited-state reaction can be explored. An example of successful application of excited-state intramolecular proto transfer (ESIPT) reaction (see Sect. 4.1.5) was reported (Sun et al. 2018). The ESIPT ligand that served as both the host and antenna for protecting and sensitizing the luminescence of Ln3+ ions was appended. Influencing on ESIPT reaction, the responses to solvents, anions, and temperature were shown. One of the advantages of this approach that will be further developed is the ability to transfer the response based on photoreactions in organic dyes and occurring on the time scale of fluorescence to time domain that is much longer (ms) and much more convenient for detection. Thus, by choosing luminescent lanthanide complexes, one may get different benefits. They demonstrate series of strongly Stokes-shifted narrow emission bands and very long luminescence lifetimes. Both these factors strongly facilitate recording of output signal and, especially, its discrimination from background emission. In addition to lifetime imaging, the lanthanide chelating units can be efficiently used as EET donors, even in combination with those acceptors that absorb light at the wavelengths of their own excitation, such as semiconductor quantum dots. Being coupled to molecules or nanoparticles with bright but short-living fluorescence, they can provide to these acceptors the property of long-lifetime emission (this issue will be discussed in Sect. 10.3). The time-resolved detection in this case allows achieving important benefits: not only the light-scattering and background emission are suppressed in such sensors. The short-living emission of directly excited acceptor does not contribute to detected signal. Chemical sensing, biosensing and immunosensing technologies can efficiently use these properties.

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6.4 Sensing and Thinking. Three Roads to Most Efficient Use of Smallest Metal-Based Emitters Three (at least) roads drive us to the most efficient use of lanthanide ions, phosphorescent complexes of rare and noble metals and clusters of several atoms. The photophysical mechanisms that provide their emission are different, and this fact determines diversity of their properties. But what is common is the necessity of stabilizing them and providing their unique properties by organic ligands. Because of that, only in complexes with these ligands the smallest metal materials must be considered as luminescent units. And since the organic surrounding is responsible to a large part for luminescence properties, it must provide the necessary efficiency for their functionalization and incorporation into sensing technologies. Anyhow, the studies of unique photophysical properties of these materials deserve to be continued. The research community involved in development of sensing and imaging technologies must benefit from this knowledge. What is essential is the fact that we possess alternative fluorophores of sub-nanometer dimensions other than traditional organic molecules. They bring new properties and offer new possibilities. The reader is asked to respond to several questions in order to check the knowledge acquired reading this Chapter: 1. 2.

3. 4. 5.

6. 7.

8.

9.

Explain how the change in size of nanoscale particles of noble metals results in change of fundamental mechanism in their interaction with light. Why scaffolds are so important for efficient few-atom noble metal clusters? What are the possibilities for their constructions using small-molecular and macromolecular units? Based on what properties of few-atom clusters their emission can be considered as being of LMCT type? If the silver cluster is stabilized by organic dyes, what emission you expect to observe? Of the dye? Of the cluster? Something different? Compare the properties of lanthanide chelates and that of complexes of transition metals. Both can generate the long lifetime emission. How do they respond to temperature and oxygen? Which of the discussed above materials generate polarized emission? How it can be used? Explain why the luminescence spectra of complexes generated by transition metals are of smooth shape and that of lanthanides are presented by series of very sharp bands? Why lanthanide ions need to be incorporated into coordination complexes? Suggest different structures of coordination shells and possibilities to induce the sensing functionality by operating with the properties of these shells. Explain how DELFIA assay works. What features did allow it to achieve superior sensitivity in comparison with competing technologies?

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10. Suggest the sensor design in which a conjugate between lanthanide complex and a dye performing the excited-state electron-transfer, charge-transfer or protontransfer reaction (check Sect. 4.1). How such sensors will work? What will be the benefits?

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Chapter 7

Fluorescent Inorganic Particles in Nanoscale World

This Chapter starts with introduction the reader to nano-world, the world of unique possibilities for sensing and imaging technologies. Here we discuss general properties of nanoparticles regarding their size, surface energy, interactions, and peculiarities of excitation energy transfer with and between them. Semiconductor quantum dots present a range of bright emitters covering whole visible and near-IR spectral range. Among nanocrystals are those that are able to display long persistent glowing, possessing trapped electronic states. Lead halide perovskite nanocrystals are the rising stars as light emitters with a great future. Finally, the upconverting nanocrystals make possible multicolor visible emission with near-IR excitation. The advantages of nanoscale emitters over fluorescent molecules are discussed in concluding section “Sensing and thinking” with a number of questions addressed to readers. One of the most important developments of recent time is the introduction of “nano” in all our life. The sensing and imaging technologies have got many benefits from that and many stimuli for further developments. This Chapter starts with the introduction to fantastic and appealing nano-world. General properties of nanomaterials that derive from their size, shape, crystallinity and order will be primarily overviewed. Interactions with and between nanoparticles and their effects in excitation energy transfer will also be discussed. The nanoscale materials satisfy successfully many requests in clinical diagnostics, monitoring the environment, agriculture and food safety control, detection of biological warfare agents and in different industries. New technologies are observed to appear together with the appearance of many new products. Many technological solutions can be found in the design of light-emissive nanoparticles. In the cases when small size of reporter unit is not important and direct response to target binding event is not requested or may be provided by the design of complex hybrid structures, the fluorescent nanoparticles have a great advantage. They can be much brighter and less susceptible to chemical damage and photodamage than individual luminophores. Assembling luminophores within the same small-scale structures we can modulate in broad ranges their relative contributions and in this way to provide

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their improvement and extend versatility. Extended surface area allows more possibilities for modifications and functionalization. These properties are achieved by different means. Essentially, the novel nanoscale materials can demonstrate very unusual photophysics, the understanding of which offers new benefits. In this Chapter the nanostructures formed of inorganic materials will be overviewed. Semiconductor nanocrystals (quantum dots, QDs) with their unique size-dependent optical properties have established their important role being used as themselves and also as the donors and acceptors in energy transfer techniques. Among nanocrystals are those that are able to display long persistent glowing, possessing trapped electronic states. Lead halide perovskite nanocrystals are the rising stars as light emitters with a great future. Finally, the upconverting nanocrystals demonstrate unique and diverse photophysical behavior. Here we overview the most important features of these nanoscale materials. The following chapters will be focused on nanoparticles made of organic materials, and diverse functional nanocomposites that allow extending the useful properties of these basic components.

7.1 Introduction to Light-Emitting Nano-world Nano-world is not only the world of small dimensions. It is the world, in which different materials exhibit new and often unexpected optical properties. Inspired by a broad range of new possibilities, many scientists have started exploring this new field (Hötzer et al. 2012; Liu and Tang 2010). For a short time these novel materials have started substituting traditionally used organic dyes in numerous practical applications, such as clinical diagnostics (Andreescu and Sadik 2004), monitoring the environment (Riu et al. 2006), agriculture and food safety control (Patel 2002), detection of biological warfare agents (Gooding 2006) and in different industries. Such versatility is further enriched by nano-bio interactions that allow incorporation of nanoparticles into biologically important systems, obtaining information on these systems and even modulating their properties (Amin et al. 2011; Suh et al. 2009; Wang 2009). The location of fluorescent nanoparticles can be precisely determined and monitored in the systems of high complexity, such as living cells and tissues.

7.1.1 Size, Shape and Dimensions of Nanomaterials Molecular clusters and nanoparticles may exist of various sizes, from a fraction of nanometer to several hundred nanometers. Many properties of nanomaterials are size-dependent, which allows selecting them within broad range of species without any change of their chemical composition, and even of the arrangement of atoms. With the decrease of particle size there increases the ratio of surface free energy over the free energy in the bulk of material. This means that the energetic, structural and, finally, optical properties of nanomaterials become dependent on interactions at their

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interfaces. In small nanoparticles, most of the atoms are exposed to the surface and participate in interactions with the surrounding. Thus, if we model a nanoparticle by a structure, in which the atoms are represented by identical balls, the minimum number of atoms to contain at least one inner-core atom is 13. The close-shell structure of 55 atoms will have 76% of them exposed to the surface and of 147 atoms—still 63% (Aiken Iii and Finke 1999). The interactions at interface are usually of different nature than the interactions within the particle core and this may strongly influence the electronic states and transitions of nanoparticles. In addition to this general statement, we indicate specific effects that depend on studied materials. We observe (see below) that excitonic effects in semiconductor quantum dots result in strong size-dependent variation in the wavelengths of their emission. Size-dependent plasmonic effects of noble metal nanoparticles do not allow their strong emission, but they induce emission enhancement of other fluorophores (see Chap. 13). These effects appear due to confinement of their electrons to a small volume. The size of nanostructures is usually much smaller than the wavelength of light, and this point is important for understanding a variety of photophysical and photochemical processes occurring on nanoscale. The electron and proton transfers are the short-range phenomena and if they are activated in the excited states of dielectric materials, they proceed on a scale below 1 nm. In contrast, excitation energy transfer (EET) and plasmonic enhancement of fluorescence may extend to distances of tens nanometers. Excitation by visible light with wavelengths of several hundred nanometers does not bring nanoscale resolution in conventional microscopy but with proper design of nanoscale emitters allows estimating proximity effects on the shortest length scale. Nanostructures can be of different shape, which can determine their optical properties. The nanoparticles that can be viewed as point emitters are defined as being of zero dimension, 0-D (Fig. 7.1). Being of spherical (or almost spherical) shape,

Zero dimensional (0-D)

One dimensional (1-D)

Point emitters (nanodots)

Two dimensional (2-D)

Anisotropic emitters (nanorods)

Three-dimensional (3-D) Emitting surfaces (nanosheets)

Fig. 7.1 The nanoscale structures of different dimensionality

Emitting nanocrystalline materials

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semiconductor nanocrystals are usually considered of zero dimensionality, which gave them the name “quantum dots”. Similarly, other nanoscale structures without strong asymmetry are often considered as “dots” (Burda et al. 2005). The ultrathin nanowires would be the 1-D analogs of these nanomaterials (Greco et al. 2008) and can be called ‘nanorods’. The examples of 2-D nanostructures are the graphene molecular sheets of atomic thickness (Park and Ruoff 2009). In addition to size, the shape of nanoparticles may influence dramatically their optical properties. The assembled nanocomposites may contain nanoparticles or clusters of different dimensionalities (Srivastava and Kotov 2009). Precise 3D self-assembly is possible with nanocomposites (Wang et al. 2006). Nanoporous solid materials may possess optical properties similar to that of nanoparticles and a fractal dimension can be attributed to them. A bright example is porous silicon (Torres-Costa and Martín-Palma 2010), which is the material with high surface to volume ratio (as large as 500 m2 cm−3 ). Its emission properties are similar to that of nanocrystalline particles made of the same material, and these nanoparticles follow the same regularity as other quantum dots—the strong size-dependence of emission spectrum (Hessel et al. 2011).

7.1.2 Variations in Nanoparticles Composition, Crystallinity and Order An intrinsic polydispersity in size and shape is frequently observed in nanoscale particles, which is connected with the ways of their synthesis. This is in contrast to dye molecules that are definite chemical entities (sometimes a mixture of several isomers). Quantum dots can be separated into fractions with narrow size distribution, but the number of their atoms is commonly not well determined (Alivisatos et al. 2005). The electronic effects govern the size-dependent switching from plasmonic noble metal nanoparticles to strongly fluorescent few-atom clusters (see Sect. 6.1). Some structures of nanomeric size can be obtained in a way that their structural uniformity can be achieved with atomic precision. They are called the “magic-size” nanoparticles, the characteristic representatives of which are the fullerenes. Such ball-shaped structures of several tens of atoms can be made of different materials. For fluorescence sensing and imaging applications the most interesting are the magicsize semiconductor quantum dots, such as (CdSe)(33) and (CdSe)(34) (Kasuya et al. 2004), the light absorption and emission properties of which are quite different from that of “conventional” nanocrystals. Any nanoparticle will be stable only if its core is stabilized by interactions between the constituents (Hoang 2007, 2012). These interactions can be of different origin (covalent, ionic, van der Waals, etc.). The strength of intra-particle bonding should be sufficient to withstand the variations of external conditions in the media of their functioning. That is why the micelles made of detergent molecules and also phospholipid vesicles being of nanoscale dimension are not considered as nanoparticles. Essentially, for generating the light-emitting properties, the nanoparticles should allow

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observing collective electronic effects resulting in formation of discrete ground and excited-state energy levels. Whereas molecules of organic dyes possess the wellspecified electronic energy levels as their basic properties, in a variety of novel nanomaterials the discrete energy levels appear as the sole effect of their small particle size. Crystals are the best atomically defined structures with relatively well-understood interactions. These interactions can be of various types: ionic, metallic, covalent, van der Waals and H-bonding. What unifies crystalline structures is the presence of longrange order based on translational periodicity of a unit crystal cell. In many important cases, the process of crystallization can be stopped on a level of tens or hundreds of atoms. Being of nanoscale dimensions, even with intrinsically perfect order, these particles usually lack monodispersity but can be presented as narrow size fractions. Translating crystal order to their surfaces, they form the “sharp edges”. Meantime, we always observe that nanocrystals cannot be considered as small pieces of bulk material, so that unusual forms of atomic disorder can be observed together with structural stiffening (Gilbert et al. 2004) and the deformation of crystal order is mostly typical for surface-exposed atoms (Jadzinsky et al. 2007). In contrast, formation of nanoparticles from soft materials results in amorphous structures, the positions of every atom in which are not defined. Their conformation is usually less stable, but they can form smooth surfaces that maximize intrinsic interactions and minimize interactions at the surfaces. The degree of their order on atomic level is variable and many of them show micelle-like behavior. Their interface energy is lower than that in nanocrystals (Navrotsky 2003).

7.1.3 Interactions at Nanoscale Surfaces For increasing the solubility of nanoparticles and their stability against decomposition or growth, their surface should often be covered with capping agents (Bagwe et al. 2006). Oligo(ethylene glycol) has proved to be such agent that induces the nanoparticle solubility in polar media without specific interaction with different types of molecules of biological origin (Herrwerth et al. 2003). Polyethylene glycol coating is friendly to cells and tissues, for instance, it prevents hemolysis produced in vivo by silica nanoparticles (Lin and Haynes 2010). Ligand exchange resulting in formation of polar surfaces is of broad utility for hydrophobic nanoparticles that allows producing their stable suspensions in aqueous media (Dubois et al. 2007; Wawrzynczyk et al. 2012). For all types of nanoparticles it can be observed that not only the stability but also the photophysical properties are strongly ligand-dependent. Core-shell structures are typical for semiconductor quantum dots (Alivisatos et al. 2005). Such composition can resolve many problems: reducing the negative effects of sharp edges of crystal cores, increasing the solubility, allowing the incorporation of functional groups for further modifications and introducing new functionalities. Their outer layer is needed

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for bioconjugation. Different techniques for providing polymeric covers were developed for that (Tomczak et al. 2009). Designing nanoscale inorganic-organic interfaces is highlighted in research papers and reviews (Balazs et al. 2006; Tomczak et al. 2009; Wang et al. 2007). Techniques of covalent attachment of organic dyes and other molecules modifying the nanoparticle surface are well developed (Hermanson 1995). Novel aspects involve application of click chemistry (Li and Binder 2011), which becomes a universal method to link reaction partners with high efficiency, solvent insensitivity and at moderate reaction conditions.

7.1.4 Excitation Energy Transfer with and between Nanoparticles The long-range interactions leading to excitation energy transfer (EET) are not limited to fluorescence and are characteristic of any emissive excited state, whatever is its origin. All the long-range phenomena (that do not require electronic coupling) can be adequately described by Förster-type inductive dipole resonance mechanism often abbreviated as FRET (see Sect. 4.3). In their description we use the term ‘dye’ in its general sense, which includes organic dye molecules and different kind of molecular complexes and nanoparticles. Definitely, there are peculiarities of energy transfer with and between the particles, the size of which is comparable with the Förster radius R0 (see Sect. 4.3). The particles of such size cannot be approximated by point dipoles and therefore evaluation of distance between them is not very definitive. Involvement of dipole orientation factor, κ2 , may also raze confusions. Moreover, the distance dependence of energy transfer may be different from that described by 1/R6 function. This is because of different mechanism of this interaction. In inductive-resonance mechanism of FRET developed by Förster, the two interacting point dipoles are considered, whereas in the case of conducting particles the donor dipole interacts with a ‘Fermi gas’ of nearly free conduction electrons. Based on such modeling it was suggested that on interaction of small organic dye and large conducting metal nanoparticle the 1/R4 function should be observed. This suggestion was confirmed in experiment (Yun et al. 2005), in which the gold nanoparticle was appended to the 5 end of one DNA strand as the energy acceptor and a fluorescein dye to the 5 end of the complementary strand as the energy donor. Analysis of the energy transfer as a function of DNA lengths (15, 20, 30, 60 base pairs) allowed testing the distance dependence in the range 5–25 nm. The result showed conformance to 1/R4 function. This is one of the results showing that for signal transduction in fluorescence sensing the application of nanoparticles may allow the extension of transduction to longer distances, up to 15–20 nm. An extended distance dependence of FRET between nanoparticles is also suggested based on quantum chemical calculations (Wong et al. 2004). The authors come to conclusion that the FRET rate at large separations exhibits several features

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not anticipated in the conventional theory: The actual rate shows much weaker shortrange distance dependence, in this version of theory closer to 1/R2 in contrast to 1/R6 value. The Förster expression overestimates the energy transfer rate by more than two orders of magnitude at short separation (300 mg/ml), so the effects of nonspecific absorption in these media must be very strong (Park and Hamad-Schifferli 2010).

7.2 Semiconductor Quantum Dots Quantum dots (QDs) is the popular name for semiconductor nanocrystals with unique ability of efficient absorption and emission of light in the visible range of spectra. They are emerging as a new class of fluorescent reporters with the properties and applications that are not available with traditional organic dyes or nanocomposite

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structures doped with them (Costa-Fernandez et al. 2006; Grecco et al. 2004; Sapsford et al. 2006). They demonstrate very high brightness, narrow emission bands and possibility of modulating the color of their emission just by the change of particle size within the same type of material. Their novel features have opened new possibilities in many areas of research and development including ultrasensitive chemical analysis and cellular imaging (Jin and Hildebrandt 2012). They have started to replace traditional organic fluorophores to serve as simple fluorescent reporters in immunoassays, microarrays, fluorescent imaging applications, and other assay platforms. As a part of nanocomposites, they are in the heart of diverse technologies, from barcoding systems in suspension arrays and multiplexed identification of infectious pathogens to tracking of intracellular drug and gene delivery (Chen et al. 2012; Zhang and Wang 2012).

7.2.1 The Composition of Quantum Dots Fluorescent quantum dots can be made of many semiconductor materials (CdS, CdSe, CdTe, ZnS, PbS), and their spectral properties depend on their composition. Within every type of these materials the dependence of spectral properties on particle size can be very strong (see below). These particles can be as small as 2–10 nm, corresponding to 10–50 atoms in diameter and in total 100–100,000 atoms within the QD volume. This is roughly the size of a typical globular protein. It has to be accounted, however, that for improving the brightness, solubility and functionality, additional layers are imposed on particle surface, which provides further increase in size (Sapsford et al. 2006). Many quantum dots are the composites made of narrow dispersed highly crystalline CdSe core coated with a shell of a few atomic layers of a material with a larger band gap, such as ZnS and CdS, on top of the nanocrystal core (Fig. 7.2). Fig. 7.2 Schematic of the overall structure of a quantum dot nanocrystal conjugate. The layers represent the distinct structural elements

Func onal unit Coa ng Shell

Core nanocrystal

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Such constructions are beneficial because the surface defects in the crystal structure act as temporary “traps” for the electron or hole, providing their nonradiative recombination and thus reducing the quantum yield. The shell also protects surface atoms from oxidation and other chemical reactions. With the proper shell design, it becomes possible to obtain photoluminescence quantum yields close to 90% and to increase photostability by several orders of magnitude relative to conventional dyes.

7.2.2 The Origin of Emission of Semiconductor Nanomaterials. The Loosely Bound Excitons The photophysical behavior of these nano-sized semiconductor particles is quite different from that of fluorescence emitters of organic origin. In previous chapters we treated light emission from aromatic molecules as fluorescence and, in special cases as phosphorescence. Because luminescence from crystalline semiconductor materials has a completely different origin than that of fluorescence, it is worthwhile to use for its description “luminescence” as the general term. To understand it, we need some knowledge of the physics of semiconductors. In contrast to common metals, the electrons in semiconductors in the ground state are not free to move and they occupy the lowest energy valence band (Fig. 7.3). Their energy is insufficient to overcome the band gap to high-energy conduction band that could allow their free motion. If by electronic excitation a sufficient energy for overcoming the band gap is provided, the electrons will be excited from the valence band to the conduction band leaving its location in the valence band that becomes

Valence band

eEmission

Band gap

e-

Excitation

Conduction band

Exciton (electron-hole pair)

Electron-hole recombination

h+ h+ h+ e

Relaxation

Fig. 7.3 Illustration of photophysical processes occurring on electronic excitation of semiconductor materials. Increase of electron energy drives it to conduction band overcoming the band gap. This creates an exciton, which is the electron pair with the positively charged hole that is made at the site initially occupied by electron. The exciton can move, relax and release energy in the form of light emission

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a positively charged “hole”. The hole originates from atoms that become positively charged. The holes do not actually move, but a neighboring electron can move to fill the hole, leaving a hole at the place that the electron occupied previously. In this way, holes can also move freely in the crystal lattice. The mobile electron-hole pair forms the quasi-particle exciton. In the conduction band the excitons are weakly bound and they can move in the crystal lattice relatively freely; they can provide conduction in the presence of an electric field. The term “exciton” was first introduced to explain the behavior of molecular crystals (Davydov 1971) and aggregates of organic dyes (Kasha 1976). In those cases, they are the strongly bound Frenkel excitons. Possessing high electron-hole affinity, they are typical for the complexes of organic molecules and conjugated polymers (Bardeen 2014); they will be discussed in Chap. 8. If these excitons are formed in dye aggregates, the electrons remain localized on molecular sites, while the excitation is coherently delocalized over many fluorophores (Scholes and Rumbles 2006). In contrast, in the case of loosely bound excitons (the Wannier-Mott excitons) the light absorption results in transition to the conduction band, which is a continuum of states where such motions are easy. This generates the motive excitons, in which the electrons and holes are attracted by Coulomb interactions, but because of dielectric screening this attraction is weak allowing these motions. The luminescence emission is coupled with the loss of energy released on electron-hole recombination. When the particle size becomes smaller than the characteristic size of free exciton motion (which is the case of QDs), the quantized energy levels appear because the wave function of the exciton becomes confined in all three spatial dimensions (Rabouw and de Mello Donega 2017). Such quantum confinement effect determines the optical properties (Takagahara and Takeda 1992). Here also the emission is the result of the loss of energy released on electron-hole recombination, but the positions of absorption and emission bands become determined by the nanoparticle sizes. The smaller they are, the fewer energy levels can be occupied by the electrons and the greater is the distance between their individual levels (Fig. 7.4). Therefore, the decrease in particle size results in blue shifts of their emission (Medintz et al. 2005; Sapsford et al. 2006).

7.2.3 The Spectroscopic Properties of Quantum Dots The properties of semiconductor quantum dots are summarized in Table 7.1. Comparison with similar data presented for organic dyes in Table 5.1 shows that the QDs features are really unique. Their absorption spectra are extremely broad, extending from long-wavelength band far into UV region with a gradual increase of absorbance, whereas their fluorescence spectra are amazingly narrow (Fig. 7.5). This property allows exciting fluorescence at any shorter wavelengths that are far aside from emission band and thus avoid the light-scattering problems. This also suggests their efficient use as EET donors to various acceptors with the possibility of selecting the wavelength, at which the excitation of the acceptor does not occur.

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Increase of emission wavelength Fig. 7.4 Schematic representation of the quantum confinement effects (Rabouw and de Mello Donega 2017). The bandgap of the semiconductor nanocrystal increases with decreasing size, shifting the emission wavelength to longer wavelengths. The discrete energy levels arise at the band-edges. The energy separation between the band-edge levels also increases with decreasing size Table 7.1 The properties of semiconductor quantum dots used in sensing and imaging technologies Parameter

Property

Size

~2–10 nm

Absorption spectra

Extremely broad, extending from the first long-wavelength band continuously to the UV

Molar (particle) absorbance, ε

Very high, up to 107 M−1 cm−1

Emission spectra

Narrow (~25 to 30 nm half-width) and symmetric, without red-side tailing

Tunability of emission spectra

Size-dependent, available in a broad range from UV to IR

Stokes shifts

Small, ~5 nm from absorption onset

Excited-state lifetime, τF

Usually longer than that of organic dyes, ~10 to 20 ns

Photoluminescence quantum yield, 

Variable, strongly depends on surface coating, can be ~90%

Photostability

Excellent

Chemical resistance

Excellent

Availability of chemical modification/functionalization

Rather poor

Cell toxicity

Disputable, insufficiently studied

Two-photonic cross-section, α

High, ~10 000 GM

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Fig. 7.5 Absorption and emission of six CdSe quantum dot dispersions differing in size (Medintz et al. 2005). The black line shows the absorption of the 510-nm emitting QDs. Below: photo demonstrating the size-tunable fluorescence properties and spectral range of the six QD dispersions possessing these spectra versus CdSe core size. All samples were excited at 365 nm with a UV source. For the 610-nm-emitting QDs, this translates into a Stokes shift of ~250 nm

Attractive is also the possibility to use for excitation the low-cost UV lamp with broad-band filter or a cheap 405 nm diode laser. With a single light source, the QDs emitting at different wavelengths throughout the entire visible range of spectrum can be excited. This allows multicolour detection of several targets. The emission spectrum can be engineered by controlling the QD geometrical size, shape, and the strength of the confinement potential. Depending on the size, the coreshell CdSe–ZnS particles can change their emission wavelength from 480 nm (2 nm in diameter) to 660 nm (with diameter 8 nm). Correspondently, for the redder emitting CdTe–CdSe QDs the variation of emission wavelengths is from 650 nm (4 nm diameter) to 850 nm (diameter 8 nm) (Medintz et al. 2005). The shift in emission spectra is accompanied by corresponding shift of absorption band origin, so that the relatively small Stokes shift remains approximately constant. The size-dependent fluorescence wavelength tunability allows obtaining the species with different emission colors. Because the electronic transitions are not coupled with atomic vibrations (in contrast to organic dyes), the emission spectra of quantum dots are symmetrical and with the width of 20–30 nm they are narrow, much narrower than that of organic

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dyes. This property together with the wavelength tunability is attractive for multiple labeling and can be realized in multiplex formats. Quantum dots exhibit excellent brightness due to high molar absorbance (reaching the values of several millions) and high quantum yields, typically of 20–70%, depending on surface coating. Such brightness cannot be easily achieved with organic dyes even with their high-density coupling with synthetic polymers and proteins or their sorption on nanoparticles. This is because of concentration-quenching effects observed in common dyes (as discussed in Sect. 8.1). In QDs these effects are absent. Excellent chemical stability and photostability are also their characteristic features. Regarding fluorescence at two-photon excitation, QDs are dramatically superior to organic dyes (Rayno and Yildiz 2007), which makes them very prospective for cellular imaging or even imaging of the whole tissues using near-infrared excitation. Meantime, their cytotoxicity is not studied in sufficient detail (Shiohara et al. 2004), it will definitely depend upon the properties of covering shells. The negative feature of QDs is their blinking (the rapid switching between their emissive and non-emissive states), which reduces their applicability in some of their applications.

7.2.4 Stabilization and Functionalization of Quantum Dots Naked QDs with their hydrophobic surfaces are insoluble in water and in other highly polar solvents. Their surface is not amenable for easy conjugation with functional groups needed for molecular sensing. Therefore, many efforts are being made for modifying this surface for making it friendlier to synthetic chemist (Dubois et al. 2007). There are several methods currently applied for QDs stabilization and functionalization (Sapsford et al. 2006): (a) The use of surface covalent modification chemistry to generate –NH2 , −SH and –COOH groups on the particle surface that could be further modified to provide attachment of target recognition molecules. This method is the most commonly used for attachment of different recognition units, such as DNA or antibodies. (b) The use of coordination chemistry that may allow with the exposed ZnS shell the interaction of Zn atom with polyhistidine and of sulfur atoms with thiols. For this purpose, the peptides containing His and Cys residues can be used. They can be further modified by attaching molecules with specific function. (c) Finally, the electrostatic interactions with oppositely charged polymer molecules can form the surface layer around the particle. Alternatively, the amphiphilic polymers can be used, which will screen hydrophobic QD surface and expose polar groups to the solvent. A simple method for the preparation of CdSe-ZnS quantum dots that are highly fluorescent and stable in aqueous solution was reported using calix[4]arene carboxylic acids as the surface coating agents (Jin et al. 2006).

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Fig. 7.6 Methods of conjugation of biomolecules to quantum dots (Goicoechea et al. 2009). a The thiol bound formation between S atoms on the QD surface and S atoms in molecules terminated with carboxylic acid, amine, or hydroxyl groups. b The formation of a complex between a Zn atom on the QD surface and polyhistidine residues by metal-affinity coordination. c The utilization of a silica overcoat which makes possible the utilization of conventional silanization techniques to functionalize the QDs. d The utilization of native TOPO ligands to functionalize the QDs using amphiphilic molecules. e The functionalization of QDs using the streptavidin-biotin affinity. f Electrostatic interactions between QDs surfaces and oppositely charged biomolecules

These principles are explored in various types of bioconjugation of quantum dots that are necessary for their efficient use in biosensor technologies (Fig. 7.6). They include designed biomolecules, particularly, peptides. Their use for coating the QDs can combine all these approaches since it allows realizing all types of interactions with the core particles plus using exposed reactive groups for additional functionalization (Iyer et al. 2006). Peptides have the advantage of being easily customized and can yield all necessary functions: (a) Protect the core/shell structure and maintain the original QD photophysics, (b) solubilize QDs, (c) provide a biological interface, and (d) allow the incorporation of multiple functions (Sapsford et al. 2006). A number of self-assembled QD-peptide conjugates were suggested for selective intracellular delivery (Delehanty et al. 2006). Formation of lipid monolayer as capping functionality can also be made for intracellular imaging and intracellular detection of nucleic acids (Dubertret et al. 2002). Thus, the aim to get the QD particles coated in such a way that would allow application of common organic chemistry to make these particles functional is achievable and can be reached in different ways. A relatively large size of quantum dots is commonly not a problem in application in different sensing technologies. Each QD particle may carry several recognition

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units represented by DNA or proteins (Medintz et al. 2004). They can be integrated into microfluidic cells and other integrated devices (Sapsford et al. 2004).

7.2.5 Applications of Quantum Dots in Sensing On the first steps of their application, the QD conjugates have started to be used as simple replacements for conventional dye labels in the cases when their unique performance characteristics allow achieving better results. Their high brightness helps lowering the limit of detection when they are used as sensor reporters. The large surface areas allow simultaneous conjugation of a number of biomolecules to a single QD particle. Advantages conferred by this approach are obvious. One may achieve increased avidity for targets due to multipoint binding, the potential for obtaining cooperative binding and the possibility of simultaneous recognition of several targets. The development of multi-color multi-analyte assays with a simple single-wavelength excitation based on QDs with essentially the same composition was realized. A number of “multiplexed” assays based on this principle were suggested for DNA and RNA hybridization (Liang et al. 2005; Shepard 2006). Such traditional trend can be seen in technologies that involve detection by antibodies. The streptavidin-coated QDs were used by their incorporation into a generic three-layer sandwich (see Figs. 2.4 and 2.5). approach using (a) an antibody against a specific target, (b) a biotinylated secondary antibody, and (c) a streptavidin-coated QD labelling this secondary antibody (Goldman et al. 2006). The same approach can be used with the ligand-binding proteins, etc. This type of application is quite similar to that of ‘nonresponsive’ dyes (see Sect. 5.2). The fluorescent particles play a passive role, in which their presence in the system is detected only. This cannot satisfy the researchers that try to find possibilities to make QDs directly sensitive to intermolecular interactions. The problem here is an insignificant knowledge of QD properties that are not studied in such detail as that of organic dyes. Still little is known on the variation of different parameters of their emission in response to different kind of intermolecular interactions that can be realized in view of various possibilities of forming their surface shells. Meantime, some fragmental research in this direction shows promise. Thus, it was reported (Algar and Krull 2007) that the spectroscopic properties of CdSe/ZnS core-shell QDs change dramatically in a pH-dependent manner when linear thioalkyl acids of variable chain lengths are used as capping ligands. The variation of luminescence of thioalkyl acid capped QDs is a complex function of dielectric constant and electrostatic or hole-acceptor interactions with ionized ligands. These effects have to be further explored (Debruyne et al. 2015). Sensing the target binding and the response in the change of QD emission intensity can be connected by photoinduced electron transfer (PET) mechanism (see Sect. 4.1). They mostly serve as electron donors to different acceptors generating analytical signal (Silvi and Credi 2015). The sensor for fatty acids was suggested as a proof-of-principle for this approach (Aryal and Benson 2006). The electron donor

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is a ruthenium complex, and the electron transfer occurs through fatty acid binding protein serving as recognition unit, so the binding results in quenching. Attachment to CdSe–ZnS core-shell QDs of strongly electron-donor indolyl group resulted in dramatic quenching of their fluorescence (Raymo and Tomasulo 2006). Via some conformational change this effect can be coupled with the target binding by the proper recognition unit. One of the promising possibilities for inducing in this way the QD reporting function has been suggested (Yiidiz et al. 2006). It is based on the electrostatic adsorption of cationic quenchers on the surface of anionic quantum dots. The adsorbed quenchers suppress efficiently the emission of QDs due to the presence of photoinduced electron transfer. Based on this mechanism a reporting signal could be generated indicating receptor-target interactions. The QD emission is restored in the presence of target receptors able to bind the quenchers and prevent the electron transfer. Quantum dots have started to be actively used in sensing technologies based on EET to or from external agent. The simplest approach here is to provide or mediate the sensor response acting as EET partner adsorbed or covalently attached to semiconductor particle. Signaling may be obtained by detecting directly the luminescence from QD and/or from the conjugate (Somers et al. 2007). A great variety of sensing technologies there appeared recently (Hildebrandt et al. 2017). It was even stated that “QDs are steadily revolutionizing the development of new biosensors along with a myriad of other photonically active nanomaterial-based bioconjugates.” Indeed, the approach based on QD donor and dye acceptor has found many applications: in DNA and RNA hybridization assays, in immunoassays, in the construction of biosensors based on ligand-binding proteins and enzymes. Other configurations such as incorporation of QDs into multistep EET processes or use of initial chemical and bioluminescent excitations are in active development (Wu et al. 2016). Quantum dots are also prospective for applications in immunoassays. As the examples, the cancer cell marker protein her2 (Huang et al. 2007), diphtheria toxin and tetanus toxin have been detected successfully using QD-labeled antibodies (Hoshino et al. 2005). Usually QDs are conjugated with sensor molecules and organic dyes and are used either for the target labeling or for the labeling of target analog in a competitor displacement assay. The donor-acceptor pairs for EET can be easily selected from QDs of different sizes and from the series of dyes possessing gradual variations of their fluorescence spectra, such as Alexa Fluor dyes. This allows composing the systems for different homogeneous assays based on the formation of complexes avidin-biotin and antigen-antibody. Competitive binding assays are efficient in such systems. An example of advanced QD-based immunosensor could be the technique for detecting explosive 2,4,6-trinitrotoluene (TNT) in aqueous media (Goldman et al. 2005). The hybrid sensor consists of anti-TNT specific antibody fragments attached to a hydrophilic QD via metal-affinity coordination. A dye-labeled TNT analogue pre-bound at the antibody binding site quenches the QD photoluminescence via proximity-induced EET. Analysis of the data collected at increasing the dye-labeled analogue to QD ratios provided an insight into understanding how the antibody fragments self-assemble on the QD particle. Soluble TNT present in the test system

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Fig. 7.7 Schematic illustration of a QD-based FRET competition assay for 2,4,6-trinitrotoluene (TNT) detection that uses attached specific antibody for TNT recognition (Goldman et al. 2005). The conjugate of TNT analogue with the quencher dye is used as the competitor. The competitor displacement removes the FRET quenching and thus produces the fluorescence response signal

displaces the dye-labeled analogue, eliminating EET and resulting in a concentrationdependent recovery of QD photoluminescence (Fig. 7.7). Given their maturity, some specific applications ranging from cellular imaging to in vitro sensing assays become of value (Hildebrandt et al. 2017; Martynenko et al. 2017; Wegner and Hildebrandt 2015). Due to possibility of selecting QDs with narrow bands distributed over the wavelength scale, the multiplexed imaging within the cell is easy to achieve and the lifetime can be an additional parameter to be used (Zhang et al. 2017a). Due to very high brightness, the QDs are ideal for imaging, and the limitation here is their relatively large size, which with the coating may reach 50–60 nm. The peculiarities of quantum dots as the components of hybrid composite structures that dramatically improve the properties of fluorescence reporters are discussed in Sect. 10.3. Due to their very high brightness but very broad excitation band, their most efficient application is as EET donors with much lesser prospects as acceptors. However, their applications as acceptors in compositions with long-lifetime donors are very beneficial in time-gated assays.

7.2.6 Semiconductor Nanocrystals of Different Shapes and Dimensions Recent studies have discovered many alternatives to classical semiconductor quantum dots with regard to shape and chemical composition. Nanostructured materials can adopt different shapes, such as nanotubes, nanorods, nanobelts, and nanowires (Huang and Choi 2007; Lim et al. 2014; Shahi et al. 2018). Such variation of shapes opens new dimension in variations of sensor properties. It was found that nanoparticles of elongated shape, “quantum rods” with diameters ranging from 2 to 10 nm and with lengths ranging from 5 to 100 nm demonstrate superior properties to that of QDs, in particular, higher brightness (Fu et al. 2007).

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In contrast to quantum dots, quantum rods emit linearly polarized fluorescence light (Lee et al. 2013) and this property can be potentially used in anisotropy (polarization) assays. Whereas QDs can allow improving the sensitivity of biological detection and imaging by at least 10- to 100-fold, quantum rods offer even superior possibilities for that. Due to their special shapes, their interactions in biological media may reveal many interesting peculiarities. The antibodies labeled with quantum rods can specifically recognize the cancer markers on cellular surface.

7.2.7 Magic-Sized Quantum Dots

Intensity

A special type of semiconductor nanoparticles are the particles with a specified number of atoms, the so-called magic-sized nanoclusters with extremely small sizes ( Br > I. The ligand exchange between these

7.4 Lead Halide Perovskite Nanocrystals

291

species occurs spontaneously in solutions within seconds, it does not require any drastic conditions. The weak points that could be derived from the current periodic literature are: (a) As in the case of Cd-based QDs, the contribution of the toxic element cadmium is essential. Another toxic heavy metal ion (Pb) plays a central role in governing the optical and electronic properties. The possibility of their substitution into another less toxic elements is under current discussion (Aldakov and Reiss 2019). (b) Poor stability and photostability of HPNCs (Wei et al. 2019). Being highly ionic, the nanoparticles can simply dissolve in water and in other polar solvents. Attempts to overcome this problem have been made (Geng et al. 2018; Wang et al. 2019). It is not clear if the stability will become sufficient for different sensing technologies and if it can be improved in further work. It is expected that proper surface coating will improve the particle stability as it was already demonstrated by incorporation of HPNCs into silica shell (Li et al. 2020). (c) The unusually prolonged blinking behavior, due to reversible transitions to trapped non-emissive states. It depends on particle size and may also be improved by proper coating (Tian et al. 2015). The applications of perovskite nanocrystals in sensing were started. Thus, coating of HPNCs with molecularly imprinted polymer was in the design of pesticide sensor (Tan et al. 2019). Their embedding into polymer matrix was also suggested for cell imaging (Zhang et al. 2017b). It is expected that existing problems with lead halide perovskite nanocrystals will be properly addressed and resolved in the near future so that the researchers will benefit from their unique optical properties.

7.5 Upconverting Nanocrystals The upconverting materials are unique in their properties that are very attractive for various sensing and imaging applications. The physical term ‘upconversion’ means the excitation from already populated excited state to a higher energy state, from which the emission can proceed being of higher energy (shorter wavelength) than the energy of excitation. In this case the term “anti-Stokes luminescence” is often used. An already excited molecule can absorb additional light quantum to be raised to a higher energy emissive state. Thus, the upconverting materials are those, in which the emissive states are reached by two quanta: first quantum excites to longliving state, in which the electrons can wait for the second quantum, and the second quantum allows reaching the state, from which the emission occurs. Such possibilities are realized in very special type of materials—the upconverting nanocrystals (Gorris and Wolfbeis 2013; Haase and Schafer 2011; Mader et al. 2010; Wang et al. 2010a). In Sect. 6.2 we discussed the possibilities for achieving the antiStokes emission by triplet-triplet annihilation within organic dyes and their metalchelating complexes. Compared to them, the upconverting nanocrystals have very

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important advantages that will be discussed here. The great interest to “upconverters” from the side of researchers is due to the fact that no other presently known natural or men-made materials that can appear in test media or living tissues can exhibit such properties. Therefore their luminescence is free from the background emission.

7.5.1 The Photophysical Mechanism of Upconversion At first, we have to draw clear distinction between the process of upconversion and of two-photon (or multi-photon) excitation. The two-photon excitation process can be applied to a very broad range of fluorophores (though with different efficiency) but on a condition that for adding the photon energies their absorption is simultaneous (see Sect. 5.4). The intermediate states do not exist, they are ‘virtual’. Such excitation requires very high photon densities and is commonly realized with the application of ultrafast light pulses. The non-linear process allows focused excitation at the sites of high photon density, which allows formation of focal plane in the two-photon microscopy. In contrast, in upconverting nanoparticles (UCNPs), the long-living intermediate states are ‘real’. They can be easily populated, so that the electrons should be ‘trapped’ for relatively long time to be excited again by the second photon. The efficiency of photon upconversion process based on UCNPs is much higher than that of nonlinear two-photon absorption, so the excitation can be performed with low-cost continuous-wave lasers rather than with the use of ultra-short pulsed lasers generating high peak energies. However, the application of upconversion effect in sensing cannot be realized with any type of dyes or nanoparticles and is limited to very special materials. Such materials are really unique. In ceramic crystalline host matrices doped with trivalent lanthanide ions, such as Yb3+ , Er3+ and Tm3+ , two important properties, a variety of electronic excitedstates and long lifetimes of these states uniquely meet. The long-lifetime excited states can operate as metastable states excited from a ground state to be excited again with the gain of energy for reaching the emissive states (Fig. 7.11). Here the site excited to lowenergy electronic state can wait until next quantum brings it to a higher excited state, from which, commonly after relaxation to lower levels, there occurs the emission of quanta of higher energies. Therefore such emission is free from restrictions imposed on two-photon excitation. It does not require very high irradiation densities and can be excited by conventional light sources. As it is illustrated in Fig. 7.11, three main types of operation with these particles can be realized: the excited state absorption (ETA), the energy transfer upconversion (ETU) and the photon avalanche (PA) (Achatz et al. 2011). In the ESA mechanism (Fig. 7.11a), a single ion is involved in the upconversion process. The ion is first excited to an intermediate metastable state, whereby a second pump photon promotes the ion from the intermediate state to a higher lying state, from which the emission of light occurs. The ETU mechanism (Fig. 7.11b) is much more efficient. It involves two neighboring ions, the sensitizer and the activator, which may be two identical or different ions. The first step involves the excitation of two ions to their respective

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Fig. 7.11 Schematic representation of the three main types of processes causing upconversion in rare earth doped materials: a excited state absorption (ESA); b energy transfer upconversion (ETU); c photon avalanche (PA). The dotted lines refer to photon excitation, dashed lines to nonradiative energy transfer, and full arrows to emissive processes, respectively. G, E1 and E2 represent ground state, excited level 1, and excited level 2, respectively

intermediate metastable levels via the correspondent absorption of two photons. This process is followed by an energy transfer from the sensitizer to the activator. Thus, on absorption of two quanta, in addition to emission occurring from a single ion, the energy can be transferred to adjacent ion. The activator ion is promoted to the upper excited state, which allows the high-energy emission and the sensitizer ion simultaneously relaxes to the ground state. Also, on exceeding some threshold light intensity, a resonant ‘avalanche’ emission can be generated (Fig. 7.11c). The Er3+ , Tm3+ , and Ho3+ ions are the lanthanides most widely used in emissive UCNPs (Zhou et al. 2015). Due to their different energy levels, they show multiple upconversion emission bands in the UV, blue, green, red, or NIR regions. By tuning the doping ions and their concentrations, the color of the upconversion emission can be properly modulated from the UV to the NIR range.

7.5.2 The Spectroscopic Properties of Upconversion Nanomaterials Presently, the most part of results on photon upconversion was obtained in inorganic crystalline NaYF4 host lattices doped with Yb3+ , Er3+ and Tm3+ . Ytterbium ion is commonly used as a sensitizer because it has higher absorbance than erbium and thulium, which are used as emitting activators. The formed nanocrystals display the ability to obtain visible emission with excitation in the near-IR (Haase and Schafer 2011; Mader et al. 2010; Wang et al. 2010a). Thus, it is possible to achieve the upconverted UV–visible–NIR emissions using a single NIR excitation source. This source can be an inexpensive commercial continuous-wave diode laser emitting at 980 nm. The emission lifetimes are long, in the range of 100 μs up to over 1 ms,

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Fig. 7.12 Energy transfer and upconversion emission mechanisms in a NaYF4 nanocrystal doped with Yb3+ , Er3+ , and Tm3+ under 980-nm excitation (Achatz et al. 2011). The dashed-dotted, dotted, curly, and full arrows refer to photon excitation, energy transfer, multi-photon relaxation, and upconversion emission. The insert shows schematically the UCNP, in which the shell incorporates Yb3+ , ions serving as sensitizers (EET donors) transferring their energy to core composed of Er3+ and Tm3+ ions that function as activators (EET acceptors) and emitters of light of different colors

which allows convenient time-gated measurements with simple instrumentation (Dos Santos and Hildebrandt 2016). As it is shown in Fig. 7.12, the excitation wavelength 980 nm is commonly used for absorption of Yb3+ ion serving as sensitizer. Its molar absorbance of 10 M−1 cm−1 for the 2 F7/2 → 2 F5/2 transition is very high considering that the absorption promotes partly forbidden f-f transitions. The absorption bands of lanthanides are very narrow, with full width at half maximum (FWHM) of about 10–20 nm. The energy conserved in the 2 F5/2 electronic state of the Yb3+ ions can be transferred to activator ions, Tm3+ and Er3+ , which act as the luminescent centers in the nanoparticles. For reducing the effect of absorption of water, which is already significant at 980 nm, the Nd3+ ion can be incorporated into the Yb3+ -sensitized UCNP structure. Nd3+ can be excited with 808 nm light, and the energy transfer between Nd3+ and Yb3+ is known to be quite efficient. The reduction of light absorption by water is necessary for working in biological media, thus avoiding appreciable heating of tissues.

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Emission intensity

7.5 Upconverting Nanocrystals

Wavelength (nm) Fig. 7.13 Room temperature upconversion emission spectra of NaYF4 nanocrystals doped with a Yb/Er (18/2 mol %), b Yb/Tm (20/0.2 mol %), c Yb/Er (25–60/2 mol %), and d Yb/Tm/Er (20/0.2/0.2–1.5 mol %) particles in ethanol solutions (10 mM) (Achatz et al. 2011). The spectra in (c) and (d) were normalized to Er3+ (650-nm) and Tm3+ (480-nm emissions), respectively. The samples were excited at 980-nm with a 600-mW diode laser

The emission of upconverting nanocrystals is represented by a series of narrow lines that are very characteristic (Fig. 7.13). Other features include long-lifetime decays, very high chemical stability, the lack of bleaching, and the absence of blinking effects (De la Rosa et al. 2005; Mader et al. 2010). The excitation energies are sufficient to convert the near-IR excitation light into emission at visible wavelengths. Therefore the ‘anti-Stokes’ emission appears to be shifted dramatically (but not necessarily with doubling of energy) to shorter wavelengths with respect to excitation. For example, this emission excited at 980 nm can be measured at different wavelengths, from green to red. The upconversion quantum yield is important parameter characterizing UCNPs. In nanoparticles based on NaYF4 host material it heavily depends on particle size and rarely reaches values above 1%. When the particle dimensions become below 50 nm (and this is needed for bioanalytical applications), all surface related effects of quenching become amplified with the involvement of surface ligands, solvent molecules and crystal defects.(Wang et al. 2010b). The surface quenching is most pronounced as a result of increased deactivation of their excited states by O–H vibrations of water molecules (Würth et al. 2017); the Ln ions really do not like water. Therefore the core-shell particle architectures are commonly used to minimize the surface deactivation by protecting the surface of the emissive UCNP core. One of the possibilities to improve the properties of UCNPs is to find for the rareearth ions more efficient than NaYF4 hosting materials. It was reported that with

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the application of CaS matrix the upconversion luminescence quantum yield can be increased to ~60% (Wang et al. 2018). However, as for other nonlinear processes the quantum yield depends on the excitation power (Kraft et al. 2018) and has to be evaluated with this account (Meijer et al. 2018).

7.5.3 Modulation of Fluorescent Signal Quite narrow excitation band and series of robust narrow emission bands do not allow in plain UCNPs the variation of spectroscopic properties in broad and sometimes necessary ranges. In addition, the brightness of these nanoparticles is low and not sufficient for many applications. Therefore, there are many efforts to improve them both from the side of excitation and the side of emission. In both cases the excitedstate energy transfer process is employed (Fig. 7.14). One of the important drawbacks of UCNPs that limit their applications is the very weak (~10–20 cm2 ) absorption cross section (Wang et al. 2017b), which is exactly the result of parity-forbidden 4f-4f transitions in conjunction with the very narrow absorption bands. Moreover, their photoluminescence quantum yields are relatively low and depend on excitation power density as well as on the surface-tovolume ratio (Würth et al. 2017), due to the multiphotonic nature of the upconversion process and surface quenching effects (Kraft et al. 2018). The major approach to overcome this problem is to apply the decoration of nanoparticles with the layer of additional sensitizers serving as light antennas. Many researchers focus on such way of luminescence enhancement by developing new nanoparticle designs and compositions incorporating organic dyes as light harvesters generating the antenna effect (Wen et al. 2019). Near infrared emitting dyes coupled to UCNP can provide a potential strategy to boost UCNP brightness (Wang et al. 2017b). The relatively broad absorption bands and the strong absorption cross section of near-IR organic dyes (see Sect. 5.4) render them ideal light harvesters and energy donors for UCNP (Saleh et al. 2019).

Excited states

hν2

Dye 1 Dye 2 Dye 3

Ground state

hν1 Sensitizer Up-converting NP Dyes with different emission color

Fig. 7.14 The possibilities for improving the spectroscopic properties of UCNPs. For increasing the brightness, the nanoparticle can be decorated with the layer rich in additional sensitizers serving as light-harvesting antennas that focus their excitation energy on UCNPs. From the side of emissive decay the dyes demonstrating different emission colors can be appended as EET acceptors

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Moreover, these dyes can also help shifting the excitation of the dye-UCNP systems from typically 980 nm for Yb-doped UCNP closer to ~800 nm, exciting Nb ions, which fits better into the biological spectral window and prevents heating effects by water absorption (Söderlund et al. 2015). This can increase the applicability of these materials, especially for biological studies. It is important to improve the UCNP brightness from the side of their emission, which due to its long duration is a subject to non-emissive losses (Wisser et al. 2018). The coupling of organic dyes can help to resolve this problem. The nonradiative decay pathways are mostly due to phonon coupling and surface quenching, and appending organic dyes in a special manner can selectively influence the radiative rate of the final emitting state. It was shown that the upconversion quantum yield enhancement was achieved by decorating the UCNP surfaces with the dyes enabling efficient energy transfer from Er3+ with the increased radiative rates of sensitized emission. The other reason to apply EET acceptors at the UCNP emission side is to use the energy transfer phenomenon for the sensor reporting signal by quantitative analysis of variation of relative intensities of upconverted and sensitized intensities (the λratiometry). Many literature data report on the success in this strategy (Su et al. 2017), and recent mechanistic analysis (Dukhno et al. 2017) allows determining the optimal conditions for that regarding particle size and quantity of dyes grafted on their surface.

7.5.4 Present and Prospective Applications Unique type of luminescence emission of UCNPs, where the low-energy near-IR light is converted to the high-energy visible emission upon sequential absorption of two or more photons is extremely attractive for various applications. In addition to their unique photon upconversion properties, UCNPs show a sharp emission bandwidth, long lifetime, tunable emission, high photostability, and low cytotoxicity, which render them particularly useful for bioimaging applications including cell tracking, tumor targeting and vascular imaging. Fascinating are the applications in sensor technologies of upconversion emitters as resonance energy transferdonors to any molecular or nanoscale acceptors (Dos Santos and Hildebrandt 2016). Due to extremely sharp and narrow emission bands characteristic of luminescent lanthanide ions, no donor emission can be detected at the wavelengths of sensitized emission of the acceptor. Also, no acceptor can be excited directly because the excitation is in the near-IR. This means that the donor and acceptor emission cannels do not overlap, which is almost impossible to achieve with organic dyes. The use of these nanoparticles as the energy transfer acceptors allows overcoming their weak point—a weak light absorbance of lanthanide ions. A conjugation of the nanocrystals with multiple strongly near-IR absorbing organic dyes acting as light harvesting surface ligands and providing the excitation energy to emissive sites allows to make them strong absorbers (Zou et al. 2012).

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Manipulation with concentrations of doping ions allows generating any color covering the whole visible range because the narrow and multiple emission lines of upconverting nanoparticles can be adjusted individually providing the large-scale manipulation with emission color (Gorris and Wolfbeis 2013). Such designed combinations ideally suit for color barcoding that is needed in homogeneous multiplex assays (Sect. 10.4). The presence of multiple emission lines together with possibility to manipulate with their relative intensities open new possibilities in λ-ratiometric sensing. The self-calibrating temperature sensor was developed based on this principle (Sedlmeier et al. 2012). The fact of absence of background emission even in the presence of many contaminants stimulates application of sensing technique to different types of heterogeneous and biological systems. This is ideal for the analysis of strongly colored and fluorescent samples, which are often of concern in clinical immunoassays and in highthroughput screening (Kuningas et al. 2005). Particularly, this approach allows the sensor applications in the whole blood, so that blood pigments do not interfere into assays. The straightforward application of upconverting nanoparticles is in cell and tissue imaging (Xu et al. 2013). With their aid, the high penetration depth of near-IR light together with the absence of light-scattering and fluorescence background allows obtaining ideal images in the visible (Chatterjee et al. 2008). This technique extends to small-animal imaging tomography (Zhou et al. 2012). Deep penetration of nearIR light into tissues allows using the upconverting particles in new schemes of photodynamic therapy of cancer (see Volume 2, Chap. 17). Recently, these UCNPs were shown to act as efficient probes for super-resolution nanoscopy (Liu et al. 2017), see Volume 2, Chap. 15. The reason for their efficient application is in their ability to establish population inversion and amplified stimulated emission at relatively low pump power. Due to these properties the low-power super-resolution stimulated emission depletion (STED) microscopy was realized with great improvement of nanometer-scale optical resolution.

7.6 Sensing and Thinking. Small Things Bright and Beautiful, What is Their Advantage? The stories described in this Chapter though diverse have something important in common. It is the collective behavior of inorganic atoms and ions. The collective effects as strong as in semiconductor crystalline QDs generating the excitons to UCNPs where the trivalent ions are connected by resonant energy transfer mechanism. All these effects can be seen and realized in different technologies in different designed, selected and improved materials, in which the crystalline order plays the key role. The number of these materials diverse and the search for better and better ones must continue.

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We also learned that the interface between the nanocrystal and its solid or liquid environment may play a role of ultimate importance, especially in small nanoparticles, possessing high surface to volume ratios. Usually this role is negative, but with smart design it can become positive and highly efficient in inducing the sensor response. Multiple groups of atoms forming the particle surface allow multiple possibilities for smart supramolecular constructions. The success in use of these materials will depend strongly not only on their fluorescence reporter properties but also on the ease with which they can be synthesized, functionalized and manipulated. Here the strategy has to be different from that for molecular emitters in view of different mechanism of their fluorescence response. We observe that the combination of smart nanoscale and molecular emitters lead to new functionally important properties and the number of these combinations becomes enormous. Fundamental research on luminescent nanoparticles continues and it is highly probable that in near future the material nanotechnology will offer to sensor community the light-emitting reporters with the advanced properties that are very difficult to predict. The questions addressed to the reader after reading this Chapter: 1.

What is the difference between nanoparticles made of crystalline and soft materials in the formation of surfaces and display of their properties? 2. In what way the classical Förster-type (FRET) mechanism of excited-state energy transfer is modified on interaction with and between nanoparticles? 3. What are the criteria for considering quantum dots as “dots”? Is it their size smaller than the wavelength of light? Or may be their isotropic properties in all dimensions? What about “quantum rods”? 4. Explain the correlation between the size of quantum dots and the positions of their optical spectra. The size is a geometric parameter and the spectra reflect the energies of electronic transitions. Why are they connected? 5. How to make the quantum dot responsive as the detection unit in direct sensing (avoiding sandwich assays)? 6. Is it easy to achieve EET between quantum dots of different sizes? What are the major problems? How these problems are resolved if, instead of quantum dots, another type of nanoparticles are used as EET partners? 7. How to achieve the long persistent luminescence in inorganic nanoparticles? What are the structural and energetic requirements for that? 8. Explain how and why perovskite nanocrystals can vary their emission spectra over the whole visible range. Suggest the applications of this property in λratiometric sensing 9. Why plain perovskite nanocrystals cannot be applied in aqueous media? Suggest efficient solution to this problem 10. What are the problems in application of perovskite nanocrystals for cellular imaging? Suggest possibilities to resolve them 11. What are the general requirements for upconverting phenomenon to be observed? How they are realized in rare earth ion doped nanocrystals?

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12. What properties of upconverting nanoparticles can be improved by application of sensitizers and the dyes acting as EET acceptors?

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Chapter 8

Fluorescent Organic Dyes and Conjugated Polymers in Nanoscale Ensembles

This chapter provides comparative analysis of properties of different organic nanoscale materials to absorb and emit visual and near-IR light with the focus on their applications in sensing and imaging technologies. These new materials are diverse. Strongly emissive nanoparticles can be formed of organic or inorganic polymers incorporating organic dyes. In dendrimers they can be attached covalently and in well-defined manner in the desired sites. Collective excitonic effects appear when the aromatic units are arranged in definite order in nanoparticles and also in conjugated polymers. The H- and J-aggregates formed in these structures generate delocalized excitons that possess the spectroscopic properties dramatically different from that of single or disorderly assembled dye molecules. Exciton diffusion can be realized easily in these nanoparticles. Operating with these collective features one may design materials of unprecedented brightness and versatility for amplified sensing and ultra-bright imaging. Nowadays, the novel nanoscale fluorescent materials appear in strong competition with traditional organic dyes. They demonstrate diverse photophysical behavior and allow obtaining diverse information when they are incorporated into sensor composites or form the images in biological systems. Among these nanoscale emitters are the structures formed of organic dyes or by these dyes incorporated into different types of polymers with the resulting dramatically increased brightness and possibility of broad-scale variation of different spectroscopic parameters. Strong fluorescence and amplified response are also characteristic for conjugated polymers. A very high brightness, which is the product of molar absorbance and fluorescence quantum yield, is the desired property of every fluorescence emitter. This result can be achieved by incorporating many dye molecules into nanostructures that can be formed by the dyes themselves, with the help of small molecules or on incorporation into organic or inorganic polymer. Due to large number of dye units housed in a relatively small volume of a single nanoparticle, the dye-doped nanoparticles present considerable advantages with respect to the same dyes used in molecular form. At the same excitation conditions, one can obtain a fluorescence signal, which in some cases is 104 to 105 times stronger (Wang et al. 2006). The benefits of such nanoparticles © Springer Nature Switzerland AG 2020 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-030-60155-3_8

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Nanoscale assemblies of organic dyes a

b

c AIE

јH

љJ

MPE

CAE PBE

Fig. 8.1 The associates of organic dyes that form nanoparticles in solutions with appreciable emission. a The H- and J-aggregates possessing the exciton coupling. b The species demonstrating the aggregate-induced emission (AIE) and cofactor-assisted emission (CAE) of nanoparticles, the cases, in which the dyes to a large extent retain their individual fluorescence properties. c Macromolecule protected emission (MPE) (by polymer, DNA, etc.) and polymer based (PBE) emission that may display both individual and collective spectroscopic effects

are clearly seen in cellular studies and in the whole-body imaging, where loading the particles with two-photonic dyes allows achieving extreme brightness and resolution (Bertazza et al. 2006). There may be different additional benefits for such incorporation. One, of course, is the isolation of the dyes from molecular contact with the tested medium and thus making them insensitive to the changes of composition in this medium. Important is the possibility to avoid degradation of dyes in aggressive media and in living cells. The dyes in solid environments usually exhibit much higher fluorescence intensity, quantum yield and lifetime than in liquid media. This is due to stronger restriction imposed by solid matrix on rotation of dye segments, intermolecular collisions and dielectric relaxations—all the factors that quench the fluorescence (Riu et al. 2006). These nanoparticles can serve as building blocks in constructing multifunctional nanocomposites (Chap. 10). Their larger surfaces may allow attachment instead of one, of several macromolecular recognition units. Attachment to their surface of various functional groups (such as avidin, biotin, antigenic peptides, ligand-binding proteins, etc.) allows their facile bioconjugation, immobilization and incorporation into various assays. In this Chapter we consider various types of nanostructures formed by assembling organic dyes with and without assistance of different low-molecular and polymeric components (Fig. 8.1). Organic dyes can form the main chain of the polymer or be appended as its side groups. Dependent on the dye properties and the mode of bond formation, they interact differently between themselves and, interacting, display different photophysical and spectroscopic behavior. Finally, we discuss the properties and applications of conjugated polymers, in which the aromatic units form the body of their structure. In all these nanoscale materials there appear collective excitonic effects, but to a different extent.

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8.1 General Effects Observed on Dye-Dye Association The materials formed of organic heterocyclic molecules are usually considered as ‘soft bodies’ due to the kinds of weak intermolecular interactions that can be realized (H-bonding, π-stacking, van der Waals forces, long-range interaction of charges), so that several types of molecular packing (crystalline polymorphs, liquid crystal phases, nanoaggregates with diverse morphologies) can be realized. Variations in this arrangement may lead to different optical properties. The strongest effects, however, are produced by electronic interactions between fluorophores that can be enhanced in the excited states.

8.1.1 Excitonic Behavior of Organized Dye Assemblies Electronic energy transfer (EET) that is often abbreviated with the reference to its dipole resonance mechanism as FRET (Förster resonance energy transfer) is presented as a binary event occurring between single donor and single acceptor. Meantime, when many such EET donors and acceptors assemble in a small particle and appear within the critical distance, they lose their individuality and new collective effects appear. For understanding these effects, we have to introduce the description in terms of excitons. By definition, excitons are the mobile neutral quasi-particles in the form of electron-hole pairs that appear in solid bodies and molecular associates upon electronic excitation (Chen et al. 2017; Davydov 1971). Excitons can be considered as the propagating collective excitations in a system of interacting molecules, in which the participating fluorophores do not behave as individual emitters and the whole system responds (emits light or is quenched) as one unit. The hole is the positively charged site, from which the electron is abstracted. The electron and the hole remain bound by Coulomb forces. Their propagation is coupled, so that they carry energy but not the charge. Depending on the strength of electron-hole bonding, the excitons can be of two major types (Knox 1972). There are the Wannier-Mott loosely bound excitons, such as that observed in semiconductor nanocrystals—quantum dots (QDs), see Sect. 7.2. They are often called the large radius excitons because, on absorbing the light quantum, the electrons are promoted to conduction band and behave as free charge carriers. They can propagate to a large distance that is limited only by a crystal or particle size, and their recombination with the holes results in fluorescence. Such behavior is only possible in the conditions of very high dielectric constant where the screening of charges is strong. This is not the case of molecular crystals and associates of organic dyes, since here the dielectric screening of charges is rather low and electron-hole interaction remains strong. Here the theory of strongly bound Frenkel excitons (often called molecular excitons) is applied.

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The theory of molecular excitons was developed by Alexander Davydov (Davydov 1971), and it has got brilliant confirmation in the studies of molecular crystals formed by organic dyes (Broude et al. 1985). In this description, the aggregates are presented by ordered structures of constituting dyes with their purely electrostatic intermolecular coupling but without significant overlap of electron density. The electronic excitation can be delocalized over many monomers in the form of excitation waves (Saikin et al. 2013) leading to new spectroscopic phenomena (Davydov 1971). In order to display them in organic dyes, the high crystal-like order in the aggregates may not be needed, but they should assemble into well-ordered intermolecular arrangements with a definite packing geometry. Possessing high electron-hole affinity, the Frenkel excitons are typical for the complexes of organic molecules and conjugated polymers (Bardeen 2014). If these excitons are formed in dye aggregates, the electrons remain localized on molecular sites, while the excitation is coherently delocalized over many fluorophores (Scholes and Rumbles 2006). In all the cases when more than one chromophore contributes to the light absorption and emission of molecular aggregate we have to consider their interactions within short distances and the transfer (or exchange) of excitation energy between them. The electronic energy transfer (EET) is the process, which occurs when the energy absorbed at one site (the donor) is transferred to another nearby site (the acceptor). Usually, in mechanistic description of these effects in multi-chromophore systems, the two limiting cases are discussed (Chenu and Scholes 2015). First is the case where there is no strong electronic interaction and the energy migrates between the dyes whose dipoles are in resonance. The transfer occurs as incoherent hopping, in which the excitation energy jumps randomly as a diffusive energy transport between molecular-type forms. Here the Förster theory of electronic energy transfer (FRET) is applicable, see Sect. 4.3. In these cases, the assumption of weak donor-acceptor electronic coupling arising from the dipole-dipole interaction (Förster limit) is satisfied. The Frenkel excitons are different. They span multiple molecules as the electronhole pairs spread in a wave-like manner over spatially separated molecules (Scholes et al. 2017). Coherent exciton delocalization is the process that creates a regime, in which the stronger electronic coupling delocalizes the excitation to produce excitonic states. In this case the energy donors and/or acceptors are not the molecules but already the excitons shared between strongly interacting fluorophores (Frenkel limit). Many examples from chemical and biological worlds show that such coherence effects exist and that they are robust to the presence of disorder and noise (Broude et al. 1985; Walschaers et al. 2016). In molecular aggregates, both Frenkel-type coherent and Förster-type incoherent energy transfers can be continuously connected to each other. The collective transition dipoles can be much larger than the molecular transition dipoles, therefore the excitonic states may accelerate dramatically the energy transfer (Brixner et al. 2017). Some authors call such acceleration a “super-transfer” (Lloyd and Mohseni 2010). The multiple steps of excited-state energy transfer can be realized according to FRET mechanism. This process is often considered as the exciton diffusion, in

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a

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b 1

2

Fig. 8.2 Different cases of excitation energy transfer in molecular chromophore systems a (1). Coherent exciton delocalization. (2) Super-transfer between the systems of delocalized excitons. (3) FRET between exciton and molecular dye, in which the dye is the energy donor. (4) FRET, in which molecular dye is the donor and excitonic system is the acceptor. b Examples of systems demonstrating exciton coupling and exciton diffusion. (1) Exciton coupling as in conjugated polymer (1) and exciton diffusion that can be observed in linear polymer with fluorophore side groups (2)

which the coherence is lost (Najafov et al. 2010). The modified version of Förster theory accounting for the role of excitonic donors and acceptors promoting the energy transfer was developed (Mirkovic et al. 2016). Essentially, the fluorophores (or excitons) participating in this Förster-type resonance excited-state process do not lose their individuality. Before transferring the excitation, the donor energy becomes thermally relaxed, the phase relationship in dipole-dipole resonance is lost and the energy migration dynamics loses the electronic coherence. Coherent and incoherent energy transfer can occur in the same molecular system are depicted in Fig. 8.2. There are many examples of systems, in which the coherence in energy transfer is preserved. They include some conjugated polymers (Collini and Scholes 2009) and the pigment clusters found in the systems of photosynthesis (Abramavicius et al. 2009; Lee et al. 2007). Excitons in these aggregates can be delocalized across many fluorophores and may show both coherent and incoherent energy transfer. When the dimension of the structure is reduced to the size comparable to the critical distance of energy transfer, its modulation can be possible by variation of nanoparticle size (but not to such extent as in the case of loosely bound excitons).

8.1.2 Modulation of Photoreactions Valuable for Sensing The aim of assembling fluorescent dyes into nanoscale structures is not only in increasing their brightness but also in enhancement of their sensor response, making it more efficient. In the conditions of close proximity between reactants and of screening from the solvent, the typical reactions of organic dyes can proceed differently than in solutions. The excited-state reactions, such as electron, charge and proton transfers, described in Sect. 4.1, can occur in improved, controlled and adjustable manner (Tabacchi 2018; Yoon 1993). Detailed analysis of these reactions, occurring with guests incorporated into silica matrices (Alarcos et al. 2017), have shown that manipulation with electron and proton donor-acceptor pairs can be very efficient.

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Excited-state proton transfer dynamics can be modulated within broad ranges in nanoporous materials (Alarcos et al. 2014), when incorporated into polymer nanoparticles (Teixeira Alves Duarte et al. 2019) and producing aggregation-induced emission (Kaur et al. 2019). It is interesting to note that the emission of exciplexes, that is usually quenched in solution, in the dye nanoscale aggregate can be found bright with proper arrangement of donor and acceptor partners (Liu et al. 2019).

8.1.3 Exchange of Excitation Energies The classical theory of Förster resonance energy transfer (FRET) described in Sect. 4.3 is hard to be applicable to the composites formed of organic dyes, even if the transfer occurs through distance according to the dipole resonance mechanism. This is because of the common occurrence of multiple transfers between identical fluorophores. The transfer between identical molecules is reversible; its efficiency also depends on overlap integral between absorption and emission bands (Sect. 4.3), which is determined by the Stokes shift. The smaller shift—the higher is the efficiency of transfer. We call this process homo-EET , in order to distinguish it from classical hetero-EET (the transfer between different types of fluorophores). Three rules can be formulated in this regard. They are important for explanation of the collective effects in the excited-state energy transfer in a system composed of different types of light emitters participating in the energy transfer process. 1. In a system composed of structurally similar dyes located in identical environments and possessing identical absorption and emission spectra and also quantum yields and lifetimes, EET (in this case homo-EET ) is just the exchange of energies between the dyes that is undetected by spectroscopic or lifetime measurements. It can only be revealed by the measurements of anisotropy and only in the case if the dyes occupy different orientations in space and their orientations do not change fast on the scale of fluorescence decay (solid or highly viscous dye environments). This rule follows from the identity of all parameters determining the EET efficiency (Sect. 4.3) and from the kinetic scheme, in which the forward and reverse transfer rates are equal and higher than the rates of non-emissive and emissive relaxations to the ground state. 2. In a system composed of structurally similar dyes possessing identical fluorescence spectra but exhibiting static quenching, the effect of quenching will be observed for the whole system. The quenching efficiency will be determined by the dye with the shortest lifetime τ. This follows from the kinetic scheme, in which for this dye the rate of non-radiative deactivation of the excited state is comparable or faster than the rate of energy transfer. If the transfer is efficient and this dye is fully quenched, then we will observe the quenching in the whole ensemble. Based on these rules one can easily describe the effect of ‘superquenching’ that can be observed in dye-doped polymeric particles, in conjugated polymers and dendrimers, as it will be shown below. Manipulating with a single trigger dye,

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one can provide efficient collective sensor response by switching on and off the whole ensemble of fluorescence emitters. 3. In ‘soft’ systems with some level of molecular disorder, the distribution of fluorophores on their weak intermolecular interactions with the environment can be observed as the inhomogeneous broadening of spectra. The energy transfer then becomes directed from fluorophores with high energy of emission to those with lower energy of emission resulting in time-dependent spectral shift to the red (Nemkovich et al. 2002).

8.1.4 Concentration-Dependent Quenching Assembling more dye molecules, should we always obtain proportionally higher brightness? Realization of this very attractive idea to assemble many molecules in a small unit meets a difficult problem. The highest density of dye molecules and direct interactions between them or exchange of their excitation energy may lead to the so-called ‘concentration-dependent quenching’ (another names ‘contact quenching’ and ‘self -quenching’). This feature was frequently observed when the protein or nucleic acid molecule is labeled by attaching an increasing number of fluorescent dyes (Donaphon et al. 2018). Then very often its fluorescence instead of linear increase passes through the maximum and falls down. For many dyes, the self-quenching is a limiting factor in the design of devices with high sensor and fluorophore density. The explanation of this effect is illustrated in Fig. 8.3. Potentially emissive dye molecules, being located at short distances, loose the properties of independent emitters. Being excited, they start to exchange their energies in accordance to homo-EET mechanism (see Sect. 4.3) that is realized between the same dye molecules serving as donors and acceptors to each other. So, they can exchange their energies even

Fig. 8.3 Illustration of homo-EET in molecular ensembles and self-quenching in dimers. a The dyes (D) exchange their excitation energy and are brightly emitting. b The presence of a nonfluorescent dye dimer (DD) acting as the trap for excitation energy results in quenching of all the dyes located at a close distance to it

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being at large distances, up to 5–7 nm (Runnels and Scarlata 1995). Normally this results in emission of one of molecules in the system (Fig. 8.3,a). With the increase of concentration of dye molecules and of the probability of interaction between them, there may occur the formation of dimers, then of aggregates. The interactions within these dye associates lead to static quenching of their fluorescence via electron transfer. Such traps of excitation energy accumulate the energy from other nearby located dyes but do not emit it (Fig. 8.3,b). This results in quenching of the whole system (Johansson and Cook 2003; Lavorel 1957). Involvement of this effect when the energy is captured by non-fluorescent species depends strongly on the properties of the dye. Fluorescein and rhodamines exhibit high homo-EET efficiency and also self-quenching, pyrene and perylene derivatives—high homo-EET but little self-quenching. Luminescent metal complexes may not exhibit homo-EET at all because of their very strong Stokes shifted emission. When the nanoparticles are composed of organic dyes or doped with these dyes, such effects can be especially important. Thus, ultra-bright polystyrene nanoparticles can be easily made by doping with BODIPY dye (Grazon et al. 2014). However, with the increase of doping rate there is no linear increase of brightness but the tendency to saturation with the gradual decrease of quantum yield and lifetime.

8.1.5 The Effect of External Quenchers, “Superquenching” The superquenching effect is the phenomenon observed when the efficient quenching of fluorescence emission of the whole ensemble of fluorescence emitters is achieved by minute amounts of quencher (e.g. analyte) interacting with only one member of this ensemble. This effect is illustrated by considering the chain of fluorophores that can exchange their excitation energies (Fig. 8.4). In contrast to individually absorbing and emitting fluorophores, we can observe the collective effect: the binding of only one quencher molecule can quench the fluorescence of the whole ensemble. In the background of this effect is the rapid energy exchange within the system. The complex of quencher with one element of ensemble plays similar role as the “intrinsic” quencher represented by dye dimer in Fig. 8.3 as the sink of excitation energy. This effect plays a very essential role in sensing technologies. In most cases, it is realized in photoinduced electron transfer (PET) reactions (see Sect. 4.1.1) but can be produced by any other event resulting in static or dynamic quenching (Sect. 3.2.3). Its efficiency is very high in excitonic systems, particularly in conjugated polymers (Zhang et al. 2020). Graphene and graphene oxide can play a role in sensor design as excitonic superquenchers reporting as PET acceptors (Yang et al. 2019). In the systems demonstrating EET between organic dyes, such as in dendrimers decorated with dansyl groups (Balzani et al. 2000b) or cyanine dyes incorporated into DNA structure (Radchenko et al. 2018) it can depend on the transfer efficiency.

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Small fluorophores Quencher

Q

Q

Conjugated fluorophore Quencher

Q

Q Fig. 8.4 Mechanism of superquenching in a system of conjugated fluorophores. Emitting species are bright yellow and those quenched are gray. The interaction of quencher Q with a small fluorophore quenches only its emission. The same fluorophores, when they are linked in excitonic structures or coupled performing efficient EET exchange, behave differently. Binding of single target molecule to a single recognition site switches off completely the fluorescence of the whole system (dye-based nanoparticle, conjugated polymer chain)

8.1.6 Variations of Anisotropy in Excitation Energy Transfer Organic dyes typically possess a high level of optical anisotropy (detected as polarization of emission when excited by polarized light) when their orientation remains unchanged during the emission (see Sect. 3.3). What happens with the emission anisotropy in dye-formed and dye-doped nanoparticles? If the dyes do not exchange their energies, their short-lifetime fluorescence emission should be highly polarized. But if there is a EET-type exchange between their energies and their orientation is not highly ordered in one direction, the emission anisotropy is lost. In densely packed multi-fluorophore structures, it is hard to avoid homo-EET, and in its presence the anisotropy of observed emission drops dramatically, often to zero level. This is because the orientations of donors and acceptors in aggregate are usually not the same, and in the case of multiple transfers and random orientation of acceptors, the information on initial polarization is lost. In an ensemble of similar emitters the drop in anisotropy is the major indicator of the presence of homo-EET. Therefore, such nanoparticles cannot be used in polarization assays. In contrast, in highly ordered J- and H-aggregates the emission anisotropy can be maintained on a high level, and this is one of the reasons to consider the highly polarized emission of carbon dots to be of excitonic origin (see Chap. 9). This seemingly discouraging result may lead to quite positive consequences. There is a possibility of manipulating with the homo-EET efficiency by variation of excitation wavelength within the absorption band generating the so-called Red Edge effect (Demchenko 2002, 2008; Nemkovich et al. 2002). In this way, the emission anisotropy can be changed from its low values observed at the band maximum to its

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b a c

Intensity

Anisotropy

λ FRET

No FRET

Fig. 8.5 The scheme explaining the Red-Edge effect as site-photoselection within the population of fluorescence emitters and the disappearance of FRET (EET) at the red edge of excitation spectrum that results in repolarization (Demchenko 2013). The system composed of similar molecules located within the distances of efficient homo-EET is shown. At shorter wavelengths (case a) and band maximum (case b) the emitters exhibiting different interactions with the environment (marked with different colors) have equal probability to absorb light and transfer the excitation energy to its neighbors, which dramatically decreases the anisotropy. At long wavelength edge (case c) the species absorbing and emitting the low energy quanta are excited only. They do not exhibit EET and their emission is highly polarized

high values (demonstrating suppression of EET) by shifting the excitation to the red (long wavelength) edge of absorption band (Fig. 8.5). This phenomenon that was first observed by Gregorio Weber in 1970, can be explained by the effect of photoselection within an ensemble of fluorescence emitters connected by EET (Demchenko 2002). Even if these emitters are the same molecules and exhibit homo-EET, their local environments and interactions with these environments differ. The recorded absorption spectrum being a superposition of individual spectra is “inhomogeneously broadened”. At the band maximum and shorter wavelengths the energy of incident light is sufficient to excite any fluorophore in the ensemble and transfer this energy to any other fluorophore, which results in low anisotropy of emission. At the ‘red edge’ the situation is different. The incident light selects those emitters that have the lowest excitation energy and, correspondingly, the lowest energy of emission that cannot be transferred to any other fluorophore. So, the species selected at the red edge are unable to serve as EET donors and they emit themselves highly polarized light (Demchenko 2002, 2008). Thus, the drop of anisotropy occurring on homo-EET is quite different from that observed on fast rotational motions, so that ‘anisotropy sensing’ becomes not possible

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in the systems exhibiting EET. Distinguishing the two causes of depolarization (rotations or EET) is possible only by variation of excitation wavelength. Fluorophore rotations should not depend on the excitation energy, but in the case of homo-EET the repolarization occurs at the red edge of excitation spectrum. This effect opens the ways of manipulating with emission anisotropy by creating or avoiding the EET conditions. It has got interesting application in fluorescence polarization microscopy for detecting the interactions between the molecules within the living cells (Squire et al. 2004). Homo-EET allows single labeling in contrast to more difficult double labeling that is normally used for observing proximity effects in a hetero-EET system.

8.2 Highly Ordered Nanoscale Associates of Organic Dyes (H- and J-Aggregates) When organic dyes associate into highly ordered structures, they attain collective properties that are described by the theory of molecular excitons that are often called the Frenkel excitons. Here due to the absence of essential dielectric screening the electron-hole pairs demonstrate strong Coulomb attraction. Therefore, being different from excitons in semiconductor QDs (see Sect. 7.2) they are also called the strongly bound or small-radius excitons. As the neutral quasiparticles, they may propagate through the aggregate, while at each instant of time the electron and the hole occupy the same molecular site. This process, by which excitons spread in a wave-like manner over spatially separated molecular dyes is called coherent exciton delocalization (Scholes and Rumbles 2006). The simple model (Fig. 8.6) linking the geometry and photophysical behavior of aggregates of aromatic molecules was suggested by Michael Kasha (Kasha 1963; Kasha et al. 1965; McRae and Kasha 1958). Based on exciton theory (Davydov 1971), he studied the effect of relative orientation of two molecules approximated as point dipoles, so that the transition dipole is placed along the long axis of the molecules. Surprisingly, this model came out to be very productive in explaining major spectroscopic effects (Bricks et al. 2017) and became the basis of many new developments. The model suggested the presence of two types of molecular associates with different orientation of constituting dipoles, in line with experimental data. These structures are known as J- and H-aggregates. On transition from monomeric forms to these aggregates their emissive state changes from monomeric to excitonic. The results demonstrated the splitting of the excited state energy into two levels occurring due to electronic state degeneracy, and the angle between the transition dipoles determines whether a transition is allowed to the higher or to lower of these levels.

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Fig. 8.6 The illustration of exciton model suggested by Michael Kasha for the variations of energy on electronic transitions of molecular dimers. The H-type dimers, formed by face-to-face association, demonstrate the strongly increased energy separation between absorbing and emitting states and, due to forbidden character, their fluorescence is low. In contrast, in J-type dimer the transition dipoles are in line with ladder-type overlap. They exhibit a strong decrease in transition energy and an increase in the transition dipole moment enhancing the probability of light absorption together with the red-shifts in the absorption and emission bands. For simplicity, the ground state energies are shown on the same levels for monomer and dimers. Note the absence of absorption to lower level of H-aggregates and to higher level of J-aggregates

8.2.1 Excitonic J-Aggregates The basic spectroscopic characteristics of J-aggregates that are observed already on the level of several coupled monomers are really unique (Fig. 8.7). They demonstrate very narrow and almost symmetric J-bands in absorption spectra (only 10–20 nm

Fig. 8.7 Schematic representation of the changes in absorption (blue) and fluorescence (red) spectra on the formation of H- and J-aggregates from cyanine dye monomers (shown above). For Haggregate the absorption spectrum shifts to shorter and fluorescence spectrum to longer wavelengths generating gigantic Stokes shift. For J-aggregates, both absorption and fluorescence spectra become very narrow and strongly shifted to longer wavelengths. Vibronic structure or broading is absent and the Stokes shift is very small

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in width) with their dramatic (often by ~100 nm) shift to the red in comparison to the spectra of monomers (Knoester 2007). If two or more narrow J-aggregate bands are found, they can be attributed to different packing of monomers in the aggregate (Kirstein and Daehne 2007). Characteristic for J-aggregates is the strongly increased molar absorbance (Eisfeld and Briggs 2007), so that with the long-chain cyanine dyes emitting in the near-IR region it can reach several millions. Two-photon absorption cross-section σa can also achieve very high values, reaching several thousands and even millions of GM units and dramatically exceeding that of the monomers (Belfield et al. 2006). Unique are also the fluorescence properties of J-aggregates (Losytskyy and Yashchuk 2010). Their fluorescence spectra match the absorption spectra and are of equally narrow width. Demonstrating nearly resonant behavior, they display very small (~200 cm−1 and often smaller) Stokes shifts and insensitivity to the environment (Würthner et al. 2011). The strong increase of absorbance is coupled with the increase of radiative rates, therefore their fluorescence lifetimes are much shorter than that of monomers and are observed on picosecond time scale (Obara et al. 2012). The latter effect is often called “superradiance” (Fidder et al. 1990). The J-aggregates demonstrate high optical anisotropy resulting in polarized fluorescence emission (Kirstein and Daehne 2007; Scheblykin et al. 1996). The sharp absorption band is polarized parallel to the aggregate axis. The emission quantum yield of J-aggregates is variable in broad ranges and in some cases it is much higher than that of constituting monomers. Table 8.1 lists the most essential spectroscopic properties of J-aggregates that are dramatically different from constituting dyes and their irregular assemblies. The excitonic nature of electronic transitions in J-aggregates is responsible for their optical properties. These properties can be conceived within the frame of Davydov’s theory of molecular excitons (Davydov 1971). Different aggregate models Table 8.1 The most essential spectroscopic properties of J-aggregates of organic dyes Light absorption

Fluorescence

– Very narrow J-bands in absorption spectra (only 10–20 nm in width) – Dramatic shift of these bands (often by ~100 nm) to the red with respect to the spectra of monomers – The spectra are insensitive to the environment – The absence of vibrational component in broadening, so these bands are very narrow and almost symmetric – Strongly increased molar absorbance, so that with the long-chain cyanine dyes it can reach several millions (M−1 cm−1 ) – Strongly increased two-photon cross-section, reaching the values of ~10,000 GM

– The spectra match the absorption spectra and are of very narrow width – The Stokes shifts are very small (~200 cm−1 and less) or even negligible – The spectra are insensitive to the environment. – Fluorescence lifetimes are much shorter than that of monomers, “superradiance” – The emission quantum yield is variable in broad ranges and is commonly higher than that of monomers – The polarization of fluorescence emission is high and can reach the limiting values – Collective behavior is present in fluorescence quenching, “superquenching”

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presented so far suggest the dye arrangement in the “staircase”-type configuration in the J-aggregate predicted by Kasha (Fig. 8.6). It allows more optimal stabilization of aggregate by intermolecular interactions and also stronger electronic coupling of collectively emitting monomers (Birkan et al. 2006; Lebedenko et al. 2009). The most typical J-aggregates are the aggregates of cyanine dyes (Bricks et al. 2017). The applications of fluorescent J-aggregates to chemical sensing and biosensing suggest new prospective solutions. Three major principles are currently realized in these applications: the J-aggregate formation/disruption, the superquenching and the operation of smart EET-based composites. All of them demonstrate dramatic targetinduced variation of an output signal (Fig. 8.8). Several examples are presented below. Dramatic spectroscopic changes on the formation-disruption of J-aggregates are seen on the dye interaction with proteins and DNA (Tatikolov 2012), and this binding was found to be target-specific (Zhang et al. 2007). Detection of different ligandbinding proteins located on cell surface was also suggested (Hou et al. 2015). One of the applied probes was efficient in imaging the tumor cells by detecting the hypoxiainduced cancer-specific biomarker—the transmembrane-type carbonic anhydrase IX. The formation by positively charged cyanine dyes of J-aggregates on cell membranes depends on the membrane charge. This allows realizing high sensitivity of fluorescence response for sensing the electrostatic membrane potential. The a

Target

Monomers

b

J-aggregate

Target J-aggregate emissive

c

J-aggregate quenched

Target No EET

EET

Fig. 8.8 The principles employed in fluorescence sensing with J-aggregates. a The dye binding to target induces the formation or dissociation of J-aggregate generating strong wavelength-ratiometric response due to switching between monomeric and excitonic emissions. b The target induces (or removes) the superquenching effect (quenching of fluorescence of the whole J-aggregate on suppressing the emission of only one monomer). c The target induces or suppresses the EET to or from corresponding molecule or nanoparticle

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hyperpolarized mitochondria (i.e. where their interior is more negative) accumulate more cationic dye, and depolarized mitochondria (interior is less negative) accumulate less dye. The very popular “slow-responding” probing of biomembrane potential by trimethine cyanines, such as JC-1 and similar dye JC-9 (Haugland 2002), is based on assembly-disassembly of J-aggregates. The linear increase in their concentration in the range 30–180 mV is observed (Mathur et al. 2000). The response time of the measurement, however, is limited by diffusion of dye through the membrane that needs, at least, seconds and even minutes. The superquenching effect is the phenomenon observed when minute amounts of analyte interacting with only one member of this ensemble quenches efficiently the fluorescence emission of the whole dye ensemble (as discussed in Sect. 8.1.4 above). Though this effect for J-aggregates is known for more than 80 years, its employment here is rather limited (Jones et al. 2001; Lu et al. 2002b), probably due to low stability of tested species. Polyelectrolyies with appended cyanine dyes forming J-aggregates were suggested for these studies (Kato et al. 2010; Lu et al. 2002a) and they may allow overcoming this problem. Other prospective ideas on increasing the J-aggregate stability is their inclusion into hollow mesoporous silica nanoparticles Chen et al. (2019). Finally, the J-aggregates may serve efficiently as donors or acceptors in different nanoscale systems employing EET (see Chap. 10). Different configurations from composed nanoparticles (Halpert et al. 2009) and nanotubular constructs (Qiao et al. 2015) to blended films (Walker et al. 2010) are possible, and the whole broad range of molecular and nanoscale luminophores can be used as the partners of J-aggregates in the formation of EET pairs. These possibilities still remain poorly explored. Among the applications for cellular and in vivo imaging, the most attractive are the near-IR absorbing J-aggregates developed for two-photon fluorescence (TPF) microscopy. They are able to be excited by light penetrating deeply into tissues and to form images on the background of low light scattering. The strong, by several orders of magnitude, enhancement of the two-photon absorption cross-section was observed on J-aggregate formation of cyanine dyes (Belfield et al. 2006), as well as of porphyrin derivatives (Ray and Sainudeen 2006). The high stability of two-photonic cyanine dyes was achieved by interaction with cationic polymer (Kato et al. 2010). Super-resolution fluorescence microscopy (nanoscopy) (Hell 2003) is still an open field for application of J-aggregates. Needed for some of wide-field localization techniques collective fluorescence blinking (“superblinking”) has already been demonstrated; it was connected with the long-range exciton migration (Lin et al. 2009). For the applications in STED-type techniques, the high photostability of fluorescence emitters is strongly desirable, and the J-aggregates seem to be much more photostable than their constituting monomers. Bright perspective of J-aggregates for in vivo imaging can be illustrated by the story with Indocyanine Green (ICG), the dye approved for application in humans and that is actively used for contrasting blood vessels. Its low chemical stability and photostability is a great problem, and the formation of J-aggregates result in considerable improvement. The J-aggregates of ICG can be stabilized by their incorporation into liposomes and polymer micelles (Shao et al. 2019).

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Self-organization of dye molecules into precisely ordered J-aggregate structures allows obtaining extraordinarily high cross-sections for absorption of one and two photons of light. Other properties are also dramatically different from that of constituting molecules, including very sharp red-shifted excitation and emission spectra with very small Stokes shifts. The uniqueness of J-aggregates is highlighted by special terms that were introduced to describe their properties: “superradiance”, “superquenching”, “supersensitizing”, “superblinking”. Deserving such description on a supreme level, they must find their proper role in technologies exploring photophysical processes and fluorescence response.

8.2.2 Emissive and Quenched H-Aggregates The H-aggregates are the highly ordered face-to-face stacked dye structures that potentially are more advantageous than the dye monomers in different applications. The energetic advantage of forming the H-aggregates in multi-chromophore assemblies is their arrangement in sandwich-type stacks stabilized by attractive dipoledipole interactions, in which the transition dipoles could be parallel and inversely oriented to each other. They can collectively act as the donors or acceptors for other groups of molecules to which they are weakly coupled. Meantime, their employment in sensing and imaging is only on the beginning step. According to Kasha model (Fig. 8.6), their emissive transition is forbidden. However, their bright fluorescence emission is observed in many cases. Many factors are responsive for that. There are the non-ideal relative location and orientation of coupled fluorophores, intramolecular vibrations and their coupling, the coupling with lattice vibrations (phonons) (Hestand and Spano 2018; Spano and Silvestri 2010). The thermal motions and dynamic disorder do not influence substantially their properties. Moreover, identity of constituting monomers is not strictly required (Bialas et al. 2016). We have to note also that in excitonic systems the emission proceeds from exciton traps, which are the states with the changed geometry in locations of atoms. Figure 8.9 allows comparing the electronic state energy diagrams for isolated fluorophore molecules and for the H-aggregates of organic dyes. In all the cases the ultrafast process of light absorption drives the electronic system to the excited state, but in H-aggregate due to excitonic effect the S1 level is split and only the upper level can be excited, as it is shown on dimer model (Fig. 8.6). Here the propagating exciton is coupled with the phonon modes. The motions of nuclei coupled with phonons result in the exciton self-trapping that occurs on a very fast (sub-picosecond) time scale with a change of nuclear geometry (Schubert et al. 2013). Thus, both electronic and nuclear sub-systems are reorganized in time on an ultra-fast scale, generating the self-trapped excitons that become the emissive species. The fate of such exciton is diverse. It can emit light, propagate in a diffusive manner, be quenched or exhibit the intersystem crossing into the triplet state T1 with the emission of phosphorescence. The result of such photophysical transformation is very unusual. The absorption is dominated by completely delocalized excitations, while fluorescence comes from

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Vibrational relaxation

Monomer

Intersystem crossing Fluorescence

Forbidden transition

Selftrapping Absorption

Fluorescence

Absorption

S0

Intra-band relaxation

Splitting

T1

Phosphorescence

S1

H-aggregate

Fig. 8.9 Simplified Jablonski diagram for the energy transformations on electronic excitation in the case of molecules in monomer forms and their H-aggregates. In monomers the fluorescence emission proceeds from S1 state after vibrational relaxation. In contrast, in H-aggregates the S1 level is split and the excitation proceeds to the upper level, which explains the short-wavelength shifts of absorption spectra. Then the intra-band relaxation drives the exciton energy to a lower level, from which the emission is formally forbidden. However, due to imperfect monomer matching, their vibrational dynamics and exciton trapping the fluorescence emission can proceed with the spectral shift to longer wavelengths and, after intersystem crossing, the phosphorescence emission becomes possible

the recombination of self-trapped excitons at the localized sites (Franco and Tretiak 2004). That is why the emission is shifted to longer wavelengths but, in contrast to J-aggregates, its spectrum is rather broad. The localized (self-trapped) states can produce highly polarized emission. Thus, the broad short-wavelength shifted absorption spectra and the broadened long-wavelength shifted emission bands are considered to be characteristic for the π- π-stacked H-aggregates (Anantharaman et al. 2017; Sharma et al. 2017). In single crystals of para-distyrylbenzene, the quantum yield of H-aggregate emission can reach 78% (Anantharaman et al. 2017). Rigid dye environment in aggregate can contribute to suppressing the non-emissive relaxation channels. It was reported that such rigidity and screening from quenching by water can be achieved by complex formation of H-aggregates with calix[4]arenes (Harris et al. 2016) or cyclodextrins (Hong et al. 2011). Both formation-disruption of H-aggregates (Mudliar and Singh 2016) and reversible transformations between H- and J-aggregates (Sahoo et al. 2017) can be used for generating the sensor response. Such response can be brightly seen not only in fluorescence but also in light absorption, which, in principle, allows the naked-eye observation.

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8.3 Aggregation-Protected and Aggregation-Induced Emission Being soft materials, organic dyes can aggregate in solutions due to weak interactions between them and under hydrophobic force that can be strong in aqueous media. Such aggregation can be observed for a much larger number of dye structures than for those forming H- or J-aggregates. By incorporating a large number of molecular fluorophores in nanoparticles, the ultra-bright nano-sized emitters can be obtained. Ideally the assembly of 1000 dyes may provide the 1000-times higher brightness than a single dye. Therefore, such form of ultra-bright nanoparticles must be very efficient substitutes of monomeric dyes that must greatly increase the range of applications of fluorescence sensing techniques. There are several possibilities to design emissive dye aggregates (Fig. 8.10), their formation is discussed below.

8.3.1 Dye Aggregates: Emissive and Quenched It is a textbook knowledge that the organic dye aggregation generally quenches the light emission (Birks 1970). The electronic interactions between closely located fluorescent dyes result in a process known as aggregation-caused quenching (Hong et al. 2011). It causes great problems dissipating nonproductively the excitation energy. As discussed above, the mechanisms of such quenching and the possibilities of its prevention in dye-doped nanostructures are important to be discussed and accounted in nanoparticle design. Some authors (Battistelli et al. 2016) suggest to distinguish the aggregation-caused quenching (ACQ) and proximity-caused quenching (PCQ). Thus, the photoinduced electron transfer (PET) quenching discussed in Sect. 4.1 should belong to ACQ and the quenching through distance by energy transfer (see

a

b

c Water

Water

d Water

-

-

-

Fig. 8.10 Creation of emissive dye aggregates. a Dye precipitation on addition of water to dye dissolved in ‘good’ solvent with generation of aggregation-induced emission (AIE). b Formation of micelles by charged or amphiphilic dyes containing hydrophobic substituents. c Self-association of dyes with modifications preventing formation of π-π stacked association. d Association of charged dye molecules produced by hydrophobic counter-ions

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Fig. 8.3) to PCQ. For avoiding the PET quenching, one has to construct the nanoparticles, in which the close presence of strong electron donor or electron acceptor groups is eliminated. The other typical mechanisms of quenching are that involving the formation of non-emissive H-aggregates in the ground state and the exciplexes, which are the intermolecular complexes in the excited states requiring definite stoichiometry (Davidson 1983). Such stoichiometry should be avoided on the dye aggregation. The fluorescence quenching can be observed even in the absence of direct intermolecular interactions at the ground or excited state, just because of simple proximity of the dyes in ensemble. We could see (Fig. 8.3) that efficient homo-EET between the dyes that can be located at a distance of several nanometers in the presence of non-emissive EET acceptor leads to complete quenching. Thus, the nanostructures not only with close packing of the dyes but also with their location within nanometer distances may not be good emitters. The aim of researcher is to avoid such energy traps. Are there exceptions to the rule that emission of dyes in their solid aggregated states should be always quenched? Hopefully there are a number of organic dyes emitting in the solid state (Anthony 2012; Shimizu and Hiyama 2010), and their great popularity is attributable to their applications in organic photovoltaics and other energy conversion devices. Those are the borate complexes of ionic dyes (Frath et al. 2014) and some dyes displaying excited-state intramolecular proton transfer (ESIPT) (Carayon and Fery-Forgues 2017; Padalkar and Seki 2016). They demonstrate attractive optical properties, such as high absorption coefficients, red-shifted emission wavelengths and high quantum yields. Obtaining and stabilizing the formed nanoparticles in solutions may involve technical problems. Their successful outcome was reported based on analysis of various chemical structures, even resulting in formation of the nanoparticles of different shapes (Carayon and Fery-Forgues 2017). Of particular interest are the nanoparticles formed of BODIPY dyes assembled spontaneously in aqueous solutions (Zhang et al. 2019).

8.3.2 Modifying Intermolecular Interactions on Dye Aggregation In the efforts for establishing the brightest nanoparticle formation technologies extendable to broader range of organic fluorophores, the scientists followed different strategies that allow excluding the contact quenching. It is important to find the ways for self-assembly of composing units in a well-controlled manner avoiding the ππ stacking and making the assembled structures stable in the conditions of their use. This aim was found to be achievable by introduction into the dye structures of the bulky substituents that induce steric hindrance and in this way reduce direct interaction between aromatic groups (Fery-Forgues et al. 2008; Yao 2010).

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At the forefront of this approach are the BODIPY dyes (Gayathri et al. 2019; Solomonov et al. 2019; Xiao et al. 2012) possessing excellent photophysical properties and high stability. The very attractive selection of their fluorescence spectra covering the broad visible-near IR range with very high brightness and photostability can be explored on nanoscale level. Also important are the chemically stable and photostable pyrene (Moorthy et al. 2007) and perylene bisimide dyes (Langhals et al. 2005). The non-planar dye conformation resulting in amorphous arrangement appears to be the best suited for the solid-state emission. Some of the dyes demonstrating bright fluorescence in the solid state can be arranged in crystalline structures (Fery-Forgues 2013). The crystalline dye arrangement can control the emission properties demonstrating strong differences from amorphous materials. Polymorphism, which is the ability of a molecule to adopt different crystal structures, provides many opportunities to tune optical properties. The molecular arrangement and conformation may be governed by the way the solid is formed and become responsible to a chemical or physical stimulus. The properties of responsive solid-state materials based on organic dyes will be discussed in more detail in Chap. 11. The bulky hydrophobic counter-ions can be used to cement the nanoparticle structures formed by ionic dyes and in this way to eliminate the direct contact between the dyes that could result in quenching (Soulié et al. 2015). It was shown that brightly fluorescent nanoparticles can be made of subjective to self-quenching aggregation cationic rhodamine dye by mixing this dye with hydrophobic anion tetraphenyl borate (Reisch et al. 2014), Fig. 8.11. Fluorinated tetraphenylborate counter-ions were found to be ideal in formation of nanoparticles of rhodamine dyes (Shulov et al. 2015). Importantly, in most of the studied cases there are no essential transformations of spectra on formation of nanoscale aggregates. This is seen, for example, in nanoparticles formed of perylene bisimide dyes, in which the π-π stacking interactions were avoided by micelle-like aggregate structure (Langhals 2008). Similar observation was made with particles made of rhodamine dyes stabilized by tetraphenylborate anion (Reisch et al. 2014). Meantime, their collective spectroscopic response was demonstrated as the two-state light-dark blinking. Being analyzed together, these observations show efficient exciton diffusion by EET mechanism but the absence of exciton coupling.

8.3.3 Aggregation-Induced Emission (AIE) When organic dyes are studied in solution, there are many different pathways for non-emissive decay to the ground state and thus the observation of strongly quenched emission (de Silva et al. 1997). That is why many conventional dyes with intramolecular flexibility are nonfluorescent in solutions or display very low intensity and ultra-short emission decay. Dynamic interactions with solvent molecules, especially involving the hydrogen bonding, excited-state reactions with the participation of solvent, and other factors, suppress their emission in solutions. On the other side, as

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Fig. 8.11 Design of dye-doped fluorescent nanoparticles using bulky counter-ions (Reisch et al. 2014). a Chemical structures of rhodamine B octadecyl ester (R18) and its different counter-ions: perchlorate (ClO4 ), tetraphenylborate (TPB), tetrakis(4-fluorophenyl)borate (F1TPB) and tetrakis(pentafluorophenyl)borate (F5-TPB). b Schematic representation of PLGA polymer nanoparticles loaded with these dye salts

we discussed above (Sect. 8.1.4), different mechanisms are involved in fluorescence quenching on dye-dye association and in solid state. In 2001 Ben Zhong Tang and colleagues have found that there are the dyes that are nonfluorescent in solutions but attain bright fluorescence upon formation of nanoscale aggregates (Luo et al. 2001). This phenomenon was called aggregation-induced emission (AIE). This discovery has led to tremendous developments in the fields of fluorescent materials (Mei et al. 2015), and design of novel sensors (Gao and Tang 2017) and probes for biology (Hu et al. 2014; Kwok et al. 2015). Initial observation of Tang was the following (Luo et al. 2001). The dye 1-methyl1,2,3,4,5-pentaphenylsilole (Fig. 8.12a) that was insoluble in neat water was first dissolved in a good solvent, such as THF. Addition of varying fractions of water first did not result in any change, but at some increased concentration the aggregates appeared, demonstrating bright emission (see Fig. 8.12d–e). This approach of particle formation is called reprecipitation by solvent exchange. Active research that followed allowed discovering many other molecules with such unexpected properties and drawing some general regularity (Hong 2016; Mei et al. 2015). Typically, the dyes demonstrating this effect possess the propellerlike structures with flexible phenyl rings. The most frequent design of so-called AIE-fluorogens involves the presence of aromatic groups connected by rotatable C– C, C–N or N–N single bonds. As isolated molecules, they undergo low-frequency torsional motions and due to strong intramolecular mobility they emit very weakly

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Fig. 8.12 Illustration of aggregation-induced emission. a Tetraphenyl silole polymer forming stable AIE nanostructures. TPAFN (b) and TPETPAFIN (c) were used as photostable organic dots for longterm cell tracking (Li et al. 2013). Their spectra for TPETPAFIN particles excited at 500 nm (d). They are formed with the appearance of strong fluorescence (inset) on the increase of concentration of water. e The increase of their fluorescence dependent on fraction of water

in solutions. In addition, the fluorescence quenching in solution may result from cistrans isomerizations, intramolecular charge transfer (ICT) or twisted intramolecular charge transfer (TICT) (see Sect. 4.1). Their aggregates show strongly enhanced fluorescence mainly due to the restriction of their intramolecular rotations in the aggregate state and elimination of all these factors. Up to present, a large number of new AIEfluorogens have been designed and synthesized, such as silole, tetraphenlyethene (TPE), triphenylamine (TPA), 9,10-distyrylanthrancene (DSA),1,4-distyrylbenzene (DSB) and their derivatives. Why their emission is not quenched in aggregate? The irregular arrangement of flexible segments in the aggregate does not allow forming their stacked arrangement leading to quenching. The emissive J- or H-aggregates are also not formed, which is evidenced from obtained spectra (Fig. 8.12d). The nanoparticles made of dyes with enhanced emission show only the changes in intensity (with the quantum yield reaching 50%), whereas the spectral changes compared to low-intensity spectra of molecular forms are rather small or even nonexistent. However, it was reported that AIE can modulate the excited-state reactions in constituting dyes, as it was shown with the excited-state intramolecular proton transfer (ESIPT) reactions (Kaur et al. 2019).

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The sensors based on such intensity changes of these dyes were suggested for exploring the aggregation-dissociation effect (Ding et al. 2013; Zhang et al. 2014). In numerous research papers and reviews (Gao and Tang 2017) it was shown that the AIE systems can find much broader range of application and their major areas were outlined. They are: (a) self-assembly with analytes to form aggregates; (b) specific recognition with analytes to restrict intramolecular motion; (c) cleavage of dissolution-promoting ligands to decrease solubility leading to aggregates formation; (d) disruption of the photophysical quenching processes. The studies of small propeller-like molecules were extended to polymers (Qin et al. 2012). Different polymer molecules that possess highly flexible fragments are soluble and swell in organic solvents. They demonstrate limited solubility when they are transferred to water (Jang et al. 2006). At high water content they form nanoparticles, so that their internal structures becomes compact with immobilization of rotating segments. With the increase of water content in mixed solvent, their fluorescence intensity increases dramatically. Thus, presented in Fig. 8.12 tetraphenylsilole polymer (a) forms stable AIE nanostructures (dots). The AIE fluorogens, TPAFN (b) and TPETPAFN (c), were designed to have high brightness in aggregate state. Bearing two extra TPE units to TPAFN, TPETPAFN has more freely rotated rotors to populate the nonradiative decay channels (e.g., internal conversion) in ‘good’ solvent and thus produces a more pronounced AIE effect upon nanoparticle formation (Li et al. 2013). It was shown that the formed dots are chemically stable and photostable in vivo. They were used for long-term cell tracking. These AIE fluorophores were usually further modified to improve their performance as imaging agents. A different mechanism for achieving the transition from nonemissive state of dye monomers to bright emission of their nanoscale aggregates was demonstrated for cyanostilbenes. Soo Young Park and colleagues using the reprecipitation approach obtained the nanoparticles formed by self-assembling of this dye. The particles of 30–40 nm in size demonstrated strong enhancement of their fluorescence, and this phenomenon was called “fluorescence enhanced by aggregation” (An et al. 2002). These molecules are nonfluorescent in solution because of twisted conformation produced by the steric effect of cyano-group. In the aggregated state this group becomes planar, which restores the electronic conjugation favoring fluorescence. Different molecules possessing cyanostilbene motif [–Ar–CH=C(CN)–Ar–] (where Ar stands for different aromatic substituents) share these unique properties. In contrast to AIE, the electronic nature of the excited state is changed. The spectroscopic data demonstrate the red shifts of both absorption and fluorescence bands, witnessing that the molecules within the particles are assembled form J-aggregates (An et al. 2002). They start to display typical excitonic properties. The role of cyano group is essential. Favoring the formation of J-aggregates, it prevents the appearance of stacked nonfluorescent H-aggregated structures. Thus, planar configuration together with J-aggregated arrangement renders cyanostilbenes in nanoparticles to become strongly fluorescent. Further developments have shown that cyano motive, if it is present in different fluorescent dyes, favors formation of aggregates of this type. Even more, through programmed self-assembly, the preparation of 1D nanowire structures becomes

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possible (An et al. 2012). This opened the new ways for different technological applications, including fluorescent probes and sensors. Future studies on the design of cyanostilbene-based intelligent materials are also predicted. The blossoming of cyanostilbene-based intelligent hybrids promises a new generation of integrated multifunctional materials, an outcome which is essential for the development of responsive materials that interface with physics, materials chemistry, biology, and medical science (Zhu and Zhao 2013).

8.4 Nanoscale Assemblies Supported by Organic Polymers and Silica Matrices The goal to be achieved here is straightforward: to create fluorescent nanoparticles containing a high density of organic dyes, to provide them high colloidal and chemical stability, to improve their spectroscopic properties and photostability and to realize better possibilities for their functionalization. This task can be realized on incorporating organic dyes into inorganic or organic polymers.

8.4.1 Compositions of Organic Dyes Based on Organic Polymers The dyes can be either located inside the nanoparticles formed by polymer chains or attached to their surfaces by physical adsorption and by covalent binding to the surface-exposed groups. The techniques for preparing nanoparticles of organic polymer are well developed. The dyes can be incorporated into them either during or after the synthesis of polymer macromolecules (Borisov et al. 2010). The porous nanoparticles are the most attractive in this respect, since they exhibit the highest surface-to-volume ratio and the cavities that are suitable for efficient dye incorporation making relatively strong multi-point contacts. These particles can be obtained based on a variety of synthetic polymers: polystyrene, polymethyl methacrylate, polyacrilic acid, polyvinyl chloride, etc. Usually this is done by micro-emulsion polymerization (Landfester 2006). Microemulsions are the specially formulated hetero-phase systems, in which the stable micro-size or nano-size droplets of one phase are dispersed in a second, continuous phase (Fig. 8.13). Each of these nanodroplets can be considered as nanoscopic individual batch reactor. Because of such confinement, a whole variety of reactions and processes can proceed resulting in both organic and inorganic nanoparticles. This procedure allows controlling the size of polymerized particles. Surfactants are used for formation of these nano-scale heterophase droplets, and ultrasound treatment helps for dispersing them. With this technique, different kinds of polymerization

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Fig. 8.13 Schematic presentation of nano-reactor formed in water-in-oil microemulsion for synthesis of polymeric nanoparticles

reactions, such as radical, anionic, and enzymatic polymerization, as well as polyaddition and polycondensation, can be carried out. Microemulsion technology is highly suited for the encapsulation into the particles of various organic and inorganic, solid or liquid materials, including fluorescent dyes. There are many advantages of polymeric nanoparticles (Reisch and Klymchenko 2016). In addition to increased brightness by many orders of magnitude, they demonstrate well-controlled surface properties and remarkable stability in biological environments. Polystyrene latexes that form stable dispersions in water and other polar media are used in research for a long time. With incorporation of different dyes, they can be made highly fluorescent. Their surface can be modified by amino and amidino functional groups introduced by the co-monomer and the initiator 2, 2 -azobis (2-amidinopropane) dihydrochloride. Fluorescent dye N(2,6-diisopropylphenyl)perylene-3,4-dicarboximide was incorporated into the copolymer nanoparticles formulated from styrene and acrylic acid or styrene and aminoethyl methacrylate hydrochloride (Holzapfel et al. 2005). The resulting latexes were stable and showed a uniform size distribution within the range 100–175 nm. These functionalized fluorescent particles are commonly used as markers for living cells, and their cell uptake was visualized using fluorescence microscopy. A simple technique of doping the nanoparticles after their synthesis can also be applied. It involves swelling the particles in organic solvent containing the dye and then drying. Being transferred to water, these particles retain hydrophobic dyes, but the restriction exists for their use in low-polar solvents due to the possibility of the dyes leaking. Preparation of polymer nanoparticles that form stable dispersions in nonpolar media can also be made. Several synthetic routs for that have been described in the literature, particularly, for the incorporation of pyrene (Tauer and Ahmad 2003). Special attention in recent literature has been paid to the production of particles that contain luminescent metal chelating complexes, such as Eu(III) complexes of beta-diketonates. As we observed in Sect. 6.3, such complexes have long decay times,

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large Stokes shifts, and very narrow emission bands in comparison with organic fluorescent dyes. Upon incorporation into polymer matrix these properties are preserved (Huhtinen et al. 2005). The template-directed polymerization that is often called ‘molecular imprinting’ (Volume 2, Chap. 6) is a very promising approach for the preparation of selective and high-affinity binding sites for different targets. In this way, the polymer nanoparticle can serve not only as a support and reporting element but also as recognition functionality.

8.4.2 Organic Dyes Incorporated into Dendrimers Dendrimers are currently attracting the interest of many scientists because of their unusual chemical and physical properties and the wide range of potential applications. Many possibilities for such applications exist in sensing technologies (Augustus et al. 2017; Bergamini et al. 2010). Dendrimers are unique molecules of high complexity and order that possess precise and gradable range of dimensions within the range of several nanometers. They are highly branched spherically shaped polymeric structures that emanate from a central core (Sowinska and Urbanczyk-Lipkowska 2014). General principle of arrangement of a dendrimer is illustrated in Fig. 8.14. Typical dendrimer contains a core monomer, from which many branches stem in all directions producing a nanoscale structure of spherical shape (Grayson and Frechet 2001). It is hard to believe that a rather simple synthetic chemistry can provide the materials of such regularity and beauty. From a topological viewpoint, dendrimers contain three different regions: core, branches, and surfaces. Each branch (dendron) can be further expanded by sequential branching with the addition of other layers of monomers, forming in this way the dendrimer of higher sequential order, a ‘generation’. Since these molecules are produced in an iterative reaction sequence, they lack polydispersity, so that each molecule has the same number of atoms and the same shape. The typical and the most frequently used dendrimer is the water-soluble poly(amidoamine), PAMAM (Soršak et al. 2015), exposing amino groups on its surface (Scott et al. 2005) (Table 8.2). Sometimes, because of different polarity of inner volume and the surface, these structures are called ‘single-molecular micelles’. Their surface amino groups are exposed for covalent attachment of fluorescent dyes (Oesterling and Mullen 2007); they are also the sites for covalent links in nanocomposite formations. There are other even more attractive possibilities to make the dendrimers fluorescent. First is by inserting selected chemical units in predetermined sites of the dendritic architecture, which can be done on the step of synthesis. Second, luminescent moiety can serve as a dendrimer core, around which the branches can grow. Dendrimers formed on the basis of Ru(bpy)2+ 3 as a core exhibit characteristic phosphorescence that is protected from external quenchers by the dendrimer branches and sensitized by attached dyes on the periphery of dendrimer (Balzani et al. 2000a).

8.4 Nanoscale Assemblies Supported by Organic Polymers and Silica …

a

333

Generations: n

N=4

N=3

N=2

N=1

Number of terminal groups: m m=6

m = 12

b

m = 24

m = 48

c

d

Fig. 8.14 Formation and structures of dendrimers. a The steps of sequential growth of a typical dendrimer by adding monomers to the surface-exposed groups. During their synthesis, each added monomer bound at one exposed terminal group exposes two new groups for binding in sequential generation. b The structure of dendrimer containing porphyrin core and benzene rings forming the surface. c The structure of 3-d generation (n = 3) of PAMAM dendrimer exposing amino groups. d Schematic presentation of the dendrimer structure and notation of its basic elements (Bergamini et al. 2010)

Table 8.2 Physical properties of PAMAM dendrimers Generation Number of surface groups Number of tertiary amines Molecular mass Diameter (Da) (nm) 0

4

2

517

1.5

2

16

14

3256

2.9

4

64

62

14,215

4.5

6

256

254

58,048

6.7

8

1024

1022

233,383

9.7

10

4096

4094

934,720

13.5

Moreover, thanks to their flexible three-dimensional structure, the dendrimers are able of encapsulating in their internal dynamic cavities the ions or neutral molecules, including fluorescent dyes (Balzani et al. 2000a; Shcharbin et al. 2007) and luminescent metal ions (Balzani et al. 2000a; Bergamini et al. 2010). It is thus possible to construct nanocomposites, in which large number of different dyes can be located in

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pre-determined sites. Therefore, coupling luminescence and dendrimer chemistry can lead to interesting photophysical properties (Bergamini et al. 2010; Campagna et al. 2011), such as (1) interactions of dendritic luminescent units in the ground and/or excited states (possible formation of excimers and/or exciplexes), (2) quenching of the whole dendrimer luminescence by external species, and (3) sensitization of luminescent metal ions or dyes encapsulated by the dendrimer. The dendrimers have started to find an application similar to that of organic polymers and silica as the hosts of organic dyes. The advantage in this case is the welldefined shape and chemical organization of these structures. They allow shielding the dye molecules from their direct contacts with each other, avoiding quenching of their emission. The flexibility of dendrimeric structures is also exploited, and this allows penetration from outside into their interior of small target molecules. In addition, a broad range of functionalization is possible for the introduction of target-recognizing groups into these structures. The dendrimers containing high amount of fluorescent label find application in DNA hybridization assays, improving or replacing the organic dye-based assays (Caminade et al. 2006). An immunoasssay based on the composed antibodyconjugated PAMAM-dendrimer-gold quantum dot complex was also suggested (Triulzi et al. 2006). Due to the possibility of easy and efficient modification of their surface, different groups recognizing small molecules and ions can be attached to this surface. In the case of their membrane permeation ability (Khandare et al. 2005), PAMAM dendrimers, especially those of higher generation, have good prospects for in vivo biological applications. There is also a new type of intrinsically fluorescent dendrimers, which is even more attractive. The dendrimers can be formed of aromatic groups as the elements with a high level of their conjugation (Astruc et al. 2010; Balzani et al. 2003). These dendrimers resemble in many properties the conjugated polymers (Sect. 8.5) with the advantage of high level of organization of their structures. Using modern techniques, it is possible to design and synthesize the dendrimers containing a variety of light absorbing and emitting groups. Even more, the effect of light harvesting (Sect. 10.1) can be easily obtained with these structures. This makes them prospective transducers and reporters in sensor technologies.

8.4.3 DNA-Based Fluorescent Nanostructures DNA molecule can serve as a template for hosting fluorescent dyes that can be intercalated between the bases or attached to its minor groove. Since the dyes can intercalate up to every other base pair, high density of fluorophores can be realized. Yet, the DNA template keeps them far enough away from each other to prevent the self-quenching (Benvin et al. 2007). Meantime, the EET efficiency between intercalated dyes and also to additional acceptor was found to be very high, which allows observing easily and exploring the effect of superquenching (Radchenko et al.

8.4 Nanoscale Assemblies Supported by Organic Polymers and Silica …

a

Fluorescence intensity

b

Absorption

Fig. 8.15 Cyanine dye forming J-aggregates on interaction with DNA (Guralchuk et al. 2007). a The structure of cyanine dye L-21. b Absorption (1) and fluorescence (2) spectra (λex = 530 nm) of this dye in the form of J-aggregates when it binds to double-helical DNA. H and J indicate H- and J-aggregates, correspondingly. M and D stand for monomers and dimers

335

Wavelength (nm)

2018). Covalent attachment of dyes to DNA bases increases the stability of such fluorescent nanostructures (Teo and Kool 2012). Both H- and J-aggregates can be assembled on DNA template (Losytskyy and Yashchuk 2010). Being attracted by negatively charged DNA, the cationic dyes interact between themselves forming such ordered associates of dye molecules. Cyanine dyes are the best formers of both H- and J-aggregates (Fig. 8.15). They can be used for DNA staining in living cells (Guralchuk et al. 2007) with the important advantage of their high brightness on two-photon excitation (Losytskyy et al. 2002). The use of DNA templates for concentration and stabilization of organic dyes has found many applications (Ranallo et al. 2018). They include immunosensors and enzyme detection systems. This is especially important, since the tailored DNA sequences can carry recognition functions (Rutten et al. 2020). Being incorporated into DNA structures the dyes serve as reporters in aptamer technologies (discussed in Volume 2, Chap. 5).

8.4.4 Organic Dyes in Silica-Based Fluorescent Nanoparticles The silica nanoparticles are attractive alternatives to organic-based templates in serving to the same purpose: confining, concentrating the dye molecules for increasing their brightness and making them functional. Compared to natural and some synthetic polymers, the advantage is their higher stability in organic solvents and at variations of pH (Yao et al. 2006). The silica particles are more hydrophilic

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and stable in biological milieu; they are not subjected to microbial attack. Due to high density of silica (1.96 g/cm3 ), their separation and fractionation is easy by sedimentation. Its interior can protect the encapsulated dye molecules without concerns of mechanical stress, swelling, porosity changes, aggregation or leakage across numerous solvent conditions. It is important that silica is photophysically and optically inert. It is transparent to light in a broad optical range and it does not participate in any energy- and electrontransfer processes. Therefore, all the spectroscopic properties of the luminescent silica nanoparticles can be attributed to the doping material and to its interactions. However, some molecular dynamics (e.g. twisting) can be suppressed (Alarcos et al. 2017). The methods for incorporation of dyes into silica nanoparticles are characterized by simplicity, low costs, versatility, and great control over the architecture of the resulting materials. They allow selecting preferential location of reporter dyes—in the rigid core, at the porous periphery or at the particle surface (Ow et al. 2005). These possibilities can be realized not only with organic dye molecules but also with their J-aggregated forms (Chen et al. 2019) and luminescent metal complexes (Lian et al. 2004; Qhobosheane et al 2001). Sol-gel matrices are typically used to provide a nanoporous support matrix, in which the analyte-sensitive fluorophores are entrapped, and into which smaller analyte species may diffuse and interact. The surface modification and bioconjugation of silica are well described in the literature (Hermanson 2013). It can be realized in a simple way: the reactive end groups can be added during the synthesis. The important for conjugation decoration of their surface with amino groups is described in detail (Liu et al. 2007). Often, an additional layer of linker molecules with various reactive functional groups (e.g., amine, thiol, carboxyl or methacrylate) is formed (Wang et al. 2008). This is typically achieved by applying an additional silica coating (post-coating) that contains the desired functional group(s). These exposed groups can be modified with all means of carbodiimide chemistry, disulfide-coupling chemistry and succinimidyl ester hydrolysis chemistry (Fig. 8.16a–d). The attachment of biological or biocompatible moieties, such as dextran, antibodies, DNA or peptides can be provided following standard covalent bioconjugation schemes. Silica nanoparticles allow realizing many possibilities for sensing, imaging and diagnosis (Bae et al. 2012; Bonacchi et al. 2011; Montalti et al. 2014). Multifunctional nanosystems can be designed by incorporating the dyes in different compositions into the particle body and also by attaching different functional elements to its surface (see Fig. 8.16). As a result of operation with emission color in the design of fluorescent nanoparticles, two interesting results can be cited. In one of them, a broad-range variation of fluorescence color was achieved by combining in different proportions of fluorescent dyes exhibiting EET (Wang et al. 2006). Three dyes can be chosen to have overlapping emission and excitation spectra in order to allow efficient fluorescent energy transfer to occur. By doping with different combinations of these dyes, barcode tags can be produced for multiplexed EET under single wavelength excitation. In other research (Xu et al. 2010), a combination of two emitters exhibiting fluorescence (FITC) and

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337

Fig. 8.16 Surface modification and functionalization of silica-based nanoparticles (Wang et al. 2008). The design scheme includes both incorporation of dyes in their desired composition into the particle body and modifications of the surface that allows covalent attachment of different functional elements

phosphorescence, tris(1,10-phenanathroline) ruthenium ion, allowed excluding EET and observing a combination of two emissions at a single excitation wavelength 450 nm. Colors of the particles can be easily tuned by varying the doping ratios. In general, multiple doping is very prospective for various applications (Bonacchi et al. 2011), especially when such transformation of reporting signal as light harvesting, wavelength converting or extending the lifetime are needed.

8.4.5 Summarizing: Dye-Doped Nanoparticles in Sensing Organic polymeric and silica-based nanoparticles as well as dendrimers and DNA scaffolds with the incorporation of different dyes have found broad range of applications (Fig. 8.17). The dramatically increased absorbance compared to individual dye molecules is their most attractive feature. Due to this feature they can be ~100 times brighter than semiconductor QDs (Melnychuk and Klymchenko 2018). They demonstrate also an increased chemical stability, photostability and protection from nonspecific interactions with quenchers in tested media. Thus, the organic polymer structures were suggested for application as fluorescent tags for DNA and in the cell imaging. It was easy to achieve the detection limits on the level of ~1 pg/ml for proteins and 6.1 × 104 copies of DNA (Huhtinen et al. 2004) with steady improvement of these values in recent studies. The application of polymeric nanoparticles in ion sensing can be illustrated by the reported fluorescent sensor for the detection of Cu2+ ions in water on the nanomolar level (Meallet-Renault et al. 2004). In this application, the sensor was constructed by associating a BODIPY fluorophore and a copper chelator (cyclam) in ultrafine

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8 Fluorescent Organic Dyes and Conjugated Polymers in Nanoscale …

Fig. 8.17 The most common applications in sensing of dye-doped particles (Borisov et al. 2010)

polymer nanoparticles, and the response was based on the ability of copper ion to quench the fluorescence emission. Fluorescent dye-doped silica nanoparticles are actively used in different sensing technologies, particularly in fluorescence immunoassays. They offer extremely high sensitivity, especially when they are impregnated with luminescent metal chelating complexes (Wang et al. 2006). When they are used for leukaemia cell recognition, the antibody was first immobilized onto the particle through silica chemistry and the cell binding was detected by an optical microscopy imaging (Qhobosheane et al. 2001). Using these antibody-coated nanoparticles, the leukemic cells were identified easily, clearly, and with high efficiency. The larger-size fluorescent polymeric microspheres are actively used in analytical assays using flow and imaging cytometry (Szollosi et al. 1998). In the case of detection of fluorescent labeled molecules, they can be used both as support and recognition units with the application of homogeneous assay methodology. Single-nucleotide polymorphism (SNP) genotyping directly from human genomic DNA samples is another area of their efficient applications (Rao et al. 2003). The dendrimers containing high amount of fluorescent label also find application in DNA hybridization assays, improving or replacing the common organic dye-based assays (Caminade et al. 2006). An immunoasssay based on the composed antibodyconjugated PAMAM-dendrimer-gold quantum dot complex was also suggested (Triulzi et al. 2006). Due to the possibility of easy and efficient modification of their surface, different groups recognizing small molecules and ions can be attached

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339

to this surface. Due to their membrane permeation ability (Khandare et al. 2005), PAMAM dendrimers, especially those of higher generation, have good prospects for in vivo biological applications. The powerful tools of synthetic chemistry demonstrate their efficient employment. The most rapid prospect is expected in designing the polymers with fluorescent groups linked as the side substituents in polymer chains in desired amounts and strictly determined positions. In their organization, such structures may resemble folded proteins. Numerous publications (Breul et al. 2013) demonstrate strong efforts to proceed in this direction. Great possibilities are demonstrated for coupling the outstanding optical properties with surface chemistry (Reisch and Klymchenko 2016).

8.5 Light-Emitting Conjugated Polymers The composites of dye molecules with intentionally reduced interactions between them may be considered as one extreme case. On the other extreme are the conjugated polymers with their strongly coupled fluorophores (Feng et al. 2010; Reppy 2010). They are polymeric molecules, in which the π-electrons of every monomeric unit are delocalized over significant part of chain sequence (Hoeben et al. 2005; Wang et al. 2018). Being electron-rich and with high level of π-electronic conjugation between each repeat unit, such polymeric chain is not only able to absorb and emit visible light but also to demonstrate strong collective effects in electronic excitation and emission. Due to their high molar absorbance (ε ~106 M−1 cm−1 ) these polymers are very good light harvesters in nanocomposite constructs (Achyuthan et al. 2005; Jana et al. 2017). Operating with substituents in the polymer chain, one can modulate their solubility and their incorporation into nanocomposites. Intensive study of organic conjugated polymers, such as poly(p-phenylene ethynylene) and poly(fluorene), and nanoparticles formed of them is stimulated by broad range of their applications in organic photovoltaics and organic light emitting diode technologies. An increased interest and many developments follow in chemical sensing and biosensing, in cellular labeling, in vivo imaging, single-particle tracking, drug delivery and other new areas of applications (Anantha-Iyengar et al. 2019). They possess the ability to form fluorescent nanoparticles without dye doping. Due to co-synthetic incorporation of target binding groups and collective response of their excited states, they can serve as efficient light harvesters, donors in EET and superquenching performers (Jiang and McNeill 2017). Depending on the application, conjugated polymers can be used as flexible molecules, as nanoparticles and as thin films.

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8.5.1 Structure and Spectroscopic Properties The conjugated polymers are polyunsaturated compounds with alternating single and double bonds (or aromatic units) along the polymer chain (Hoeben et al. 2005), Simple chemical structures together with many possibilities to modify it by attaching functional groups makes a number of conjugated polymers very popular. Their core elements are hydrophobic (see Fig. 8.18). Attachment of side groups allows modulating the polymer charge and hydrophobicity in broad ranges. Biosensing in water needs good polymer solubility, which is provided by introduction of charged groups. Such modification makes them the fluorescent polyelectrolytes. The key element that determines the optical properties of conjugated polymers is their polarizable π-electronic system extended along the conjugated backbone. This brings to these polymers the property of electronic conductance and even allows considering them as ‘molecular wires’. Being electron-rich and with high level of πelectronic conjugation between each repeat unit, such polymeric chain is able to very efficiently absorb and emit visible light. Upon excitation, there appear the conductionband electrons forming together with the positively charged nuclear sites the neutral collective excitations called molecular excitons (see Sect. 8.1.1 above). The excitons are the Frenkel-type molecular excitons, and in some cases the signatures of interchain H-aggregates and intra-chain J-aggregates are clearly observed (Baghgar et al. 2014). Meantime, due to structural disorder this picture is not as clear as for molecular dyes spontaneously assembled into these structures in solutions (Meng et al. 2017). Excitons can migrate along the polymer chain and, in densely packed aggregates, between the chains. The mechanism of this migration involves both the through-space dipolar coupling and strong mixing of electronic states. Fluorescence quenching is essentially the termination of this migration producing collective effect (Fig. 8.19). It involves not only the site of quencher location but also the whole polymer or its significant part (Thomas et al. 2007). The situation, in which a large number of otherwise brightly fluorescent monomeric units are simultaneously quenched by one quencher molecule, is often referred to as ‘superquenching’. Such effect appears when one element of such system binds a quencher with the result of quenching the

Fig. 8.18 Chemical structures of several most frequently used conjugated polymers

8.5 Light-Emitting Conjugated Polymers

hνF

341

hνa EXCITONS

Path length

-

-

-

Conduc on band LUMO

+ Recombina on with emission

+ Excita on

+ Recombina on with quenching

Valence band

Fig. 8.19 Diagram illustrating the mechanisms of electronic excitation, exciton transport along the conjugated polymer chain, fluorescence emission and the quenching by electron-hole recombination. Excitation promotes electron to a high-energy conduction band with the generation of positively charged ‘hole’ in the ground state. Electron-hole pair generated on excitation forms the exciton that can migrate along the polymer chain with an emissive transfer to a ground state. External quencher can easily interfere into this process and induce the non-emissive recombination if its LUMO (lowest unoccupied molecular orbital) band is located lower in energy than the excited state of a polymer

fluorescence of the whole system, demonstrating a highly amplified response to the binding. This effect is interesting. With the increase of chain length, the fluorescence intensity becomes higher (and therefore the effect of any external quencher stronger) until certain limit is reached that is determined by the rate of exciton migration and its lifetime. With further increase of the chain length, the intensity does not grow and the quenching effect, if it exists, decreases. This means that superquenching effect stops at some particle size of polymer chain. Electron acceptors are commonly very strong fluorescence quenchers in these systems. Therefore, the presence of only one such group interacting with one element of polymer chain may quench fluorescence of the whole chain (or of its significant part if it is too long), as it is depicted in Fig. 8.4. If a thin film is made of such polymer, one may observe a dark spot on the film, due to exciton migration both within and between the chains, which manifests the superquenching. In comparison with corresponding ‘molecular excited states’, that could be observed with uncoupled monomers, a greater than million-fold amplification of the sensitivity to fluorescence quenching can be achieved (see also Sect. 10.1). Such reaction is reversible if the polymer and the quencher are free to dissociate. The emission of conjugated polymers depends on the chain conformation, which makes them very different from other brightly luminescent nanostructures discussed above. The changes in chain conformation from extended to bent or tilted one may change both absorption and fluorescence spectra. Planar conformation extends the chain π-electronic conjugation and favors the high fluorescence quantum yield,

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8 Fluorescent Organic Dyes and Conjugated Polymers in Nanoscale …

a

HIGH INTENSITY

b

LOW INTENSITY

HIGH INTENSITY TARGET

Fig. 8.20 Scheme illustrating the influence of conformation of conjugated polymer on its fluorescence. a Planar conformation. The extent of conjugation is high. Fluorescence intensity is high, so if the quenching exists it extends to significant length (superquenching). The spectra may be shifted to longer wavelengths (J-aggregate effect). b Non-planar conformation. The extent of conjugation is low and the emission can be localized to a significant extent. The intensity is low and the quenching effect is weak. If a planar conformation is re-established in sensing event (e.g. by binding to DNA) then the high intensity can be restored

providing a broad dynamic range for realizing strong superquenching (Fig. 8.20). This property has found application in homogeneous DNA assays, in which extended conformation is achieved for cationic conjugated polymer on its electrostatic binding with DNA. More on these assays can be found in Volume 2, Chap. 11. The change in chain conformation may produce also the shifts in emission wavelength. This is because the planar conformation favoring extended conjugation length allows achieving the lowest in energy exciton trap and the strongest Stokes shift. When the planarity is destroyed as a result of conformation change, the spectra shift to shorter wavelengths.

8.5.2 Conjugated Polymer Nanoparticles (P-Dots) There is a clear tendency in designing the nanoparticles made of conjugated polymers with compact structures that are stabilized by covalent linking or by hydrophobic interactions, the polymer dots (P-dots) (Du et al. 2017; Feng et al. 2013; Wu et al. 2010). Their size is usually small, in a range of 5–30 nm. They are commonly prepared by miniemulsion (or microemulsion) method (see Fig. 8.13) and by reprecipitation. Miniemulsion consists of addition of dissolved polymers in waterimmiscible organic solvent to an aqueous solution of appropriate surfactant with subsequent ultra-sonication and solvent evaporation. In re-precipitation method, hydrophobic conjugated polymer is dissolved in a good for the polymer solvent and then added into a miscible poor solvent (e.g., water), under stirring. With the

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343

Fig. 8.21 The structures of different conjugated polymers forming stable P-dots in aqueous media due to their hydrophobic tails: poly(9,9-dihexylfluorene) (PDHF), poly(9,9-dioctylfluorene) (PFO), poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV), poly[2-methoxy-5-(2ethylhexyloxy)-2,7-(9,9-dioctylfluorene)] (PFPV), poly(p-phenylene ethynylene) (PPE), poly(9,9dioctylfluorene-2,7-diyl-co-benzothiadiazole) (PFBT)

appended hydrophobic tails (Fig. 8.21) they can be stabilized by hydrophobic forces. Post-synthetic modification strategy makes them water-soluble. The photophysical properties of these particles depend on the constituting elements and the particle size (Wu and Chiu 2013). They have broad absorption spectra with narrow emission bands and high fluorescence quantum yields (50–60%). Because of very high absorbance they are among the brightest fluorescent nanoparticles developed to date. Roughly, their single-particle brightness and cell-labeling brightness are more than one order of magnitude higher than those of inorganic QDs of comparable size and about three orders of magnitude greater than that of typical organic fluorescent dyes. Changing the composition of P-dots allows achieving the tunable emission wavelengths within the whole visible range. The propagating excitons are strong in these particles in contrast to unfolded polymers in solutions, where the exciton diffusion is the primary mechanism of excitation energy transfer (Jana et al. 2017; Saikin et al. 2013). Such transition can be seen when the polymer dissolved in good solvent is transferred to water making the compact P-dot structure. The spectra may shift to red with the band narrowing, demonstrating the appearance of J-aggregates. However, in some polymers the blue shift is observed showing the H-aggregate formation. Simple yet powerful approaches were found for introducing functional groups into P-dots and controlling their surface chemistry. The P-dots are attractive for two-photon fluorescence applications. The reported two-photon action cross sections were as high as 104 to 105 GM. These values are three to four orders of magnitude higher than the values for conventional fluorescent dyes, and an order of magnitude higher than that for inorganic quantum dots. Even higher values, ranged from 106 to 107 GM, can be found in the literature (Pecher et al. 2010). These studies consistently support the potential of P-dots for two-photon fluorescence cell imaging. Depending on the polymer, the fluorescence lifetime of P-dots generally varies from 100 ps to 1 ns (Wu and Chiu 2013). The particle size does not influence significantly the spectroscopic properties, and no aggregation-induced quenching is commonly observed. Regarding their photostability, quite favorable for P-dots is their

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comparison with monomeric organic dyes. They demonstrate very low phototoxicity, which is important for cellular imaging and studies in vivo. The extremely bright P-dots were found to be useful for single-particle tracking, the powerful methods for investigating a wide variety of cellular processes, such as molecular transport, membrane dynamics, and the motion of motor proteins (Yu et al. 2009). By recording ~200 000 photons detected per particle per 20 ms exposure they can follow the local partially confined diffusion and reversible and irreversible binding to cellular components in cells. Their tracking can be recorded by video rate fluorescence microscopy (Yu et al. 2012).

8.5.3 Conjugated Polymers as Fluorescence Reporters in Designed Sensors Conjugated polymers are very interesting and prospective tools for designing fluorescence sensors. A key advantage of sensors utilizing these polymers over the devices using the thin films or nanoparticles composed of monomeric dyes is their strong potential to exhibit collective properties. The greatly enhanced sensitivity of the polymer over the monomeric compound is due to facile energy migration along the polymer backbone that involves great number of monomeric elements. These properties are sensitive to minor perturbations produced on occupation by the target of a very small number of binding sites. Due to conjugation of many fluorophores linked in chain, they provide the effect of superquenching. The quencher could be the target itself, the competitor or a designed group in the sensor unit. Its simplest application can be the introduction of external quencher dye that allows sensing the binding of a target by coupling or decoupling of this dye with the polymer. The frequently used term ‘superenhancement’ is in fact a ‘super-dequenching’, since the polymer does not allow any additional enhancement over its natural emission. The strong increase in emission intensity is achieved simply by removal of quencher and allowing displaying the natural brightness. The aggregates formed by non-covalent association of polymer chains show weak or quenched fluorescence, making them suitable fluorescent sensors for detecting signals that can influence the state of aggregation (Zhao and Schanze 2010). The other possibility that can be realized with the conjugated polymers is to modulate the extent of conjugation. In this case, the quencher can be present permanently. If the conjugation is high, its effect will extend over a large distance. Then the conformational change in polymer chain may reduce the conjugation, so that the quenching effect will be reduced with resulting fluorescence enhancement. Moreover, with the change in conjugation the color of fluorescence can change (due to formation-disruption of H- and J-aggregates). This effect can be recorded in the form of λ-ratiometric response. There is a whole range of possibilities for introducing different functional elements into the conjugated polymer structures. For instance, attachment of crown ether

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345

groups makes them potent ion sensors and of cationic groups—the DNA sensors. These results will be discussed in Volume 2. If the DNA or RNA is the target or when the recognition element changes its conformation, this change can be detected by electrostatic binding of polycationic conjugated polymer. Conjugated polymers allow realizing different signal transduction and reporting mechanisms being active participants in different supramolecular constructs. They induce the ability to exhibit collective effect in PET and EET quenching. A designed combination of this polymer with another partner serving as a quencher or EET acceptor opens many interesting possibilities in sensing. One of them is the combination of conjugated polymer with monomeric dye. The dye can be either directly attached to the polymer chain or co-incorporated into nanostructure with the collective effect in energy transfer that can be very efficient when it is directed from the excited polyanionic conjugated polymer to a cationic cyanine dye (Tan et al. 2004). Different nano-scale support media, such as silica, can be used for assembly of polymer-coated particles (Wosnick et al. 2005), in which different routes of reporter signal transduction can be activated. The analytical assays using flow and image cytometry (Szollosi et al. 1998) use the larger-size fluorescent polymeric microspheres for detection of fluorescent labeled molecules. They are efficient as support and recognition units with the application of homogeneous assay methodology. Single-nucleotide polymorphism (SNP) genotyping directly from human genomic DNA samples is another area of their efficient applications (Rao et al. 2003). An exceptional ability of gold nanoparticles to quench cationic conjugated polymer (Fan et al. 2003) is one more possibility for realizing them in sensing. During fabrication, the gold particles can be stabilized with citrate, so they can contain the negative charge. In the bound form, they produce the quenching effect due to PET, so that one gold nanoparticle could ‘superquench’ several polymer chains. A promising field of application of conjugated polymers is the sensing technology based on thin films, especially in gas sensing and development of artificial ‘noses’ and ‘tongs’ (Volume 2). To date, the conjugated polymers emitting in the near-IR range are poorly presented in the literature, and a range of novel π-conjugated oligomers is expected to fill this gap (Li and Pu 2019; Wang et al. 2019). The design of P-dots with these properties was suggested with their conjugation with organic dyes and forming composites with semiconductor QDs (Chan et al. 2012). Formation of such hybrids helps solving another problem—the broad widths of fluorescence spectra of P-dots, which complicates their applications in multiplexing techniques. Still existing problem of obtaining P-dots of small (~5 nm) size and small size distribution (Wu and Chiu 2013) is a complication for in vivo applications. Summarizing, emissive conjugated polymers demonstrate considerable potential as the basis for sensors designed for both biological and chemical targets. These macromolecular fluorophores demonstrate the features arising from the delocalized electronic states (excitons) created by extended conjugation of the aromatic backbone. They lead to extremely high brightness with single-photon and two-photon excitations and possibilities to modulate this brightness in different sensing technologies. Additional possibilities arise on variation of conformation and aggregation

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state of these polymers. The nanoparticles formed of them (P-dots) are in a strong competition with other organic-based nanoparticles, and not only them. However, to a large extent these very attractive features have to be empirically selected without strong support from the basic knowledge.

8.6 Sensing and Thinking. The Struggle for Brightness and Signal Amplification in Sensor Design The problem of limited brightness of fluorescent dyes in sensing and imaging stimulated development of amplification mechanisms that convert a single molecular recognition event into response equivalent to hundreds and thousands of fluorescent dyes. The simplest idea was to assemble the dyes into nanoparticles. But its realization was not so simple and success did not always follow. As a result, the highest brightness was achieved with different organic dyes and in different ways. Moreover, J-aggregates formed by some dyes allows achieving the brightness much higher than that of constituting dyes, and with AIE and related techniques becomes possible to convert nonemissive or low-emissive dyes into bright emitters. In addition to accomplishment of extremely bright emission allowing 100–10,000fold increase over common organic dye molecules, some additional beneficial properties have been achieved (see Fig. 8.22). Among them is high and, if necessary, finely modulated solubility and stability in different media and, notably, their photostability. The dyes have got full or partial screening from the solvent, protecting from external quenching together with exposure of some groups for specific modifications. Due to large surface areas these chemical modifications can be multi-point and multi-functional. If the chemistry of dye incorporation is determined by the host particle composition, these functions can be the same with the use of different dyes. The brightest response in sensing is the switching of emission intensity between brightest light and full darkness. Such efficiency in response is easy to realize by employing the effect of superquenching. Meantime, realizing other types of fluorescence response (anisotropy, lifetime, spectroscopic changes) is more difficult and may need some additional smart design. Fig. 8.22 The most successful achievements in use of dye-doped and dye-assembled nanoparticles, and also of P-dots, extending dramatically the range of applications of materials based on organic dyes

Protection

ASEMBLING ORGANIC EMITTERS

Brightness

Solubility

λ

Excitonic effects

Functionality

8.6 Sensing and Thinking. The Struggle for Brightness and Signal …

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Respond to the following questions: 1.

2.

3. 4.

5. 6.

7. 8. 9.

10. 11.

12.

13. 14.

The idea of assembling the dyes into nanoparticles for increasing their brightness is trivial. Explain, why in its straightforward application it does not work or meets important difficulties. Demonstrating weak intermolecular interactions, organic dyes can be assembled into the structures of different molecular order. Explain what kinds of inter-dye interactions can be realized here and to what spectroscopic effects this can lead. How the absorption and fluorescence spectra are transformed on formation of H- and J-aggregates? What happens with anisotropies and lifetimes? In Sect. 7.2 we discussed the spectroscopic properties of semiconductor quantum dots that demonstrate the excitonic origin of their emission. The photophysical properties of dye aggregates and conjugated polymers are also excitonic or contain excitonic component. What is the difference? Explain the difference between exciton coupling and exciton diffusion. How they can be recognized? Can they be coupled? Consider the system which exhibits the self-quenching being composed of dyes incorporated in high density into polymeric matrix. What will be the result of introducing EET acceptor in small concentration into this system? Draw a kinetic scheme explaining this case. What is common and what is different between superquenching effect in conjugated polymer and concentrational quenching of organic dyes? What is the role of hydrophobic counterion in making fluorescent nanoparticles of organic dyes? How they resist to concentrational quenching? What are the requirements for obtaining the nanoparticles with bright aggregation-induced emission in terms of structures, dynamics and interactions of constituting dyes? Explain the difference in spectroscopic behavior and photophysical mechanisms between AIE-fluorogens and cyanostilbene-type fluorescence enhancers. How to incorporate the dye into organic polymer and into silica nanoparticle? What are the possibilities for dye location and interactions? Explain the benefits offered by dendrimer structures. Explain the mechanism of fluorescence emission in conjugated polymer. What are the means to modulate their emission in order to generate the sensor response? Compare the efficiency of P-dots as the nanoparticles formed of “active” polymer with that formed of dyes embedded into “passive” polymer. Unlike in solution, fluorescence in the solid state is a collective phenomenon of molecules that are commonly modulated through controlling molecular packing and the electronic conjugation of fluorophores. Nanoparticles are the solid bodies of nanometer dimensions. Analyze systematically the ways to enhance or to suppress this control for making the brightest emitters and responsive sensors.

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Chapter 9

Fluorescent Carbon Nanostructures

Nanoscale structures formed of inorganic carbon in the form of nanodiamonds, graphene and graphene oxide pieces and the so-called carbon dots joined quite recently the family of fluorophores offering use on incredibly broad scale, from optoelectronics to sensing and imaging. Their role is versatile, from very efficient quenchers to very bright emitters and image-formers. Their unexpectedly discovered unusual and still not fully understood optical properties raise many discussions that are highlighted and analyzed here. Their collective effects observed in the studies in solutions and on single-paricle level find explanation within the molecular exciton theory. Conceptual model based on involvement of H-aggregates is presented. The unique properties of these nanoscale emitters make the background for broad range of important implication, particularly in biosensing and bioimaging. The questions at the end of this Chapter may help stimulating the reader’s creativity. Discovery and investigation of unusual properties of pure carbon realized in smart nanoscale materials (Agnello and Messina 2020; Georgakilas et al. 2015; Jelinek 2017; Khan et al. 2018) attract increasing attention of scientific world. It was marked by thousands and thousands publications with their exponential growth in recent years and resulted in revolutionary new technologies. Prominent advances in research in these areas were marked by two Nobel prizes. One was awarded for the discovery and study of fullerenes, the structures of carbon atoms in the form of hollow spheres (Smalley 2003). Later fullerenes found their use in photovoltaics (Li et al. 2012a), as scaffolds in designing fluorescence nanocomposites (Dmitruk et al. 2008; Palma et al. 2009) and electron-transfer quenchers (Ito and D’Souza 2012). The other Nobel award was for graphenes (Novoselov 2011), the two-dimensional sheets of atomic width (Huang et al. 2011) that also became known as very potent electron-transfer fluorescence quenchers (Lu et al. 2009). The emission of their own nanoscale fragments has recently been found (Yan et al. 2011) and their successful applications in sensing was reported (Scholes and Rumbles 2006). Graphene oxide (GO) nanoparticles with modified surface and edges of graphene sheets with polar substituents were found to display stable bright emission, their modifications and stabilizations became a subject of rapid development (Cao et al. 2012a). Carbon © Springer Nature Switzerland AG 2020 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-030-60155-3_9

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Nanodiamond

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Fig. 9.1 The schematic structures of nanoscale carbon materials

nanotubes can be considered as folded into tubes forms of graphene. They are known as potent fluorescence quenchers, but they exhibit their own emission in the near-IR, which is already used in sensing (Barone et al. 2005). Their short oxidized fragments are fluorescent in the visible range (Bottini et al. 2006). Attention was also directed to other nanostructured materials that became attractive for nanosensor design. Quite recently, great interest was attracted to two special types of fluorescencent carbon nanomaterials with bright and stable emission in the visible—nanodiamonds (NDs) (Schrand et al. 2009) and carbon nanoparticles of lower graphite-type order—“carbon dots” or “C-dots” (Baker and Baker 2010; Jelinek 2017). They differ in arrangement of carbon atoms, so that the mechanism of their emission is quite different. Whereas the light emission from nanodiamonds occurs due to point defects in their sp3 hybridized carbon structures (Krueger 2008), the carbon dots share many properties with other electron-rich carbon materials with sp2 hybridization, including their photocatalytic action (Li et al. 2010). Fluorescent carbonic nanostructures represent new types of materials (Georgakilas et al. 2015), in which the high surface to volume ratio is a strong determinant of their properties (Radovic 2012). They may contain different combinations of basic structural elements (of graphene-like sp2 and diamond-like sp3 hybridization types) that can be assembled and additionally modified with polar groups. These groups are introduced during or after the synthesis of these nanoparticles, and because of that they can be dissolved in polar solvents, including water. Basic graphene 2-D and diamond 3-D structures together with carbon nanotubes, fullerenes, graphene oxide GO-dots, and C-dots are schematically presented in Fig. 9.1.

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The two-dimensional graphene and three-dimensional diamond are basically nonfluorescent. Graphene emission appears on reduction of its size to several nanometers and it is enhanced by attachment of polar groups to its edges. In graphene oxide the polar groups are bound throughout the plane, disrupting the π-electronic conjugation within the plane and forming the elements of structure that are fluorescence emitters. Short carbon nanotubes attain the same fluorescence properties as graphene oxides after attachment of polar groups. Carbon dots are composed of graphene and graphene oxide sheets fused by diamond-like insertions and containing amorphous structural elements. Their surface can be richly modified with polar groups.

9.1 Light-Emissive Nanodiamonds We start our detailed discussion from the properties of nanodiamonds, which are the nano-sized crystals with strong potential for sensing and imaging applications. On nanoscale, they represent the diamond form of carbon, which with the density of atoms of 1.76 × 1023 cm–3 is the densest material on Earth. The sp3 hybridized carbon atoms are located in tetrahedron configuration at distances of 1.54 Å forming a dense network of very short and strong covalent bonds, so that the atoms are connected by high interaction energies of 83 kcal/mol. The diamond nanoparticles are commonly obtained in large quantities on detonation of carbon-containing materials in the conditions of high temperatures and pressures. The highest density and strength of diamond being due to sp3 hybridized carbon is preserved on the nano scale, though their surface may possess some elements of amorphous structure. The light emission of diamond particles is due to the presence of color centers— fluorescent crystallographic defects embedded within the diamond lattice. The impurity atoms incorporated into the diamond lattice interact strongly with the electronic structure of the carbon atoms and generate highly localized electronic states. Absorption of light by such defects results in excitation of electrons from one well-defined energy state to another and the emission of photons upon relaxation of the electron to the ground state (Shenderova et al. 2019). These defects are formed by irradiation with high-energy particles to displace carbon atoms and create vacancies that become primary constituents of color centers. Thus, the treatment by high-energy beams of protons or electrons generates the so-called (N–V)− defects (Krueger 2008; Xiao et al. 2015) that are formed due to inclusion into the structure of atomic nitrogen (Schirhagl et al. 2014). Due to their presence, nanodiamonds absorb light at about 560 nm and emit at ~700 nm, see Fig. 9.2. Until recently, the production of fluorescent diamond particles was mostly centered on formation of nitrogen-vacancy centers displaying red/NIR emission. Exploring other defect centers has started recently resulting in making of particles with vibrant blue, green, and red fluorescence (Nunn et al. 2019). Their molar absorbance is definitely lower than that in organic dyes if the estimation is made based on equal masses, but quantum yield may be high enough for various applications. They exhibit relatively long lifetimes, 10–20 ns (Nunn et al. 2019). The

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b Emission intensity

a

Wavelength (nm) Fig. 9.2 Luminescent lattice defect in nanodiamond structure: a the structure of an N–V centre embedded in the diamond lattice; b luminescence spectrum of a diamond containing neutral N–V0 and negatively charged N–V− centers. The zero phonon lines of both types are clearly distinguishable in the spectrum at 575 and 637 nm

spectra are broad and there is a strong Stokes shift, which allows simultaneous excitation of particles with different color. There is no variation in absorption or emission bands between these nancrystals of different size. Compared to other nanoscale luminophores, nanodiamonds demonstrate extremely high photostability. These features determine the range of applications of fluorescent nanodiamonds, in which they can successfully compete with quantum dots (Alkahtani et al. 2018). In sensing and imaging, fluorescence can be excited at the same wavelength as for many other emitters (e.g. organic dyes) but with emission spectrally separated due to stronger wavelength shifting. The lifetimes much longer than autofluorescence of living cells is beneficial for microscopic studies (Kuo et al. 2013). Meantime, the efficiency in two-photonic excitation is rather poor. Ultra-small size (4–10 nm) of nanodiamonds resulting to very high surface to volume ratio (300–400 m2 /g), and uncompensated valences on their surface result in their high ability for sorption of different compounds that allows many possibilities for their functionalization. These particles are well tolerated by living cells (Alkahtani et al. 2018). Being loaded with proteins, they can be used for cell labeling and targeting (Chang et al. 2013). Due to their extremely high chemical stability in biological media, they are attractive for in vivo applications (van der Laan et al. 2018).

9.2 The sp2 Forming Structures: Graphene Sheets, Nanotubes, Fullerenes Graphene sheet is the two-dimensional elementary unit of graphite of atomic width (Yang et al. 2018a). It can fold into balls forming fullerenes or into nanotubes that can be viewed as one-dimensional rods. In all these cases the sp2 hybridization of carbon

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atoms allows forming strong bonding with their in-plane neighbours allowing πelectrons to be delocalized over the whole structure. Regarding electronic transitions, an extended graphene sheet with two-dimensional π-electronic network is known as having zero band gap (Neto et al. 2009). This property makes it electron conducting but unable to attain electronic excited states that could be able of optical excitation and visible emission. The bandgap appears when the graphene two-dimensional structure is cut into small pieces of several nanometers in size, so the smaller is the structure the greater is their resemblance to aromatic hydrocarbons (Yan et al. 2011). Thus, the spatial confinement in such graphene fragments (G-dots) is important for achieving the light absorbing and emissive properties. Single-wall and multi-wall carbon nanotubes are studied very actively. Deformation of conjugated π-electronic graphene structure and folding it into tubes results in a small band gap responsible for light absorption and fluorescence in the near-IR region, which can be strongly quenched on tube-tube interactions (Lin et al. 2005; Minati et al. 2011). Nanotubes generally have a large length-to-diameter ratio close to 1000, so they can be considered as nearly one-dimensional structures (Saifuddin et al. 2012). Their short oxidized fragments are fluorescent in the visible range (Bottini et al. 2006). It was observed that better functionalization with increased density of polar substituents improves not only the nanotube dispersion, but also makes the energy trapping sites more emissive thus diminishing the quenching due to intertube interactions (Lin et al. 2005). Fullerenes are poorly dissolved in polar solvents and are ready to form associates. Therefore they are commonly used after their modifications (Mchedlov-Petrossyan 2013). When studied in “good” solvents they demonstrate fluorescence in the red spectral region but of a very low intensity. Surprisingly, being symmetric, they demonstrate emission anisotropy (Nascimento et al. 2007). Due to low brightness, their application for fluorescence probing was limited. In the sensors based on molecular assemblies and composites they perform the function of strong electron-transfer acceptor generating the quenching (Ito and D’Souza 2012). Generally, not only fullerenes but also graphenes (Lu et al. 2009) and nanotubes (Satishkumar et al. 2007) are known as very potent electron-transfer fluorescence quenchers. Quenching fluorescence of organic dyes allows realizing different applications based on sorption-desorption of labeled analytes or competitors on their surface. Since fullerenes, graphenes and carbon nanotubes are known as strong electron acceptors and the major mechanism of quenching is the photoinduced electron transfer (PET) (Wróbel and Graja 2011; Zhang and Xi 2011), this explains the absence of spectral sensitivity in quenching and allows calling carbon nanomaterials the universal quenchers. An example presented in Fig. 9.3 demonstrates the ability of carbon nanotubes to sense specific DNA sequences by hybridization with sensor DNA sequence labeled with fluorescent dye (Zhu et al. 2010). The latter can be adsorbed on nanotube surface, and its fluorescence is quenched. Upon hybridization, the formed dsDNA segment dissociates with restored fluorescence signal. In a similar way, the DNA aptamer reacting with protein target can be used. Its fluorescence is quenched when it binds to nanotube, but it is enhanced on specific interaction with the target. Similar

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Fig. 9.3 Scheme for signaling biomolecular interactions using an assembly between carbon nanotube and dye-labeled ssDNA or thrombin-binding aptamer (Zhu et al. 2010). In the bound form fluorescence is quenched, but the formation of complex with the target results in desorption of molecular sensor and restoration of its fluorescence

techniques for targeting specific nucleic acids and proteins were developed with the use of graphene and graphene oxide (Loh et al. 2010).

9.3 Fluorescent Graphene and Graphene Oxide Nanoparticles Graphene dots (G-dots) are commonly viewed as small pieces of 2D graphene with perfect honeycomb lattice structure (Chen et al. 2018b) (Fig. 9.1). It has to be noted that the G-dots studied in experiment are often the nanoparticles composed of several small graphene sheets fused together forming ‘nano-graphite’. Some of the edge groups in G-dots are covalently modified with polar groups (commonly, hydroxyls and carboxyls) for induction of their solubility. This is important, since they are usually a subject of experimental research in water and other polar solvents and must possess proper solubility (Qu et al. 2012). In the family of G-dots one may observe very strong absorpbance in the UV region, around 230–280 nm, and a long tail extending into the visible range (Li et al. 2013), Fig. 9.4. The broad UV absorbance in the region 230–320 nm can be attributed to π-π* electronic transitions in the aromatic carbon rings, and its long-wavelength tail to n-π* transitions of carbonyls or other connected groups. The calculated energy gaps when counted based on small ring number suggest the size dependence of spectra (Eda et al. 2010). Meantime experimental data did not show the dependence

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of absorption peak position on the size of G-dots, and their fluorescence spectra are also size-independent (Tang et al. 2012). Thus the ‘quantum confinement effects’ that determine the optical properties of semiconductor quantum dots do not operate in this case, and the photophysics is not governed by loosely bound excitons (Sect. 7.2) but molecular excitons (Sect. 8.1). Surprisingly, the excitation of G-dots in the range of very strong UV absorption does not lead to fluorescence emission at all (or this emission is quite rare). In contrast, the major emission band in the visible is commonly excited at the wavelengths 320– 370 nm. It is the far edge of the major absorption band where the absorption is low and the signature of excitation band maximum is undetected or observed as the sholder at the main absorption band. Exciting at these wavelengths, a variety of fluorescence colors can be observed in different preparations. Nitrogen-doping and oxygen enrichment of the periphery of G-dot structures does not change the character of their light absorption and emission but can provide variation of emission color. A variety of fluorescence colors can be observed in different preparations of G-dots with excitation at 320–370 nm. The dominant emission bands are in the bluegreen range, and the dependence of emission spectra on excitation wavelength often demonstrates the presence of intra-band heterogeneity. Spectroscopic heterogeneity can be seen as the shifts of fluorescence spectra as the function of excitation wavelength (Fig. 9.4), see for example ref. (Pan et al. 2009; Zhang et al. 2012). Meantime, some authors demonstrated the absence of such spectral heterogeneity for blue (Dong et al. 2012a) and green (Zhang et al. 2012) emitting G-dots. The quantum yield of G-dots varies in broad ranges, and there is a common tendency of its increasing on passivation with organic ligands (Qu et al. 2012).

Fluorescence intensity

Absorption

Emission

Excitation

Excitation

250

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450

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Wavelength (nm) Fig. 9.4 Typical spectroscopic behavior of G-dots demonstrating two types of unconnected light absorbers. Those possessing strong absorbance in the UV demonstrate dramatic quenching. The excitation wavelength range shown between 300 and 400 nm is vagely presented at the low-intensity edge of absorption spectrum. Possessing very low absorbance in the near-UV and visible range, the emission can be very bright and multicolor. Within the excitation range the shift of excitation wavelength results in proportional shift of fluorescence spectrum with the decrease of intensity indicating spectroscopic heterogeneity

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Graphene oxides differ from graphenes by the presence of oxygen-containing groups not only at the sheet edges but also within the sheet planes (see Fig. 9.1). When graphene oxide nanoparticles (GO-dots) are synthesized through chemical processing of graphite, they contain a number of such groups, including epoxy and also hydroxyls, and carboxyls. If these groups are located on the basal plane, they disrupt the extended regular conjugation, which leads to the presence of isolated sp2 domains. These insertions can be the sites of various chemical modifications (Qu et al. 2012). The spectroscopic properties described for GO-dots are quite similar to that of G-dots (Cao et al. 2012a). It was reported that their light emission can be blue (Hu et al. 2013), green (Zhang et al. 2012; Zhu et al. 2011) or red (Gokus et al. 2009) when the excitation band is shifted from its typical position at 320–370 nm to longer wavelengths. The switching between these colors can be achieved by dopants and other factors or just by variation of temperature on their synthesis (Peng et al. 2012). Their spectrally heterogeneous emission can be revealed by variation of pH (Li et al. 2013).

9.4 Fluorescent Carbon Dots (C-dots) Fluorescent carbon dots (Fig. 9.1) are versatile and less defined nanocarbon materials that share similar properties with the G-dots and GO-dots discussed above, such as the presence of sp2 domains forming the core and of the polar groups at their periphery (Sun 2020). They attracted many researchers just because of their simple syntheses (Esteves da Silva and Gonçalves 2011) achievable by many approaches from a variety of starting materials. They can be made by simple burning (Liu et al. 2007a) or microwave heating (Zhai et al. 2012) of organic matter (Chandra et al. 2012) and even of waste natural products, such as coffee grounds (Hsu et al. 2012) and soy milk (Zhou et al. 2012a). But their synthesis based on well-defined molecular precursors is preferable since it is the way to achieve the functional carbon materials with controlled surface chemistry, mesoscopic morphology, and microstructure (RondeauGagné and Morin 2014).

9.4.1 Carbon Dots in the Family of Light-Emissive Nanomaterials The C-dots can be obtained of different size. Usually they are between 2 and 10 nm, which is the size of a typical protein. On the steps of their synthesis the obtained particles may incorporate hydroxyls, carbonyls and carboxy groups that become exposed in the formed nanostructures. Importantly, the one-step synthesis allows

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locating on their surface the reactive amino groups, and this possibility strongly facilitates their functionalization (Dong et al. 2012c). It is surprising that the results of structural analysis by the methods of NMR, Raman and IR-spectroscopy demonstrate strong similarity between the C-dots obtained from different sources and by different methods. They witness for high amount of carbon-carbon bonding predominantly in graphene-type sp2 hybridization, but reveal relatively high amount of the diamond-type sp3 hybridization (or disorder) of carbon atoms. This suggests that the π-electronic conjugation must exist along the graphene sheets, but this conjugation may be irregular, especially in view of the presence of hydroxyl and carbonyl groups (Yang et al. 2011). Based on these data, the C-dot can be viewed as a highly defected composition of coexisting aromatic and aliphatic regions, the elementary constituents of which are graphene, graphene oxide and diamond that are assembled in proportions and with variations of surface groups that depend on the conditions of their synthesis (Demchenko and Dekaliuk 2013) (Fig. 9.5). A detailed structural analysisis performed by high-resolution transmission electron microscopy (HRTEM) showed remarkable divergence of structures, even within

Fig. 9.5 Schematic representation of three types of C-dots with different surface groups and their typical fluorescence excitation and emission spectra (Dekaliuk et al. 2014). The “violet” C-dots (a) are with nitrogen and oxygen containing groups, “blue” (b) with oxygen containing groups and cations, and “green” (c) C-dots contain oxygen groups only. Excitation wavelengths λex = 315 and 350 nm (a), 350 nm (b), 425 nm (c). Emission wavelengths λem = 405 nm (a), 440 nm (b), 530 nm (c)

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the same sample. One of two typical types of particles shown in Fig. 9.6c–f, demonstrates a lattice spacing of around 0.35–0.36 nm, which agrees well with the plane of graphitic carbon (Baker and Baker 2010) (we call it the crystal structure I). The second type of C-dots is characterized by fully crystalline structures, with 2–5 nm in diameter (Fig. 9.6g, h). The lattice spacing of nearly 0.24 nm was also observed in different studies (Zhang et al. 2017) and corresponds to the crystal plane of graphite (Baker and Baker 2010) (crystal structure II). All the observed C-dots are composed of multiple layers of graphene, as it was also found in other studies using alternative top-down or bottom-up synthetic methods (see (Baker and Baker 2010; Li et al. 2012b) and citations therein). Some of the C-dots, which possessed an onion-like shape, exhibited an amorphous carbon shell.

Fig. 9.6 High resolution transmission electron microscopy (HRTEM) images of individual Cdots possessing various crystal structures and shapes (Ghosh et al. 2014). These nanoparticles are localized as bright spots in fluorescence images (a, b). The images (c–h) were obtained in the areas where the bright spots corresponding to individual luminescent nanoparticles were detected by fluorescence microscope. The white bars at the right bottom corners are the 5 nm scale bars

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Thus, one must account the versatility of these carbon core structures and their intra-particle ordering in view that the function of carbon core in generating fluorescence emissive states is highly disputable. Some authors suggest its major role (Fu et al. 2015), but this notion does not agree with a number of experimental data. We observe that the particles with different core structures, from graphene-like to amorphous, possess similar fluorescence (see Fig. 9.6). Recently (Ganguly et al. 2019) the structural models for the C-dots core were classified in the following categories: (i) graphite structure with sp3 defects, (ii) diamond-like structure, (iii) amorphous carbon structure, (iv) graphite oxide structure, and (v) graphitic cluster structure. The C-dots possessing sp3 diamond core were found not to differ in fluorescence from those with graphitic one (Zhang et al. 2013a) and beta carbon nitride core (Messina et al. 2016). These data present arguments for only passive role of carbon core in photophysical behavior of carbon nanostructures. The role of attraction between graphene layers cannot be important for spontaneous formation of C-dots in solutions (Hoeben et al. 2005), since the π-π stacking forces are inherently weak (being interactions between molecular quadrupole moments) (Sao et al. 2017). The appearance of dipolar aromatic fluorophores and their assembly may be the primary factors guiding the ordering within the nanoparticle by their own π-stacking interactions with the contribution of electrostatic attraction. The known (see Sect. 8.2) self-assembled structures of organic dyes (H- and J-aggregates) are formed in this way (Koch et al. 2017; Würthner 2016). In such multichromophore systems, a relative orientation of the dipolar dyes pointed preferably in opposing directions compensates for their individual dipole moments resulting in strong attraction forces. In C-dots, those may be the aromatic fragments with carboxylic and hydroxy groups (Qu et al. 2012) that with the inclusion of nitrogen may be complemented by pyrole and pyridine derivatives (Dong et al. 2013) and with the participation of sulfur—by thiophenes (Dong et al. 2013). All of them possess strongly asymmetric electronic charge distribution and thus the tendency to aggregate in stacked manner. If the C-dots are formed in this way of stacking the aromatic dipoles as a primary process, then the carbonization with the formation of particle core may proceed later, as it was observed in thermal treatment of citric acid + amine where the aromatic fluorophores and their associates were found to appear well before the carbonization occurs (Ehrat et al. 2017; Kasprzyk et al. 2015; Krysmann et al. 2011; Schneider et al. 2017; Zhu et al. 2016).

9.4.2 The Spectroscopic Properties of C-dots The absorption and emission spectra of C-dots obtained by various methods and in different labs are remarkably similar and strongly resemble that observed for other species of nanocarbon family (G-dots and GO-dots). When excited at 330–450 nm, a bright fluorescence of C-dots is usually observed in the blue or green spectral range. These nanoparticles can be fluorescent if they contain carbon and oxygen only.

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However, their properties can be modified dramatically if different heteroatoms are included into their structures as the components of precursors during their synthesis (doping) or by binding after the synthesis (passivation). Doping with nitrogen (Dong et al. 2013; Zhu et al. 2009), sulfur (Xu et al. 2015) and, less frequently, by phosphorous (Zhou et al. 2014) and boron (Bourlinos et al. 2015; Choi et al. 2016) atoms is shown to increase dramatically the fluorescence quantum yield. The most popular nitrogen-doped structures are formed of citric acid as a carbon source with the addition of ethylene diamine, urea and amino acids as the source of nitrogen. In this way, multicolor C-dots could be obtained by different oxidative or reductive treatments (Hu et al. 2015). The bright red emitting C-dots were obtained from citric acid and ethane diamine in formamide (Ding et al. 2017). Synthesis of blue, green and red C-dots was reported by changing the solvent in the solvothermal reaction of citric acid and urea from water to ethanol and then to dimethyl formamide (Zhu et al. 2018). The strong Stokes shifts in the C-dots emission (see Fig. 9.5) can be seen as the property independent of their structure, precursors, conditions of synthesis and spectroscopic studies, excitation and emission wavelengths. It is a general regularity for the particles emitting light over the whole range of spectrum, from blue to red (Tetsuka et al. 2015). The excitation spectra shift according to the shift of fluorescence bands, demonstrating the Stokes shifts similar in magnitude. This feature is quite similar to that observed for G-dots and GO-dots, suggesting the same photophysical mechanism of their emission. The fluorescence quantum yields of C-dots were already reported on a relatively high level of 30–40% (Dong et al. 2012c; Zhai et al. 2012) and step-by-step they are improving together with extending the variation of emission color. Being fluorescent emitters, they exhibit the nanosecond lifetimes (Baker and Baker 2010). The two-photonic cross-sections σ of carbonic structures was found to be outstanding! Already on initial steps of studying the C-dots, their σ values were estimated to be of the order of 40,000 GM (Goeppert-Mayer units, 1 GM = 1 × 10−50 cm4 s/photon) (Cao et al. 2007). Similar or even higher σ values were reported later (Liu et al. 2013b; Tong et al. 2015) and the high two-photon absorption efficiency of G-dots and GO-dots was also demonstrated. Moreover, these values can be further increased by doping with nitrogen (Liu et al. 2013b) or boron (Bourlinos et al. 2015) atoms. These values have made the carbonic nanoparticles to be competitive to the best performing semiconductor QDs (Gui et al. 2017) and considerably better than the best performing molecular organic dyes (He et al. 2008). Thus, regarding the dyes commonly used in microscopy (Makarov et al. 2008), this difference is by about three orders of magnitude (for fluorescein and rhodamines σ ~30–50 GM). Compared with traditional one-photon fluorescence microscopy, the two-photon imaging has the advantage of generating the high-energy visible fluorescence from low-energy excitation in the near-IR region. The nonlinear effect of dependence of light absorption on illuminating light intensity allows forming the very sharp images in the microscope focal plane.

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9.5 The Origin of Fluorescence Emission of Carbon Nanostructures Despite strong variability in size, composition, structural order, solubility and other reported parameters, the photophysical and spectroscopic properties of G-dots, GOdots and C-dots demonstrate remarkable similarity (Cao et al. 2012a; Zhu et al. 2015) with the features that are quite different from that observed in organic dye molecules (Chap. 5) or nanoparticles of inorganic origin, such as quantum dots (Chap. 7). This fact raised many difficulties in fitting them into well-established set of knowledge about light absorbing and emitting materials. Resolving them is not easy since here it is impossible to start from monomeric dyes and observe all the steps of their assembly. The fluorescent nanoparticles are obtained from nonfluorescent starting materials in empirically chosen conditions and it remains to discuss why they are fluorescent and how to make their fluorescence optimal for particular application. The difficulty here is not only in diversity and complexity of the studied systems but also in terminology used by the researchers. One type of description comes from the solid state physics and operates with exciton lenghs and band gaps. The other comes from organic photochemistry with their convenient terms of electronic states, transitions between them and of molecular orbitals. As a result, the scientists stop to understand each other and the suggested theories do not address the whole range of observed phenomena. There is a significant drive to identify a unified emission mechanism that could allow attaining reliable control over the photoluminescence properties of these nanomaterials.

9.5.1 Arising Questions to Be Answered What are the features that make the fluorescent carbon nanoparticles so unusual? Specifying them, we may find the ways for establishing the photophysical mechanisms of their light-emissive behavior. We have to find ansvers to a number of specific questions. (a) Why the carbon nanoparticles are so emissive? One of the mysteries of carbon nanomaterials is their bright unquenched fluorescence emission in the visible range. Graphite, graphene, carbon nanotubes and also fullerenes are known as very potent electron-transfer quenchers for other fluorophores (Lu et al. 2009). The quenching of emission by graphene (de Miguel et al. 2012; Matte et al. 2011) and graphene oxide (Wang et al. 2010; Zhao et al. 2016) of the dyes attached to them is so efficient that it is used in different sensor techniques. Moreover, the emission of C-dots (Liu et al. 2007b; Wang et al. 2009). demonstrates the electron transfer quenching on interaction with other carbon materials—graphene oxide and carbon nanotubes (Yu et al. 2014). So, why very small particles made of carbon can be strong absorbers and emitters of light in the visible range of spectrum and do not quench themselves? The direct answer is lacking, and one of suggestions is based on the absence of excitation bands

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for visible emission in the UV range of strongest absorbance (Dekaliuk et al. 2014). Thus the two excited states, UV and nearUV-visible, are uncoupled, probably because of strong difference in their energies (Demchenko and Dekaliuk 2013), see Fig. 9.4. There is no indication of Förster resonance transfer of excitation energy (FRET) from light absorbers of the first type to emissive species of the second type (witnessed by the absence of additional maxima in the UV range of excitation spectra). The former is quenched and it also quenches external emission. The other explanation that does not disregard the first one is that the structural separation between the light absorbers at the carbon core (quenched) and the particle periphery (fluorescent) is so large that the overlap of their electronic wave functions is small, and this does not allow the electron-transfer quenching. (b) Why there is no self-quenching of fluorescence within the carbon nanoparticle excited in the visible? This effect also known as concentration quenching is a very general phenomenon typical for dye aggregates (Sect. 8.1.4). It is commonly present in π–π-stacked dye ensembles. The aggregation-induced emission (AIE) of organic dyes is well-known (Sect. 8.3), but in that case the π–π-stacking is avoided by random orientation of flexible molecular segments frozen in aggregate (Hong et al. 2011). A variety of excitation energy traps could appear on the formation of carbon nanostructures, similar to that on association of organic dyes (Davidson 1983), such as on formation of excimers and exciplexes. Avoiding them is the problem in constructing the nanoscale fluorescence emitters made of organic dyes (Reisch et al. 2017). So the question is why the quenching does not happen in carbonic nanostructures. The possible explanation is the formation of excitonic states, in which the mechanisms of energy propagation and quenching are quite different from that provided by molecular states of aromatic constituents. (c) How the emissive particles are formed from non-emissive starting materials? These pathways are different in the cases of “top-down” and “bottom-up” particle formation. In the former case the carbogenous material is cut into pieces. Aromatic heterocycles are there, but for inducing solubility the polar groups have to appear at particle surface (or edges) inducing the charge distribution in these heterocycles and favoring strong interactions with their neighbors. This may result in integrating different types of electronic transitions within the nanoscale composition. Therefore it is quite possible that new low-energy electronic states appear on introduction of polar groups (Li et al. 2012c; Tang et al. 2012). Then the optical properties can be closely associated with the structural defects and with the interactions at the surface. In the “bottom-up” case several steps of molecular transformations may proceed. The associated molecular fluorophores are formed first and then they attain the carbonic core with dramatic change of the emission (Ehrat et al. 2017; Krysmann et al. 2011; Liu et al. 2017). All that suggests that thermodynamic and kinetic drive exists for ordering, resulting in quite discrete supramolecular forms possessing discrete electronic states. What is essential, even before the carbon core formation the particles attain their characteristic features, such as superquenching and large Stokes shifts. (d) What determines the excitation and emission peak positions? It is not clear if the analysis elaborated for organic molecules is applicable here. Carbon nanomaterials demonstrate incredibly broad range of fluorescence emitting colors. Nowadays

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they cover the whole visible spectral range down to near-IR (Shamsipur et al. 2017; Wen et al. 2012). They emit bright fluorescence with band maxima observed in yellow (Liu et al. 2018; Lu et al. 2018), orange (Jiao et al. 2018; Jing et al. 2019), red (Yang et al. 2018b; Zhang et al. 2017; Zhu et al. 2019b) and near-IR (Ding et al. 2019; Li et al. 2018). This diversity is achieved with only a small change of starting materials and conditions of particle formation. Can this diversity in electronic transition energies be achieved by variation in conjugation extent of sp2 domains within the carbon core? In single-particle experiments (see Fig. 9.6) the species with crystalline and amorphous core were studied in the same sample and they demonstrated rather similar characteristics with respect to their fluorescence (Ghosh et al. 2014). The key role of surface-exposed groups is outlined in a number of recently published reviews (Park et al. 2016; Reckmeier et al. 2016; Sciortino et al. 2018; Yan et al. 2018; Zhu et al. 2015). The dramatic change of fluorescence parameters can be achieved by their passivation, modification, oxidation, reduction or ion binding without altering the electronic states of the core. However, assuming the concept of central role of surface-exposed molecular fluorophores (Xiong et al. 2018) it is still not easy to predict and modulate the emission color based on determination of surface groups and their chemical modifications. The working principle of highly delocalized surface states involving a large number of functional groups (Sciortino et al. 2017) and of the involvement of sites for emissive recombination of excitonic electrons and holes (Xiao et al. 2017) was not based on solid theoretical background and our efforts for optimizing the optical properties remain empirical. (e) What factors determine the particle size and how the size is connected with parameters of fluorescence emission? In most cases the particle size varies between 2 and 10 nm, irrespective of conditions of their production. If they are produced by “bottom-up” synthesis, some energy-entropy balance prevents the particle growth on prolongation of treatment. The particle size is definitely determined by arrangement of surface groups and their interactions with solvent. The most efficient arrangement of these groups may leed to ordering generating molecular excitons. Two or more discrete particle forms with recognizable spectroscopic properties can be obtained on synthesis in one pot (Chen et al. 2017; Fuyuno et al. 2014; Shamsipur et al. 2017) and these properties are retained in the solid state (Chen et al. 2017). The single-particle studies (van Dam et al. 2017) have demonstrated that different resolved spectral forms can be attributed to individual particles. Interesting and not understood in full are the observations on the concentration-dependent tuning of fluorescence emission (Mu et al. 2016; Shi et al. 2019) that in some cases was found to be very strong, from 404 to 583 nm (Wang et al. 2019) and even from 400 to 630 nm (Meng et al. 2017). This means that carbonic nanoparticles may interact in solutions and this interaction decreases substantially the energy of emitted quanta. (f) Why the strong Stokes shifts are systematically observed? Now we refer to unusual very strong and regularly observed Stokes shifts in carbonic nanoparticles. The substantial shifts of the visible emission with respect to the excitation band, reaching or even exceeding the level of 3000–6000 cm−1 , are quite common for all fluorescent carbon nanomaterials. They cannot be generated by molecular forms of aromatic hydrocarbons if they do not create strong molecular dipoles interacting

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with polar solvent in the excited states or participate in some excited-state reaction, such as charge transfer or proton transfer. These reactions require specific arrangement within a fluorophore of specific groups of atoms and in view of diversity of studied spontaneously formed structures it is hard to imagine them to be of general value. It was reported that such strong Stokes shifts are almost polarity independent when studied in solutions (Wei et al. 2019). Moreover, the Stokes-shifted emission was found to be retained in solid state (Ren et al. 2018; Tetsuka et al. 2015) or in cryogenic conditions (Malyukin et al. 2018; Yu et al. 2012), in which the solvent environment is removed or immobilized, as well as in single-molecular studies of isolated nanoparticles immobilized on support (Ghosh et al. 2014). Therefore, they cannot be related to molecular relaxations that are known to occur only in polar environments with high molecular mobility. The excited-state energy transfer flow from high-energy to lower-energy emitters within a nanoparticle is a possibility but it is not supported by experiment (Dekaliuk et al. 2014). (g) What is the origin of spectral heterogeneity and tunability? The spectroscopic heterogeneity of most nanoscale carbon samples is clearly evidenced by the broad unstructured emission bands, the dependence of their positions on excitation wavelength, the non-exponential kinetic traces in fluorescence decay (Khan et al. 2015a). Continuous shifts of fluorescence emission as a function of excitation wavelength are usually accompanied with a great loss of intensity (LeCroy et al. 2017). These features are present in different extent, from being very strong to their complete absence (Choi et al. 2016; Sharma et al. 2016; Zhu et al. 2013). Shifting the excitation wavelength, one can produce photoselection of species whose fluorescence is the brightest at this wavelength, and correspondent emission spectrum becomes also wavelength-shifted. It can be the result of structural heterogeneity of emitters within the nanoparticles or between them. Though different opinions exist (van Dam et al. 2017), the single-particle experiments (Ghosh et al. 2014) together with the facts that purification and fractionation removes such heterogeneity (Vinci et al. 2012; Wen and Yin 2016) are against the idea on its intra-particle origin. (h) Does the interaction with solvent influence the spectroscopic properties of carbonic nanoparticles? The experiments show that their spectral positions may not be significantly affected by the solvent change (Zhou et al. 2013) or by drying the sample and its studying in solid state (Feng et al. 2017; Ren et al. 2018; Tian et al. 2017). The study of single particles applied on support also show the spectra close to that measured in solutions (Ghosh et al. 2014). However, in solutions if the surfaceexposed groups are involved in emission they should interact with the solvent and we should see some evidence for that. Indeed, solvent effects were noticed modulating the positions of emission spectra that can be plotted as a function of polarity (Mei et al. 2019; Ren et al. 2018; Sciortino et al. 2016). With the increase of solvent polarity the red shifts of these spectra were observed, and the shifts reported by some authors were really dramatic, e.g. from green (530 nm) in toluene to red (620 nm) in water, and similar change of color was achieved on their incorporation to polymer matrices of different polarities (Ren et al. 2018). Such effects are similar or even more substantial than that in charge-transfer organic dyes chosen as polarity indicators (see Sect. 5.3). Dielectric polarizability and hydrogen bonding with protic solvent molecules may

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contribute to this spectral change (Sciortino et al. 2016). Regarding the hydrogen bonding, its effect is local and should involve only small proton accepting/donating surface groups. In contrast, the dielectric polarization is a collective effect, it requires the dynamic generation of reactive field in fluorophore environment in response to the appearance of its high dipole moment due to strongly increased charge separation in the electronic excited state (Demchenko and Yesylevskyy 2011). How then such dipole moment is formed? In organic dye molecules this happens by redistribution of π-electron density, but it is hard to comprehend how the electron density is shifted in the case of carbon dots. Does that occur over the whole nanoparticle or only at its local emissive site? No answer by now. Many other questions can be raised, and answering them we achieve deeper understanding the properties of these new materials.It is surprising that with all diversity of structures, interactions and excited-state reactions, all types of fluorescent carbon nanomaterials tend to show several recurrent properties, such as a bright and broad (60–100 nm in width) fluorescence emission demonstrating unusually strong Stokes shifts that are never observed in unmodified aromatic hydrocarbons. The similarity between the different types of nanocarbon extend to fluorescence lifetimes that are always in the nanosecond time range, usually longer than that of typical monomeric dyes (Wang et al. 2014). The fluorescence quantum yields were already reported to be relatively high, 30–40% (Dong et al. 2012c; Zhai et al. 2012), and some recent data report on a much higher level (Hou et al. 2016; Zheng et al. 2015). On the other hand, random formation of particle surface groups, their modification and heteroatom doping, the interaction of these heterogeneously formed surfaces with solvent must result in microscopic heterogeneity, the issue raised by many researchers. Variation of starting material composition and of the conditions of synthesis (e.g. temperature, exposition time, pressure) generates innumerable number of structural variants. Nowadays these factors are still selected by empirical trials with the aim to achieve the optimal parameters for various applications.

9.5.2 Carbon Nanoparticles as Collective Emitters The involvement of collective properties in the behavior of carbon nanostructures is recognized by many researchers. The collective excitations (excitons) may propagate within the whole particle volume (Shang et al. 2012; Sun et al. 2008) or through the surface (Liu et al. 2013b; Zhang et al. 2013a). In this case the emission should be observed at exciton-hole recombination sites (Xiao et al. 2017; Yu et al. 2012) that could be the localized surface traps (Bao et al. 2011) or specific edge states (Pacurari et al. 2016). The experimental data presented below provided strong evidence to exciton concept. Collective behavior (response as one unit) can be seen in the effects of quenching on interaction with external quenchers, e.g. ions. When any quencher regardless of the mechanism of quenching interacts with any unit of the excitonic system, not only this unit but all the system becomes quenched (De Boer and Wiersma 1989; Lu et al.

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2002). This effect explains very efficient quenching ability of all types of carbon nanostructures and is explored in different chemical sensor applications. Thus, the detection limit for sensing mercury cation (Barman and Sadhukhan 2012; Hou et al. 2015) as well as of many other metal ions (Dutta Chowdhury and Doong 2016; Gao et al. 2016) reached several nanomoles! The Stern–Volmer plots (see Sect. 3.2) with strong upword curvature witness for superquenching effect and suggest the involvement of excitonic H-, J- and HJaggregates (Devi et al. 2020). The ion binding that quenches the emission occurs at the particle surface. The fluorescence quenching data reveal that pyridinic nitrogenbases have a dominant influence on J-like emissive aggregates. Similarly, oxygencontaining functional groups play a significant role in constructing the H-aggregates and the most intense emission is contributed by the J-like assembly of H aggregates. The disruption of electron–hole radiative recombination resulting in quenching occurs locally, but the whole nanoparticle may provide the mechanistic framework in harvesting of photo-generated electrons for productive purposes (Xu et al. 2012b). The observed superquenching effect is likely a result of amplified energy transfer occurring in C-dot due to exciton propagation, in analogy to another systems exhibiting the Frenkel excitons, the conjugated polymer nanoparticles (Jiang and McNeill 2016; Tian et al. 2010) and J-aggregates of cyanine dyes (Jones et al. 2001; Lebedenko et al. 2009). The methods of single-molecular spectroscopy allow obtaining the information about the photophysical behavior of individual nanoparticles. Thus, the averaging of characteristics of many particles can be avoided. The particles can lose their fluorescence reversibly (blink) and irreversibly (bleach) and the long-time observation allows detecting them. A focused search of single particle could respond to a question if it performs bleaching or blinking as one unit or in multiple steps demonstrating the presence of a number of fluorescent sites. The experimental data obtained with single C-dots deposited on cover glass plates (Ghosh et al. 2014) demonstrate the characteristic single-step transient blinking and photobleaching (Fig. 9.7). They suggest the presence of single excited-state levels in each particle that can be reversibly or irreversibly inactivated in one step. A correlation was found between the brightness and bleaching rates (Fathi et al. 2019). Due to these properties, the Cdots were successfully applied in different versions of super-resolution microscopy (nanoscopy) (Ghosh et al. 2014; Misra et al. 2018; Verma et al. 2018). Interesting is the blinking statistics. Derived from experiments on blue-green (Ghosh et al. 2014) and yellow–red (Khan et al. 2017) emitting single C-dots with amorphous core structures, it follows a power law function (Khan et al. 2017). This regularity is similar to that for semiconductor QDs and suggests that the stochastic dynamics of intermittency is governed by photo-induced energy fluctuations generating the exciton traps. One of the mysteries of carbonic nanomaterials is the fact that they generate highly polarized fluorescence emission and, in fact, are not zero-dimensional “dots” (Dekaliuk et al. 2014). When studied in ensemble-averaged conditions, the emission anisotropy reaches its limiting value in the solid state (Xiao et al. 2017), and in viscous solutions it decays on the time scale of emission (ns) starting from high

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Fig. 9.7 On–off intermittency and bleaching of carbon dots. a The photoluminescence time traces (1–2) of two individual CDs with “green” emission (Ghosh et al. 2014). The emission intermittency varies on a scale from short off-states near several milliseconds, to longer ones of the order of hundreds of milliseconds. b Representative traces of a reduced particle showing one emission intensity level and single-step bleaching (1) and another one demonstrating multiple fluorescence intensity levels (2) before bleaching (Das et al. 2014). c The traces observed for CDs showing singlestep bleaching in the presence of ascorbic acid, an electron donor (1) and of methyl viologen, an electron acceptor (2) (Khan et al. 2015b). Reproduced from Ref. Demchenko and Dekaliuk (2016)

initial level (Das et al. 2018). Anisotropy demonstrates rather homogeneous spectral behavior being almost independent of excitation and emission wavelengths (Dekaliuk et al. 2014; Xiao et al. 2017). The high values of anisotropy across the spectra were attributed to localization of excitons (Demchenko 2019). For possessing high optical anisotropy, the whole-particle quantum emitter should possess the permanent dipole moment that does not change its original orientation in the excited state. Experiments provided on a single particle level have shed more light on this issue (Ghosh et al. 2014; Khan et al. 2017). Figure 9.8a–e shows the excitation patterns of individual fluorescent C-dots, for which the transition dipole moment (TDM) is oriented along the plane of support (Ghosh et al. 2014). The image of every nanoparticle is represented by two nearly bright spots of elliptical shape, which corresponds to a fixed linear excitation TDM,

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Fig. 9.8 Fluorescence images of the individual CDs spin coated on the glass surface. Above a–e: fluorescence excitation patterns: double white arrows show the orientation of the excitation transition dipole moment (TDM). Below f–j defocused images and assigned orientation (double white arrows) of the emission TDM (Ghosh et al. 2014)

marked by arrow. Fixed orientation of the pattern indicates that the TDM does not undergo reorientation during the acquisition time. Comparison of the excitation patterns and defocused images of the same individual C-dots showed that both excitation and emission transition dipoles of the particles are oriented parallel to each other. This finding implies that each nanoparticle has only one optically active fluorescent site, which is directly excited by the laser light without any energy transfer from other optically active groups (Ghosh et al. 2014; Jing et al. 2017). No change in orientation of the dipole moment was observed on blinking, which demonstrates that the structural anisotropy is the inherent property of these nanoparticles and it is responsible for the existence and orientation of dipoles. The studies of emission anisotropy of individual nanoparticles are of special importance. Collective properties can appear due to exciton diffusion within the nanoparticle by excitation energy transfer (EET), see Sect. 8.1. In that case, however, the anisotropy induced by polarized light excitation should be totally lost in emission. The picture that we observe is different. The presence of high emission anisotropy should be attributed to resonant propagating excitons and emission by exciton recombination in energy traps. Of special interest is the finding of explanation to gigantic two-photon absorbance. It cannot appear just by assembling individually emitting molecular fluorophores (two quanta arriving simultaneously excite single fluorophore) just because their σ values are lower by several orders of magnitude. The analogy with the deeply theoretically (Spano and Mukamel 1991) and experimentally (Collini 2012) studied excitonic associates of organic dyes suggests that here also the enhancement of two photon absorption is due to the generation of excitons. Such behavior is dramatically different from the behavior of organic dyes assembled in detergent micelles (Maurin et al. 2009) or trapped in polymer conjugates (Cepraga et al. 2013), in which the high performance is achieved due to only local concentration effects. Within the

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frame of exciton theory the intensive nonlinear optical response could be explained by the occurrence of the large oscillator strength generated by one or few delocalized excitonic states.

9.5.3 The Exciton Model In this Section we address the physical mechanisms of emission of carbon nanoparticles based on the theory of molecular excitons and try to explain on this basis their most essential properties. Exciton in molecular aggregates is considered to be a collective quantum emitter in a sense that constituting chromophores lose their individuality and respond to electronic excitation as one unit (Sect. 8.1). The ability of forming the Frenkel-type excitonic states must be determined by molecular structures that allow realizing the electronic nature of the π-conjugated systems. Due to the lack of sufficient structural information, on the present step only a conceptual model can be suggested (Demchenko 2019). The role of the carbon core is one of the key issues discussed in current literature. In G-dots the sheets forming the core are the necessary element of structure (Gan et al. 2016). In other carbonic nanostructures these core elements may be present or not, and if present, they can be seen in crystalline or amorphous forms (see Fig. 9.6). Therefore, it can be expected that the primary role of the core is in stabilizing the structure by forming the planar sheets interacting between themselves by van der Waals interactions. The magnitude of these weak interactions between the planes should provide a stabilizing effect. These sheets being analogous to nanographenes (Stepien et al. 2016) may extend to the particle surface and terminate by the chargetransfer fluorophores that combine nonpolar and polar fragments. In this way the planar layered core structure may guide the planar arrangement of the latter. The key role in generating and modulating the optical effects should be attributed to the particle shell that is the target of many modifications and solvent-induced perturbations changing dramatically the spectroscopic behavior (Ding et al. 2015; Liu et al. 2019; Park et al. 2016; Sciortino et al. 2018). In the course of synthesis and structural modifications the shell can be formed of structurally asymmetric amphiphilic charge-transfer fluorophores (Galande et al. 2011; Wang et al. 2012). This role can be attributed to the coupled π-electron system with carbonyl groups (Liu et al. 2019). For being at the interface between nonpolar core and polar solvent, their most energetically favorable configuration can be achieved if they are planar and extend the polar groups to the surface with or without the structural guidance by the particle core. These surface-exposed fluorophores should be arranged in stacks, which is typical for the self-assembly of organic dyes (Koch et al. 2017; Würthner 2016), The dense stacking arrangement may be stabilized by stronger dipole-dipole interactions (Chen et al. 2009; Würthner 2016) due to the charge transfer properties of the partners (Kumar et al. 2014) and particularly by the electrostatic complementarities between

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the electron-rich and electron-deficient region of the stacked aromatic groups and complementary charges on the neighbors (Guldi and Prato 2004). This is the most energetically favorable configuration for the formation of the H-type excitons. Here the analogy can be drawn with H-aggregates formed in solutions, where planar aromatic molecules arrange in 1-D face-to-face stacks with the appearance of their overlapping electronic π-orbitals (Würthner et al. 2015). In the case of “top-down” synthetic procedure, such preferential arrangement of surface-exposed fluorophores must be realized by after-treatment of already formed core that can operate as a template for the surface modification by polar groups (Gonçalves and da Silva 2010). Regarding the “bottom-up” (e.g. solvothermal) synthesis, the stacks of aromatic heterocyclic fluorophores may appear on the first steps of synthesis (Das et al. 2017; Xiong et al. 2018), ahead of carbonization and core formation. This core-shell (or shell-only) configuration of fluorophores stabilized by complementary positive and negative charges (Würthner 2016) are the sites of binding-release of protons and ions of different sign. The essential feature of excitons is their ability to absorb light collectively as one unit formed by π-conjugated network but to emit light locally at the electron-hole recombination site. Our model schematically presented in Fig. 9.9 depicts the most general sequence of events occurring on light absorption and emission in H-aggregate forming carbonic nanostructures. These events could be the following. Light absorption by an H-aggregate located within the particle shell possesses all the features of collective excitation that propagates over the whole excitonic unit that can be of different size—from coupling two neighboring fluorophores to integrating the whole particle surface. It displays the characteristic features, such as the strong one-photon absorbance and gigantic

Fig. 9.9 The proposed model of excitonic H-aggregate as the light-emissive functionality of carbon nanoparticles illustrating the mechanism of their emission (Demchenko 2019). The fluorescent Haggregate is formed by side-to-side stacking of aromatic groups resulting in their π-electronic coupling at the particle shell. The major sequence of events is (1) absorption of light quantum by H-aggregate as an integral quantum system, (2) exciton propagation and transition to self-trapped state and (3) the emissive electron-hole recombination at the self-trapped site

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absorbance of multiple photons, short-wavelength position of the absorption band and high transition dipole moment. The propagating exciton has many possibilities to decrease its high energy. If it is quenched, it can exhibit the superquenching effect, as well as blinking and bleaching (see above). It can also demonstrate the intersystem crossing resulting in conversion to triplet with the appearance of phosphorescence emission). However, the fastest and the most efficient will be the intra-band relaxation provided with the aid of collective vibrations (phonons) creating a dynamic defect. Such relaxation proceeding without activation barrier allows achieving a high efficiency of energy conversion and its release in the form of emission. As a result of exciton-phonon coupling, the exciton appears in the state of the lowest excited-state energy, and some part of this energy is released in the form of ultrafast local heating. This brings a local distortion of structure that breaks the Haggregate stacking configuration with the neighbors and results in a change of angle α between their dipoles (see Fig. 9.9). As it was suggested (Malyukin et al. 2018), the distorted local fluorophore arrangement attains the properties of J-aggregate [Such H- to J- transformation is known in molecular aggregates (Hestand and Spano 2015)]. This opens an allowed transition for the emissive decay from this localized site, and therefore the fluorescence can be highly polarized and with a high quantum yield. Since the emission of J-aggregates is always shifted to longer wavelengths, this explains the strong Stokes shift. Thus, this model suggests mechanistic realization of proposed earlier surface energy traps, the existence of which was noted in the pioneering work on fluorescent C-dots (Sun et al. 2006) and postulated in many subsequent studies (Sciortino et al. 2018). The essential features of this mechanism include the “antenna effect” in light absorption, the excitation energy transformation involving conformational variables coupled with collective vibrations and the emission from the trap site. The H-aggregates are able to generate the excitons serving as the structurally determined antennas for absorbing and transforming the light quanta and for coupling these processes with molecular and lattice vibrations. These vibrations (phonons) may be considered as noise, but there are those that allow the H-aggregates to demonstrate bright fluorescence emission. This view is similar to the mechanism described for molecular aggregates of organic dyes and features strong resemblance to that in photosynthetic antenna systems (Scholes et al. 2017; Zhu et al. 2019a). Similar level of coherent effects is expected to play an important role in carbonic nanostructures. Many new discoveries are expected with carbonic nanoscale materials. We have entered a new exciting world of knowledge full of intriguing and nontrivial questions for experimental and theoretical work. The prospect is bright but the road is difficult for a unified understanding of complex quantum phenomena that are in the background of carbonic nanoscale emitters. Based on this understanding, a clear vision should appear and the new areas opened for technological developments and applications.

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9.6 Analytical, Bioanalytical and Imaging Applications of Carbon Nanomaterials Unusual situation is observed in the physics and chemistry of nanocarbon structures. It is not the theory and deep understanding of background mechanisms that drives the applications but numerous, versatile and very successful applications are the stimulus for the development of theory. The extended application areas of carbonic nanostructures include their use in photoelectronic (Li et al. 2015) and light harvesting devices (Choi et al. 2018), photocatalysis (Wang et al. 2017) and other related areas (Chen et al. 2018b). Their connection with sensing and imaging becomes more and more evident. Bioanalytical and bioimaging applications of carbon nanomaterials are presented in books (Hui et al. 2019; Sun 2020), numerous reviews and innumerable number of research papers. Results in these domains grow so rapidly that the reviews focused on them become outdated soon after their appearance. Most of the papers selected for citation below belong to the first romantic period, in which most of the technologies have started featuring explosive development.

9.6.1 General Strategy The ability of dispersions of carbon nanoparticles to be very strong absorbers of UV radiation on a low light scattering level and to demonstrate non-linear effect at high radiation densities suggested their applications in different light protecting devices (Liu et al. 2009; Sun et al. 2000). For such application commonly known as optical limiting, carbon nanotubes (Vivien et al. 2002) and nanodiamonds (Vanyukov et al. 2019) are applied together with other forms of nanocarbon (Huang et al. 2018). Their efficiency is enhanced when they are used as composites with upconversion (Chen et al. 2018a) and noble metal (Xu et al. 2018) nanoparticles. The extreme efficiency of carbon nanomaterials as fluorescence quenchers (see Sect. 9.2) that is much higher than that of noble metals (Kasry et al. 2012) has found many applications. Different emitter-quencher configurations can be realized in sensor design (Balapanuru et al. 2010b; Chang et al. 2010; Lu et al. 2010). Their interaction with peptides and proteins quench fluorescence of Tyr and Trp residues (Li et al. 2012d) and it was observed that the quenching efficiency depends strongly on the distance between carbon nanoparticle and the dye and on the flexibility of the linker (Kasry et al. 2012). The reversible protonation of attached fluorescent dye can be used as pH sensor (Melucci et al. 2012). Due to their own strong fluorescence the carbon particles can function in molecular sensing as the labels covalently attached to recognition units. The response in this intrinsic fluorescence must be based on quenching or on excitation energy transfer, in which they can be both donors and acceptors (Dong et al. 2010; Wei et al. 2012; Zhang et al. 2011). They demonstrate prospects for photon correlation spectroscopy and single particle tracking (Jin et al. 2009), the techniques for which the fluorophores

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of high brightness and stability of output signal are in strong demand. Displaying different solubilities and affinities, they can be very efficient image formers in cell microscopy. Very attractive are the applications in two-photon and super-resolution microscopy. Forming nanocomposites with other fluorescent ions, molecules and nanoparticles is the powerful strategy open for further development. C-dots can host lanthanide ions generating narrow spectra and long lifetimes, which allows time-gated optical imaging (Sun et al. 2013a).

9.6.2 Chemical Sensing with Carbon Nanostructures The properties of strong absorbers of UV light, efficient quenchers of fluorescence of other emitters and their own emission abilities can be explored and efficiently combined in different sensor designs. However, like in many other types of fluorescent nanosensors, the possibilities in choosing the parameters of their response are often become limited to variations of quantum yield, which is commonly detected as the change in intensity (enhancement or quenching). They lack remarkable reporting properties of different organic dyes based on λ-ratiometric solvatochromic and electrochromic response. Since the intensity has no absolute value, it always needs proper calibration (see Sect. 3.2). It was shown that reversible binding of mercury ion results in dramatic fluorescence quenching of G-dots, and this effect was suggested for determining mercury in test media (Chakraborti et al. 2013). A simple method for the determination of iodide in aqueous solution was reported. Iodide anion can displace mercury ion from its complex formed with carbon dots leading to fluorescence enhancement (Barman and Sadhukhan 2012; Du et al. 2013). The same principle of substituting the bound mercury ion was applied for determining biothiols (Zhou et al. 2012b), and of bound lanthanide ion for determining inorganic phosphate (Zhao et al. 2011). Static quenching is observed on interaction of Sn(II) ions with C-dots, which was used for determining these ions in micromolar range (Yazid et al. 2013). In contrast, interaction of graphitic nanoparticles with Fe3+ ions leads to dynamic quenching, which can be used for detecting these ions with nanomolar sensitivity (Zhang et al. 2013b). More complicated detection systems can be constructed on the basis of carbon dots. A complex of C-dot with lysine and bovine serum albumin was used for selective determination of Cu2+ ions (Liu et al. 2012b). In another work these ions were determined by fluorescence quenching of C-dots decorated with polyamine polymer (Dong et al. 2012b). Cu2+ ions are known as the strong fluorescence quenchers operating by electron transfer mechanism, and this approach is hard to expand for detecting other analytes. Therefore specific ligands have to be developed to every analyte and coupled to fluorescence reporter. An example is the work, in which the ion binding to crown ether produces modulation of resonance energy transfer between carbon dots and graphene (Wei et al. 2011).

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In these classical works we observe the tendency to move from smartly prepared one-pot synthesized nanoparticles based on combination of reagents to their efficient functionalization in the course of after-treatment. Regarding detection technique, the trend that started to be realized recently is instead of intensity sensing to try to apply more prospective and reliable λ-ratiometry (Demchenko 2010), which is already observed in temperature (Macairan et al. 2019) and heavy metal ions (Yarur et al. 2019) sensing with C-dots.

9.6.3 Applications in Biotechnologies Fluorescent carbon nanoparticles were successfully incorporated into two major families of biosensor technologies. One is based on application of antibodies and their gene-recombinant fragments. The other uses the aptamers, the target binders selected from large nucleic acid libraries. In both cases the fluorophores are needed for reporting on the binding event. In antibody-related techniques, they are used mostly for labeling. The response in aptamers, in contrast, can be coupled with target-induced conformational change that allows obtaining wavelength-ratiometric response (Demchenko 2010) leading to ‘direct’ fluorescence sensing (Altschuh et al. 2006). Accordingly, carbon fluorophores can play different roles in these strategies. Carbon nanoparticles have started to be applied in different immunoassay formats, particularly in lateral flow and microarray immunoassay tests (Posthuma-Trumpie et al. 2012). It was claimed that they provide a less expensive but more sensitive and stable alternative to other commonly applied labels. The aptamer-functionalized C-dots can be a sensory platform for protein detection. The presence of target protein can induce the aptamer-modified C-dots to form a non-fluorescent sandwich structure with aptamer-functionalized silica nanoparticles through specific protein-aptamer interaction. This assay when realized for sensing thrombin shows high specificity (Xu et al. 2012a). Another approach for the detection of thrombin in human plasma (Wang et al. 2011) is based on target-induced disruption of EET from upconverting phosphor to linked GO-dot. This strategy offers a suitable approach for detection of other proteins in biological, pharmaceutical and nanomechanical applications. In different molecular recognition events the resonance energy transfer quenching by GO-dots of semiconductor QDs fluorescence can be efficiently used (Dong et al. 2010). Particularly, an assey for protease activity was established when these partners are connected by protease-cleavable peptide (Li et al. 2011). GO-dots modified with glutathione can be obtained with a high fluorescence quantum yield. They can be used for detecting different metabolites inducing the quenching effect (Liu et al. 2013a). It was reported that ‘blue’ fluorescent C-dots can be used for protein staining after the separation in gel electrophoresis with the advantage of higher sensitivity over convenient staining with organic dyes (Na et al. 2013).

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There are many possibilities realized in DNA sensing. The binding to GO-dot of ssDNA labeled with fluorescein derivatives completely quenches the fluorescence of these dyes. When the complementary DNA strand hybridizes with the dye-labeled DNA to form dsDNA, the dye fluorescence is restored (Lu et al. 2010). The complexation of the dye with GO-dot can be noncovalent and mainly electrostatic, such as of cationic pyrene derivative (Balapanuru et al. 2010a) that results in quenching of its emission. The quenched fluorescence is turned on because of disrupting its complex with GO-dot if the dye interacts with an analyte DNA more strongly. It was shown that the dye-GO-dot sensing platform for DNA is highly selective over a broad range of common surfactants and biomolecules, including RNA. Rapid detection of bacteria in sewage water was achieved with fluorescent C-dots (Mandal and Parvin 2011). They are easily conjugated with bacterial cell membrane and allow counting the number of these cells.

9.6.4 Cellular Imaging Applications Fluorescent nanocarbon materials have found many applications in cellular imaging and in chemical sensing and biosensing inside the living cells and the whole organisms. Being of similar size to biological macromolecules and much smaller than the cell organelles or whole living cells, they can be decorated with different functionalities. They can enter the cell interior, produce informative image by their distribution and report about particular interactions in the studied systems in the form of fluorescent emission (Huo et al. 2019; Sun et al. 2013b). The C-dots are mostly localized in the cell membranes and cytoplasm without entering the cell nucleus, which is evident from excitation wavelength dependent cell images (Hsu et al. 2013). Their distribution strongly depends on the modifications of their surface, so if hydrophobic C-dots are covered with amphiphilic polymer, they are preferentially located in cytosol (Callan et al. 2012). By modifying their surface, one can modulate their intracellular distribution in broad ranges. The pH sensitivity of fluorescence response of carbon nanomaterials was reported in different studies, which is due to the presence of ionizable groups on their surface (Lin et al. 2012). This allows developing the pH sensors in bioimaging (Shen et al. 2013) and, if needed, avoiding the pH-dependence in a broad range (Chen et al. 2013). Broad and rapidly expanding area is mapping the disctribution of different metabolites by observing the targeting and distribution of carbon nanoparticles within cells. The carbonic nanoparticles demonstrate highly improved photobleaching and blinking behavior, but the results of different authors differ and probably depend on the studied structure. The single-step transient blinking behavior of C-dots (Ghosh et al. 2014) illustrated in Fig. 9.7 allowed their successful application in superresolution optical fluctuation imaging (SOFI) (Chizhik et al. 2016). Figure 9.10 illustrates the striking improvement in image details and a reduction of background in the second-order SOFI images over common diffraction-limited wide-field optical imaging.

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Fig. 9.10 Comparison of fluorescence wide-field and second-order SOFI imaging (Chizhik et al. 2016). In Saos-2 cell, (a and d), the two subareas of the cell were selected and compared for wide field (b, c) and SOFI (e, f) images, respectively. Scale bars 10 μm. The dotted lines in images b and e indicate cross-sections shown in figure g that demonstrates the presence of well-resolved fine structure. h Comparative cross-sections though a single bright spot in the wide-field and SOFI image

The high brightness, relatively high photostability and the ability to be reversibly or irreversibly inactivated in one step have opened the ways for application of carbon nanoparticles in other super-resolution microscopy (nanoscopy) technologies. Those are the applications in stimulated emission depletion (STED) nanoscopy (Leménager et al. 2014), STORM nanoscopy (Verma et al. 2018) and single particle detection technique (Misra et al. 2018). The studies on correlation of nanoparticle structure and composition with their brightness and blinking-bleaching behavior has got a new impuls (Fathi et al. 2019). Possessing very high two-photon absorbance and the ability to penetrate easily into the cells, the families of C-dots (Cao et al. 2007), GO-dots (Qian et al. 2012) and G-dots (Liu et al. 2013b) were found to be very attractive and efficient in highcontrast two-photon microscopic imaging of living cells and tissues. Recent success

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in synthesis of synthesis C-dots absorbing and emitting in the near-IR region allows using directly the excitation by femtosecond pulsed laser and achieve in bioimaging the deeper penetration into living tissues (Pan et al. 2016). The aability of carbon nanoparticles to serve as indicators of pathological cell transformations can be illustrated on the model of apoptosis. It is a genetically encoded cell death program that involves different processes occurring on molecular and sub-cellular levels. It was found that apoptotic cells demonstrate an increased accumulation of unmodified C-dots and their changed distribution within cell interior, which can witness on altered mechanisms of their translocation through the membrane (Dekaliuk et al. 2015). The images of apoptotic cells were much brighter and demonstrated different distribution within the cell cytoplasm, still without penetration into nucleus. Only when the cells are fixed and treated with detergent, their nucleus is also labeled. Flow cytometry allows distinguishing the sub-populations of living and apoptotic cells in their cultures and suggests a very cheap and easy way to characterize them (Fig. 9.11). The application of such cheap and easily accessible nanoparticles provides more opportunities to simplify the popular methods of cell labeling and detection. Clever functionalization of particle surface with desired ligands may extend these possibilities (Pirsaheb et al. 2019). Thus, cancer cells can be identified in culture by their targeting with folate-functionalized carbon dots specifically labeling folate receptors on cell surface (Liu et al. 2015; Zhang et al. 2018). On a general scale, carbon nanomaterials were found to be nontoxic or low-toxic and biocompatible when studied both in vitro and in vivo (Madannejad et al. 2019; b

Side scatter

a

Forward scatter

Fluorescence intensity (log scale)

Fig. 9.11 The results of comparative studies of normal and apoptotic cells labeled with C-dots (Dekaliuk et al. 2015). a The flow cytometry plot of a sample containing both normal and apoptotic HeLa cells showing that both types of cells are labeled with C-dots. The cells with smaller size and higher side scattering signal were gated as apoptotic cells (black); the cells with larger size and lower side scattering signal were gated as living cells (red). b The histogram representing relative fluorescence intensity (logarithmic scale) of cells normalized to their number: autoflourescence of living cells (black), autofluorescence of apoptotic cells (red), living cells stained with C-dots (blue), apoptotic cells stained with C-dots (green)

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Wang et al. 2013). Among the exceptions are the positively charged C-dots that can penetrate into the cell nucleus (Havrdova et al. 2016) and carbon nanotubes that can provide mechanical damage to cells (Firme III and Bandaru 2010). These data are in contrast with the findings for cadmium and selenium-based quantum dots, the toxicity of which is a concern (Sharma et al. 2017). With the advantage of low toxicity and successful competition in brightness with semiconductor QDs the carbon nanomaterials can be successfully used for in vivo applications, which is witnessed by numerous results obtained on animal models (Liu et al. 2012a; Sun et al. 2013b). In order to increase the brightness of fluorescence emissions, the carbon nanoparticles may be surface-doped with an inorganic salt, such as ZnS (Cao et al. 2012b).

9.7 Sensing and Thinking: The Values of New Discoveries in the Science of Carbon Some people consider chemistry as the science that passed already its era of great discoveries. This notion was spread over its branches—material chemistry and photochemistry. But the surprising stories on discovery of novel carbon materials say about the opposite. Before 1995 the chemistry of inorganic carbon new only diamond and graphite as its crystalline allotropes plus amorphous forms, such as soot and charcoal. Unusual forms of carbon were reported only by astronomers in the atmospheres of red giant stars, and these stellar data encouraged Harry Kroto to simulate their appearance on earth (Kroto et al. 1985). In this way, the experiments on condensing carbon vapor have led to discovery of fullerenes (Smalley 2003)! Next after that, the scientists started to look for new forms of carbon, and they were awarded. In 1991 carbon nanotubes were discovered in carbon arc chambers similar to those used by Kroto to produce fullerenes (Iijima 1991). Fullerenes and carbon nanotubes may be considered as the “folded” forms of graphene. For many years theoretical physicists predicted its flat form. It was well known that the layers in graphite structure are connected by only weak forces, in contrast to very strong forces connecting carbon atoms in-plane, and in centuries people used graphite pencils without knowing that on writing they remove graphene sheets. But only in 2004 the sheets were isolated and characterized (Novoselov et al. 2004). And this was done by surprizingly simple way—by removing them from graphite surface and transferring to inert support by using Scotch tape! Who could predict that these new 2-D materials of single-atom thickness will be then produced in square meter quantities and their market will reach the $ 100 million level? And what about carbon dots, the rising star in carbon nanotechnologies? They were discovered in 2004 by the property of their major use at present, by fluorescence (Xu et al. 2004). The scientists occasionally found them in garbage that remained after the synthesis of carbon nanotubes… I recollect the words said by Louis Pasteur: Dans les champs de l’observation le hasard ne favorise que les esprits prepares (In the field of observation, chance favors

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only the prepared mind). Perhaps we find ourselves at the mere beginning with many exciting discoveries ahead of us. Let us not miss them. By responding to these questions the reader may check the understanding the information presented in this Chapter and generate his/her own ideas. 1. The discovery of novel carbon materials was made in the course of very unusual (for syntheyic chemist) treatments. What are they and what is common between them? 2. With nanodiamonds one cannot achieve high level of light absorbance. Why? What is then their advantage in cellular imaging? 3. Fullerenes and carbon nanotubes are known as very potent fluorescence quenchers but they possess their own fluorescence. If they assemble in a solid body or in surface layer, will they quench each other? 4. Why we consider elementary chromophores within carbon nanoparticles (Gdots, GO-dots and C-dots) as interacting? What kinds of interaction and what extent of excited-state energy migration is possible? Analyze the nanoparticle of 5 nm in diameter regarding the possible distances and orientations of potential molecular emitters. 5. What are the grounds to consider carbon nanoparticle as collective quantum emitter? What theory can be applied to describe its behavior? 6. The large Stokes shifts for C-dots is retained in cryogenic conditions, down to 10 K. What can this result witness regarding the fluorescence mechanism? 7. What are the peculiarities in applications of carbon nanoparticles in comparison with organic dyes? Refer to Chap. 3 and explain what sensor technologies can or cannot be realized in these cases. 8. Compare fluorescent bcarbon nanoparticles with semiconductor QDs considering the parameters that are essential for sensing and imaging.

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Yang W, Zhang H, Lai J, Peng X, Hu Y et al (2018) Carbon dots with red-shifted photoluminescence by fluorine doping for optical bio-imaging. Carbon 128:78–85 Yang ZC, Wang M, Yong AM, Wong SY, Zhang XH et al (2011) Intrinsically fluorescent carbon dots with tunable emission derived from hydrothermal treatment of glucose in the presence of monopotassium phosphate. Chem Commun 47:11615–11617 Yarur F, Macairan J-R, Naccache R (2019) Ratiometric detection of heavy metal ions using fluorescent carbon dots. Environ Sci Nano 6:1121–1130 Yazid SNAM, Chin SF, Pang SC, Ng SM (2013) Detection of Sn (II) ions via quenching of the fluorescence of carbon nanodots. Microchim Acta 180:137–143 Yu P, Wen X, Toh Y-R, Lee Y-C, Huang K-Y et al (2014) Efficient electron transfer in carbon nanodot–graphene oxide nanocomposites. J Mater Chem C 2:2894–2901 Yu P, Wen X, Toh Y-R, Tang J (2012) Temperature-dependent fluorescence in carbon dots. J Phys Chem C 116:25552–25557 Zhai X, Zhang P, Liu C, Bai T, Li W et al (2012) Highly luminescent carbon nanodots by microwaveassisted pyrolysis. Chem Commun 48:7955–7957 Zhang C, Yuan Y, Zhang S, Wang Y, Liu Z (2011) Biosensing platform based on fluorescence resonance energy transfer from upconverting nanocrystals to graphene oxide. Angew Chem Int Ed 50:6851–6854 Zhang J, Zhao X, Xian M, Dong C, Shuang S (2018) Folic acid-conjugated green luminescent carbon dots as a nanoprobe for identifying folate receptor-positive cancer cells. Talanta 183:39–47 Zhang M, Bai L, Shang W, Xie W, Ma H et al (2012) Facile synthesis of water-soluble, highly fluorescent graphene quantum dots as a robust biological label for stem cells. J Mater Chem 22:7461–7467 Zhang T, Zhu J, Zhai Y, Wang H, Bai X et al (2017) A novel mechanism for red emission carbon dots: hydrogen bond dominated molecular states emission. Nanoscale 9:13042–13051 Zhang X-F, Xi Q (2011) A graphene sheet as an efficient electron acceptor and conductor for photoinduced charge separation. Carbon 49:3842–3850 Zhang X, Wang S, Zhu C, Liu M, Ji Y et al (2013) Carbon-dots derived from nanodiamond: photoluminescence tunable nanoparticles for cell imaging. J Colloid Interface Sci 397:39–44 Zhang Y-L, Wang L, Zhang H-C, Liu Y, Wang H-Y, et al (2013b) Graphitic carbon quantum dots as a fluorescent sensing platform for highly efficient detection of Fe3+ ions. RSC Adv 3:3733–3738 Zhao HX, Liu LQ, De Liu Z, Wang Y, Zhao XJ, Huang CZ (2011) Highly selective detection of phosphate in very complicated matrixes with an off–on fluorescent probe of europium-adjusted carbon dots. Chem Commun 47:2604–2606 Zhao Y, Li K, He Z, Zhang Y, Zhao Y et al (2016) Investigation on fluorescence quenching mechanism of perylene diimide dyes by graphene oxide. Molecules 21:1642 Zheng C, An X, Gong J (2015) Novel pH sensitive N-doped carbon dots with both long fluorescence lifetime and high quantum yield. RSC Adv 5:32319–32322 Zhou J, Shan X, Ma J, Gu Y, Qian Z et al (2014) Facile synthesis of P-doped carbon quantum dots with highly efficient photoluminescence. RSC Adv 4:5465–5468 Zhou J, Sheng Z, Han H, Zou M, Li C (2012) Facile synthesis of fluorescent carbon dots using watermelon peel as a carbon source. Mater Lett 66:222–224 Zhou L, He B, Huang J (2013) Amphibious fluorescent carbon dots: one-step green synthesis and application for light-emitting polymer nanocomposites. Chem Commun 49:8078–8080 Zhou L, Lin Y, Huang Z, Ren J, Qu X (2012) Carbon nanodots as fluorescence probes for rapid, sensitive, and label-free detection of Hg2+ and biothiols in complex matrices. Chem Commun 48:1147–1149 Zhu H, Wang X, Li Y, Wang Z, Yang F, Yang X (2009) Microwave synthesis of fluorescent carbon nanoparticles with electrochemiluminescence properties. Chem Commun, 5118–5120 Zhu J, Bai X, Bai J, Pan G, Zhu Y et al (2018) Emitting color tunable carbon dots by adjusting solvent towards light-emitting devices. Nanotechnology 29:085705 Zhu S, Meng Q, Wang L, Zhang J, Song Y et al (2013) Highly photoluminescent carbon dots for multicolor patterning, sensors, and bioimaging. Angew Chem Int Ed Engl 52:3953–3957

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Chapter 10

Smart Luminescent Nanocomposites

Assembling organic or inorganic luminophores into nanoparticles and nanocomposites results in essentially new features. In this Chapter we describe how to optimize their design for the best functional performance. Different possibilities are demonstrated allowing not only to increase dramatically their brightness but also to explore superenhancement-superquenching effects in sensing. Moreover, the wavelengthshifting to any desired wavelength with desired modulation of emission lifetime can be provided. Essential for these constructions is the proper choice of donor and acceptor pairs in excitation energy transfer (EET). In contrast, λ-ratiometric sensing with the reference, multiplexing assays and multicolor optical coding require suppressing EET—the fluorophores in nanocomposite must emit light individually. The last “Sensing and thinking” section discusses these issues together with questions addressed to the readers. In previous Chapters of this book we discussed different properties of fluorescence emitters in order to understand and optimize them, adapting for different sensing and imaging technologies. New unlimited possibilities are offered by assembling them into nanocomposites formed of different types of emitters. They display a variety of novel useful features and thus extend dramatically the information content of output data. Researcher can design new materials selecting the building blocks from different types of luminophores of molecular and nanoscale origin (Fig. 10.1), manipulating with their connection, interaction, formation of new surfaces and interfaces. Their fluorescence response can be modulated by electronic conjugation or by excitation energy transfer and coupled with other functionalities on nanoscale level. The collective spectroscopic effects appear when the fluorophores are located at close distances. Depending on functional role in nanocomposite they can be both strongly positive and negative. In addition to superenhancement-superquenching effects, one can explore the designed shifting of emission wavelengths and increase the lifetimes by several orders of magnitude. The quenching-dequenching or wavelength shifts can be reversibly modulated by light. In sensing applications, the controllable broad-scale variation of fluorescence parameters can be used with great benefit. The structures can be designed, in which the excitation providing the light harvesting © Springer Nature Switzerland AG 2020 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-030-60155-3_10

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Organic O dyes Conjugated polymers

Lanthanide chelates Nanodaimonds

Quantum dots

UpconverƟng NPs

Fluorescent nanocomposite

Metal clusters Metal NPs

GFP-type proteins

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Fig. 10.1 The diversity of molecular and nanoscale luminophores that can be used as the building elements of smart nanocomposites

at the beginning of wavelength conversion cascade bring to final emitter very strong emission intensity with its depolarization and long lifetime (Hildebrandt et al. 2017). These possibilities are realized by exploring the mechanisms of excitation energy transfer (EET). The most important of them, in addition to electronic conjugation, is the Förster resonance energy transfer (FRET), since it can propagate to nanometer distances and couple most (or all) fluorophores in nanocomposite. The transfer through overlapping electron densities occurs at close distances. Located in close proximity, a number of dye molecules or various nanoclusters and nanoparticles, exhibiting both homo-EET and hetero-EET, allow realizing many interesting ideas that will be discussed below. Broad range of possibilities appear for successful development of sensing technologies based on these effects with a broad choice of molecular and nanoscale emitters used as building blocks (Fig. 10.1). If the EET mechanism is involved, the researcher can choose from this assortment of structures the optimal donors and

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acceptors. One has to account (or explore!) that in nanocomposite the fluorophore individuality disappears and that the emission parameters of acceptors may become different from that observed on their direct excitation. However, there are sensing technologies, which require the presence in emission of several bands belonging to individual fluorophores, and, therefore, EET is not desirable. Those are the techniques of λ-ratiometry with reference dye and of multiplex assays that are based on several emissions for several sensors. Optical barcoding that is needed for suspension array technologies involves multicolor labeling with dyes possessing their recognizable pattern of spectral bands, and this needs avoiding EET that brings wavelength conversion to single wavelength of the acceptor. Thus, the luminescent molecules and nanoparticles can serve as building blocks for achieving versatile functions that are not observed in separate emitters alone. This opens enormous possibilities in sensor design.

10.1 Broad-Scale Manipulation with Fluorescence Parameters in Nanocomposites The luminescent materials presented schematically in Fig. 10.1 can be viewed as building blocks for achieving versatile functions that are hard to observe in separate emitters. In these larger nanocomposites the fluorescent intensity can be strongly enhanced (Fig. 10.2), and this can allow decreasing the limit of detection in analytical assays and the probe concentrations applied for imaging. The fluorophore brightness is defined as the product of absorbance by quantum yield of emission, and both these factors are equally important for achieving enhancement of emission intensity. We will discuss the existing possibilities for that.

10.1.1 Assembling, Screening, Immobilization The nanocomposites of increased brightness can be achieved by assembling many organic or inorganic emitters into nanostructures formed by organic or inorganic polymer (see Chaps. 7–9). As it is depicted in Fig. 10.2, different factors can be responsible for the enhancement of fluorescence emission in dye assemblies. The most important of them is trivial. It is an increase in the absorption cross-section that can be made by greater number of assembled luminescent dyes (Wang et al. 2006b). Additional advantages of such incorporation could be the screening of dyes from direct molecular contact with the test medium and avoiding the dye degradation in aggressive conditions. The results of screening the fluorescent dyes and luminophore moieties within nanoparticles from direct contact with polar solvents, especially water, can be dramatic. Low emission observed on dye contacting with solvent can be due to

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Screening from solvent Assembly to form dense par cles

Plasmonic enhancement

Suppression of internal mobility

Excitonic effects

Fig. 10.2 Illustration of possibilities for enhancement of fluorescence emission upon the assembly of nanocomposites. See text

many different factors that are well described in the literature (de Silva et al. 1997). One of the most important of them is observed with the interplay of nonpolar and electron-rich emitters. Water molecules (and alcohols to a lesser extent) stochastically form in the bulk solvent the clusters with strong solvating power for electrons (the electron traps). The electron transfer from excited dye to such traps results in complete quenching of emission (Moore et al. 1985). This effect explains very low fluorescence intensity in water of many polarity-sensing charge-transfer organic dyes. When the contact with water is removed, the dyes become shining brightly. Many dyes possess flexible molecular structures. In solid environments they usually exhibit much higher quantum yields and lifetimes than in liquid milieu. This is due to stronger restrictions imposed by solid matrix on rotation of dye segments, to intermolecular collisions and to dielectric relaxations—all the factors that quench fluorescence (Riu et al. 2006). Moreover, the nanoparticles were designed that upon assembling the dyes demonstrate the aggregation-induced emission, AIE (see Sect. 8.3). For this purpose, those dye molecules were selected that contain the internal structure elements rotating in solutions and thus possessing the pathways for non-emissive electronic energy decay of the excited states. Being immobilized inside the nanoparticles, they lose such mobility. When the ultra-fast non-emissive decay associated with these motions is suppressed, the particles display bright emission.

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Different organizations of fluorophores in nanoparticles allow realizing these principles. Thus, the dendrimer scaffolds allow selecting preferential location of reporter dyes—in the rigid core, at the porous periphery or at the particle surface (Ow et al. 2005) see Sect. 8.4. These possibilities can be realized not only with organic dye molecules but also with luminescent metal complexes (Lian et al. 2004; Qhobosheane et al. 2001) and ions (Balzani et al. 2000; Shcharbin et al. 2007) (see Chap. 6).

10.1.2 Plasmonic Enhancement Plasmonic enhancement can be considered as a general mechanism of increasing the brightness of different emitters. It operates on their interaction with plasmonic nanoparticles made of silver, gold and copper. The physical background of this effect and its different applications will be discussed in Chap. 14. Therein we will also overview the rather stringent requirements regarding the distances, orientations, spectral fitting and other factors influencing the enhancement. Here we discuss in short the enhancement effect that can be realized in nanocomposites. Based on this effect, the composites can be constructed, in which fluorescent signal enhancement comes from the interaction between fluorophore and the surface plasmon generated in metallic nanostructure. Its realization can be achieved with different configurations of composite (Stewart et al. 2008). For instance, quantum dot can be attached to metal particle covered with polymer layer (Ma et al. 2011), or the metal particles can be bound via covalently interfacing the QD surfaces (Fu et al. 2009). Efficient are also the core–shell structures, in which the dyes are located in the particle core, so that the shell is the plasmonic metal cover (Miao et al. 2010). Gold and silver nanoparticles are also known as extremely potent fluorescence quenchers (Dulkeith et al. 2005). This ability can be used in combination with strong emitters such as the dyes and fluorescent nanoparticles. An immense variety of nanocomposites exhibiting plasmon-enhanced emission or efficient quenching can be constructed (Geddes 2017). This allows achieving important goal to increase the output signal and thus to reach the lower limits of target detection in sensing and the highest contrast in images.

10.1.3 Excitonic Effects. Coherent and Incoherent Energy Transfer Excitons are the mobile quasi-particles in the form of electron–hole pairs that appear in solid bodies and molecular associates upon electronic excitation. In conjugated polymers, the excitons can migrate along the polymer chain and between the chains (see Sect. 8.5). It was shown that the mixing of high and low band gap polymers in one nanoparticle can be very efficient in producing the enhancement effect. Due

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to exciton migration and energy transfer, the composites with excellent absorbance (3.0 × 107 cm−1 M−1 at 488 nm), wavelength-conversion and production of efficient deep-red emission (quantum yield = 0.56) were reported (Wu et al. 2011). Excitons in large dye aggregates can be delocalized across many fluorophores and may show coherent or incoherent energy transfer (Abramavicius et al. 2009). They can form ordered assemblies of various types, the limiting cases of which are the H- and J-aggregates (Sect. 6.3). Their properties can be described on the Kasha dimer model (Fig. 8.6). H-aggregates are formed by side-by-side association, their absorption spectra are shifted to shorter wavelengths, and fluorescence can be almost absent. In contrast, the absorption spectrum of J-aggregates are characterized by the presence of very sharp and strongly enhanced intensity of emission bands that are shifted to longer wavelengths and demonstrate very bright and often sharp bands in fluorescence emission (Würthner et al. 2011). Their absorption and fluorescence bands are narrow because of motional and exchange narrowing that dynamically average the inhomogeneous distribution of energies and also because the interaction between electronic and vibrational modes does not occur. The absence of solvent effects also contributes to very small Stokes shifts of J-aggregate bands. Very efficient excitonic enhancement effects can be observed on aggregation of cyanine and squaraine dyes (see Sect. 8.2). Many of these dyes are low soluble and almost nonfluorescent in aqueous solution. Thus, a very weak emission is observed for dye CN-MBE (1-cyano-trans-1,2-bis-(4 -methylbiphenyl)ethylene) in solution, but the intensity is increased by almost 700 times in the nanoparticles (An et al. 2002). Such strong enhancement was attributed to the synergetic effect of intramolecular planarization and J-type aggregate formation (restricted excimer formation) in nanoparticles. Cyanine dyes are known as the best J-aggregate formers (Bricks et al. 2017). However, much larger types of structures of organic dyes can make them on a scaffold, and as such scaffolds could be polymer nanoparticles, quantum dots (Halpert et al. 2009), macromolecules of DNA (Losytskyy et al. 2002), or protein molecules (Xu et al. 2010). It was shown that fluorescence intensity (at λem ≈ 690 nm) of a squaraine dye increases by a factor of up to 200 upon addition of bovine serum albumin (BSA) due to formation of J-aggregates, suggesting this effect as a tool for protein detection. In the case of quantum dot—J-aggregate composites, the effect of excitation energy transfer was clearly seen, resulting in complete quenching of fluorescence of the donor and the appearance of very narrow and very bright fluorescence band characteristic of J-aggregate (Halpert et al. 2009). The J-aggregates of cyanine dyes can be also formed with the aid of plasmonic gold nanoparticles inducing their assemblies (Lim et al. 2006). Thus, a new route in designing nanocomposites that combine plasmonic and excitonic effects is opened (Saikin et al. 2013).

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10.1.4 Exciton-Exciton and Plasmon-Exciton Interactions Among different possibilities for assembling fluorescent molecules and nanoparticles into composites, the combination of emitters with different types of collective excitation is the most interesting. Their interaction can be realized in direct way, extending spatially through electronic coupling between the components. Of particular interest are the systems based on plasmon interactions with excitons generated in the dye J-aggregates (Wiederrecht et al. 2004) that are known to produce very sharp bands in electronic spectra (Zheng et al. 2010). The emission of highly mobile excitons in conjugated polymers can also be strongly enhanced on interaction with plasmonic nanoparticles, and different hybrids have been suggested that display this effect (Tang et al. 2011). It has found application in DNA sensing (Geng et al. 2011). For different exciton-generating species, interaction with metal nanoparticles may present a new interesting case of exciton–plasmon interactions that goes beyond plasmon enhancement (Cao et al. 2018). The size and assembly of nanocomposites are the important parameters in nanoscale sensor design, since in quantum dots, conjugated polymers or organic dye aggregates the exciton size depends on the physical dimension and organization of the nanoscale system. Energy variables are also important, since the exciton migrates to the sites with the lowest band gap, and this must result in red-shifted emission. It was shown that mixing in a single nanoparticle of high and low band-gap polymers is very efficient, resulting in high absorbance and fluorescence quantum yield of red-shifted emission (Wu et al. 2011). Hetero-composites of conjugated polymers and quantum dots (Chan et al. 2012) demonstrated high brightness in the red and near-IR regions. Concluding, we indicate that the coupling of localized excitations with collective effects in excitation (excitons and plasmons) has great potential for modulating the excitation and emission properties of hybrid nanoscale materials in the desired direction. This area is open to active research and soon we expect many interesting results and important applications.

10.2 Realization of Electronic Energy Transfer (EET) in Nanocomposites In this Section we focus on the abilities of nanocomposites to alter the fluorescence emission parameters of constituting components. Light harvesting, wavelength shifting (conversion), lifetime extension, red-edge repolarization, these are the effects that sole or together can change fluorescence outputs in desired direction. This opens new avenues in optimizing the imaging and enhancement in sensor response.

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10.2.1 Homo-Transfer and Hetero-Transfer Usually in textbooks, electronic energy transfer (EET) is presented as a binary event occurring between single donor and single acceptor. The theory of electronic energy transfer developed by Theodor Förster and known with the abbreviation FRET refers to this case. Meantime, when many such EET donors and acceptors assemble in a small particle and appear within the critical distance, they lose their individuality and new collective effects appear (Demchenko 2013). One of these effects, the concentrational quenching (self-quenching), we already discussed in Chap. 8 in relation to nanoparticles formed by assembling organic dyes in polymeric and silica matrices or by their own self-assembly. In these cases we observe clear examples of homo-EET , the excitation energy transfer between fluorophores of the same structure. Like for classical hetero-EET (the transfer between different types of fluorophores), for homoEET the overlap between emission spectrum of the donor and absorption spectrum of the acceptor is the necessary condition (Sect. 4.3). Since in homo-EET the donor and acceptor molecules are the same, its efficiency is determined by the Stokes shift that determines this overlap The smaller shift—the higher is the efficiency of transfer. When the fluorophores are close enough, the spectral overlap becomes unimportant and the transfer of excitation energy occurs according to different rules. Location in close proximity of a number of dye molecules exhibiting both homoEET and hetero-EET allows realizing many interesting possibilities that are discussed below. One of them is shifting to the red of the emission spectrum by proper choice of acceptor dye without altering the light absorption properties. This may be useful for multiplex assays (single excitation and several emissions for several sensors). Moreover, impregnation of nanoparticles with a number of different dyes serving as both donors and acceptors transforms the particles into efficient wavelength converters with emissions over the whole visible range, extending to near-infrared. The other possibility is to increase the brightness of the acceptor by transferring the excitedstate energy from the donors. In this case, the donors may serve as ‘antennas’, lighting-up the acceptor and increasing in this way the sensitivity of its response.

10.2.2 General Rules for Collective Effects in EET We begin this Section with formulation of three rules that are important for explanation of the collective effects in the excited-state energy transfer in a system composed of different types of light emitters participating in the energy transfer process. 1. In a system composed of structurally similar dyes located in identical environments and possessing identical absorption and emission spectra and also quantum yields and lifetimes, EET (in this case homo-EET ) is just the exchange of energies between the dyes. It is undetected by spectroscopic or lifetime measurements. It can only be revealed by the measurements of anisotropy and only in the case if the dyes occupy different orientations in space and their orientations do not

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change fast on the scale of fluorescence decay (solid or highly viscous dye environments). This rule follows from the identity of all parameters determining the EET efficiency (Sect. 4.3) and from the kinetic scheme, in which the forward and reverse transfer rates are equal and higher than the rates of non-emissive and emissive transfers to the ground state. 2. In a system composed of structurally similar dyes possessing identical fluorescence spectra but exhibiting the effect of static quenching (see Sect. 3.2), the quenching will be observed for the whole system. This effect is called superquenching (Sect. 8.1). The quenching efficiency will be determined by the dye with the shortest lifetime τ. This result follows from the kinetic scheme, in which for this dye the rate of non-radiative deactivation of the excited state is comparable or faster than the rate of energy transfer. If the transfer is efficient and this dye is fully quenched, then we will observe quenching in the whole ensemble. 3. In a system composed of structurally dissimilar dyes possessing different excitation and emission spectra, the transfer is directional from the dyes possessing short-wavelength to that possessing long-wavelength absorption and emission spectra (with the account of rule 2). This is because of overlap integral J for the transfer being larger if the donor emission is at shorter and acceptor absorption is at longer wavelengths than in the opposite case in the conditions of FRET (see Fig. 3.13), and this regularity is also observed in the case of the presence of electronic coupling between fluorophores. Thus, the energy will flow from blue (short-wavelength) to red (long wavelength) emitters. The latter case is the case of hetero-EET . Based on these rules one can easily describe the effect of ‘superquenching’ that can be observed in dye-doped polymeric particles, in conjugated polymers and dendrimers (Chap. 8). Moreover, manipulating with a single trigger dye, one can provide efficient collective sensor response by switching on and off the whole ensemble of fluorescence emitters. In a general case, the transfer and the quenching efficiencies are determined by the interplay of rate constants of all transformations of the excited states, starting from that of initially excited donor (Varnavski et al. 2002).

10.2.3 Light-Harvesting (Antenna) Effect One of collective effects realized in nanoparticles is the light harvesting. It can be observed when a large number of strong light-absorbing EET donors, being excited, transfer their energy to a much smaller number of acceptors generating their multiple times brighter emission (Scholes et al. 2001). The antenna effect is illustrated in Fig. 10.3. The necessary conditions for its efficient implementation are the high molar absorbance of antenna dyes, their large number, efficient energy transfer to acceptor dye and high quantum yield

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b hνa

hνa

a

D

D D D

D D

A D

Low-intensity emission

D D hνF

D

D D D

D D

A D

Ultra-bright emission

D D hνF

Fig. 10.3 Collective effects observed within the system of multiple fluorophores connected with efficient EET. a Direct excitation of the acceptor. Acceptor demonstrates its own excitation and emission properties and the fluorescence is relatively weak. b Light-harvesting. Many donors (D) serving as antennas donate their excitation energies hνa to small number of acceptors (A) that emit light of energy hνF much brighter than it should be on their own excitation

of emission of the latter. Such systems of signal transduction allow providing the response in sensor operation on several steps: the energy exchange (homo-EET) and quenching in antenna system, the energy transfer to the acceptor and quenching of the acceptor emission. The possibility to manipulate with reporter properties of acceptor is retained. It may be the environment-sensitive two-band or wavelength-shifting dye or the dye forming exciplexes (Chap. 4). The acceptor fluorescence properties should not be different from those observed on its direct excitation but can be seen on a much brighter level. It is interesting that this principle of light-harvesting is used in the natural systems of photosynthesis that collect an enormous amount of solar energy by exciting the so-called antenna pigments and re-directing it to a small number of reaction centers where it is converted into redox chemical energy. So, this principle is very general and it may be used for optimizing fluorescence (and, in general, luminescence) of many molecular and supramolecular systems, from dimers of organic dyes to coupled conjugated polymers (Wu et al. 2011) and complex nano-composites (Chen et al. 2005; Frigoli et al. 2009). For instance, such ‘antenna effects’ are also in the background of dramatic increase of luminescence of weakly absorbing lanthanide ions when they form coordination complexes with strongly light absorbing ligands (Sect. 6.1). With new fluorescent materials the choice of donors, acceptors, their relative concentrations, etc., if it is properly made, can be very efficient. An example of such solution can be found in the work on blended conjugated polymer nanoparticles (Huang et al. 2007). A 90% of conversion of blue light into green, yellow and red emissions was achieved by addition of less than 1% of polymer emitting at

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these wavelengths. It was stated that because of such composition, the fluorescence brightness of the blended polymer nanoparticles can be much higher than that of inorganic quantum dots and dye-loaded silica particles of similar dimensions. A dramatic increase of fluorescence emission intensity of the acceptor dye due to ‘focusing of antenna’, compared to direct excitation, some authors call an ‘amplification’. It should be stressed, that in contrast to cases discussed in Sect. 7.1 there is no amplification on the level of acceptor dye, since its absorbance and quantum yield do not increase. All what happens is that via antenna it becomes excited much more frequently than on common direct excitation. This is the reason of its highly increased apparent brightness. Photostability of acceptor dyes is important (Forde and Hanley 2005), since the increase of the frequency of the acts of photoexcitation-emission results also in their accelerated photodegradation (Demchenko 2020).

10.2.4 Wavelength Converting. Cascade Energy Transfer Manipulating with hetero-EET, one can design the efficient ‘wavelength converter’. One can choose the primary donor with the excitation spectrum that ideally fits the applied light source and the final acceptor with position of emission maximum at the desired wavelength. This wavelength can be chosen throughout all visible range, down to near-IR. The transfer, in which several different dyes provide the chain of transfer events for achieving a very significant shift in emission wavelength, is called a ‘cascade ’energy transfer’ (Serin et al. 2002). If the goal is in obtaining bright emission of final acceptor, it is important on this pathway to avoid the self-quenching interactions of the dyes, the ‘light traps’. The ‘intermediate’ dyes serving as both donors and acceptors may be needed to fill the gap in the energy transfer chain. By fusing organic dyes (Ziessel and Harriman 2011) or semiconductor nanoparticles (Rogach et al. 2009), cascades with different wavelength-converting properties have been devised. From the description of FRET effect (Sect. 4.3) one can derive that, though this process is reversible, it is always shifted towards the decrease of excitation energy resulting in the appearance of acceptor emission spectra at longer wavelengths. (Check the effect of overlap integral with the account of Stokes shift). The reverse transfer is low probable, since, according to FRET theory, this requires the overlap integral between long wavelength emitting donor and short wavelength absorbing acceptor. In an ensemble of fluorescence emitters, the result is in directional energy flow from short- to long-wavelength emitters (Stein et al. 2011). Thus, by selecting the dyes with optimal spectral overlap one can excite fluorescence at short wavelength and obtain emission at much longer wavelengths. Operating with such ‘wavelength converter’ (Varnavski et al. 2002) and combining several connected by EET emitters displaying sequentially shifted excitation and emission spectra, one can shift fluorescence spectrum of the terminal acceptor over the whole visible range, maintaining the short-wavelength excitation (e.g. near-UV)

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in the desired range. This can be achieved in cascade manner in a sequence of dyes, in which an energy acceptor on one step can serve as the donor to the dye on another step. The scheme (Demchenko 2013) and a typical structure formed from the chain of organic dyes (Schwartz et al. 2010) are the illustrations of such processes (Fig. 10.4). For constructing such ‘cascade energy transfer’ a required combination of organic dyes can be easily assembled within silica nanoparticles (Rampazzo et al. 2014, Wang et al. 2006a). Any other scaffold can be used, from DNA template to plain surface. Recent literature contains many examples for constructing the ‘cascades’ (Haustein et al. 2003), including those made of quantum dots (Rowland et al. 2017). Usually they are made by covalent linking of monomer dyes, which allows strict control of their stoichiometry (see Fig. 10.4b). Such are the pyrene-BODIPY molecular dyads and triads (Ziessel et al. 2005). Efficient energy flow was reported in a purposebuilt cascade molecule bearing three distinct fluorophores attached to the terminal acceptor (Harriman et al. 2008). A combinatorial approach with the selection of the

Fig. 10.4 The cascade system operating as the wavelength converter based on multi-step EET a. The primary donor (D) is excited by the short-wavelength energy quanta hνa . This energy is transferred without emission to intermediates (I1 ) and (I2 ) that serve as both donors and acceptors transmitting their excitation energy to final acceptor (A). The later emits light of very different energy hνF with a strong red shift. b Sequential energy transfer across four fluorophores tethered to a single-stranded acyclic backbone

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best hits can be applied using the assembly of fluorescent oligonucleotide analogs (Gao et al. 2002). Removing and adding the intermediates in a cascade EET process and in this way modulating the emission color, one can produce strong signals in λ-ratiometric detection (Watrob et al. 2003). Such multi-color measurements can be efficiently applied in flow cytometry (Fábián et al. 2013). One can combine the light-harvesting and cascade-transfer wavelength shifting. Moreover, the generation of superquenching effects can be easily produced in cascades and under the action of light harvesters. So, if the EET is efficient, the quenching of final acceptor quenches the whole ensemble (see Fig. 8.3). This opens enormous possibilities in sensor design.

10.2.5 Extending the Emission Lifetimes Fluorescence probing and sensing with emitters exhibiting long lifetimes has a great advantage by allowing the rejection of a typically shorter background emission (see Sect. 3.4). This is very beneficial, particularly, for increasing the contrast in cell imaging. To address this demand, different nanocomposites were suggested, in which the long-lifetime emitter (e.g. lanthanide complex) is the EET donor, and the strongly responding fluorophore (e.g. organic dye) is the acceptor. The fact that in cascade energy transfer the acceptor, changing the wavelength of its emission, retains the emission lifetime of the donor, τD „ is illustrated in Fig. 10.5. In this combination the acceptor can emit light with an unnatural very long lifetime, retaining its own spectroscopic response (Bunzli and Piguet 2005).

Life me

Fig. 10.5 The cascade system similar to that in Fig. 10.4. The emission passing through the energy transfer chain is red-shifted on a wavelength scale, but these transformations occur with the unchanged lifetime τD of the donor. In this way the final acceptor A can emit light with strongly prolonged lifetime

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Such changes in emitter properties occur because in a donor–acceptor system connected by EET the donor lifetime determines the lifetime of the acceptor (Hildebrandt et al. 2007). This follows from kinetic scheme of this reaction (Charbonniere et al. 2006b) describing continuous energy transfer during the lifetime of the donor. Such transfer results in emission of the acceptor on a much longer time scale than on direct excitation. The fluorophores, that are not at a proper distance or do not exhibit necessary for the occurrence of EET spectral or electronic coupling, will not extend their lifetimes. This allows rejecting the background emission (Morrison 1988). These attractive features were put in the background of time-gated EET that demonstrates strongly increased popularity (Zhao et al. 2018).

10.2.6 Variations of Anisotropy Organic dyes, carbon dots and anisotropic semiconductor nanostructures typically possess a high level of optical anisotropy detected as the polarized emission when the emitter is excited by polarized light. The exploitation of changes in polarization of emission in nanoparticles is much more limited than in the case of monomeric organic dyes. The polarized incident light excites selectively the fluorescent species in particular orientation, and they emit polarized light (see Sect. 3.3). The necessary condition for that is the absence of rotational mobility of a fluorophore or of the whole structure to which it is firmly bound during the emission lifetime. The dyes in nanostructures can be rigidly fixed in them and the rotations of the whole particles are usually rather slow on the nanosecond time scale, so a high level of anisotropy can be observed. This cannot always be so, since the other condition is the absence of EET, which depolarizes the emission. In densely packed multi-fluorophore structures it is hard to avoid homo-EET, and in its presence the anisotropy of observed emission decreases dramatically, often to zero level. This is because the orientations of donors and acceptors are usually random, and in the case of multiple transfers and random orientation of acceptors, the information on initial polarization is lost. In any ensemble of similar emitters the drop in anisotropy is the major indicator of the presence of homo-EET. Section 8.1 provides extensive discussion on this subject. This seemingly discouraging result may lead to quite positive consequences. There is a possibility of manipulating with the homo-EET efficiency by variation of excitation wavelength within the absorption band generating the so-called Red Edge effect (Demchenko 2002, 2008). In this way the emission anisotropy can be changed from its low values observed at the band maximum to its high values (demonstrating suppression of EET) by shifting the excitation to the red (long wavelength) edge of absorption band (see Fig. 8.5).

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10.2.7 Modulation of Luminescence by Excitation Light Switching of emission in energy transfer cascade can be achieved by incorporation of photochromic dyes, whose intrinsic property to absorb and emit light is controlled by external light sources. They can also serve as key components of the systems operating by EET mechanism (Sect. 5.4). In these composites, different fluorophores (organic dyes, conjugated polymers, fluorescent proteins, quantum dots, etc.) can be used as the donors and photochromes as acceptors. Key role is played by these EET acceptors, which can be switched reversibly between colorless spiropyran and colored merocyanine forms upon alternating irradiation with UV and visible light (Klajn 2014). In such composed photochromic systems, by the same excitation of the donors the emission can be switched reversibly between a fluorescent ON and a non-fluorescent OFF state (see Fig. 5.23). Since the process is reversible, the fluorescent light intensities can oscillate between the EET donor and acceptor emissions providing ON–OFF switching at each act of photoinduced transformations by light (Giordano et al. 2002; Jares-Erijman and Jovin 2003; Tian et al. 2009). Among different photochromes, diheteroarylethenes present many advantages (Giordano et al. 2002) that have to be realized in nonpolar media. Therefore, for making them useful in biological imaging, the water-soluble nanoparticles with amphiphilic properties hosting them were created (Díaz et al. 2013). Lucifer Yellow dye was used as the donor and diheteroarylethenes as acceptors. Both of them were covalently bound in multiple copies to an amphiphilic polymer to create dual polarity particles in the form of polymersomes. Quantum dots were incorporated into the core of composed nanoparticle, serving as a stabilizing template. The designed architecture was used to increase the efficiency of the donor dye and to provide dual-color self-referenced emission at single excitation wavelength (Tian et al. 2009). In photochromic composites, quantum dots can play a more active role being EET donors providing excitation energy to photochrome groups coating them. The advantage here is their large ultraviolet–visible absorption cross-section and broad range of wavelengths that can be used for excitation (Díaz et al. 2015). There are many implications of such photochromic systems. For instance, the photochrome can be incorporated at each site of wavelength converter cascades described above, which allows switchable by light termination of change in emission color with restoration of donor emission. Moreover, the whole process may start in the near-IR by using up-converting nanoparticles (Zhang et al. 2012). Major applications of such nanocomposites are expected in fluorescence microscopy, particularly with superresolution capability (Kozma and Kele 2019).

10.3 Superenhancement and Superquenching In this section, we discuss in brief already presented above (Chap. 8) possibility to modulate the response of fluorescence emitters arranged into ensemble in much broader ranges than it is possible with a single reporter unit. Our aim is in achieving the most efficient signal transduction and the most intensive signal output. We try

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to explore collective properties in fluorophore ensembles that are absent in the composed units. Several strategies have been proposed and realized to generate amplified fluorescence response in sensor systems, which can be the transitions from the highest possible brightness to its extremely low level, or the reverse. Basic mechanisms of achieving the fluorophore brightness can be realized by assembling them into nanoparticles. They were discussed in Chap. 8 regarding the dye assemblies and conjugated polymers. Here, by assuming that the highest brightness is achieved, we discuss how it could be manipulated for generating the most efficient analytical signal. We will operate with the term”superquenching” that will indicate the quenching of the whole ensemble of coupled emitters in a single event. The term “superenhancement” could mean the restoration of a bright fluorescence emission of this ensemble, “superdequenching”. Here we consider superquenching as a general effect that can be realized in any assembly of fluorophores connected by electronic conjugation or EET. If the system of similar emitters is connected by homo-EET, then the energy can migrate between them and it can be emitted by any different emitter within this system. Then if the quencher is present in the system, it becomes the trap for this energy. Such quencher can be non-fluorescent EET acceptor, metal nanoparticle or molecular complex exhibiting static quenching. The dynamic range of change in intensity is much greater than on quenching of individual fluorophore (see Fig. 10.6a). Thus, ‘superquenching’ is the collective effect, which, with the introduction of fluorescence quencher, results in the quenching of the whole ensemble of emitters (Demchenko 2013; Lu et al. 2002). Essential is the fact that the bright emission can be provided by the whole ensemble of dye molecules and that a single additional

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Fig. 10.6 Mechanisms of ‘superenhancement’ and ‘superquenching’ in a system of fluorophores connected by EET. a The fluorophore D emits light intensity on level 2 and its interaction with the quencher Q results in emission decreased to level 1. When the system of a large number of fluorophores D is excited, its emission is much higher, on intensity level 3, and the interaction of only one fluorophore with the quencher results in quenching of the whole ensemble of these emitters. The vertical arrows indicate the dynamic ranges of sensor response in both cases. b The same, but the FRET acceptor A is present in the system that collects all excitation energy from donors D and emits light at a red-shifted wavelength. When the quencher is present and A is alone, the dynamic range of response as the difference between intensity levels 2 and 1 is small. It becomes very significant when the system of donors D is excited and transfers its energy to acceptor A

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molecule can report on intermolecular interaction by inducing the effect of quenching in the whole system (Johansson and Cook 2003). This strongly amplified collective response can be seen, for example, in the dye-doped nanoparticles (Kim et al. 2006). It has the same origin as the ‘concentrational quenching’ in dye aggregates (Johansson and Cook 2003), discussed in Sect. 7.1, but in this case it can be used for controlled sensor response. We can consider a different realistic case, in which in contrast to that considered above (Fig. 10.6a) the excitation energy is collected not by the quencher but by light emitting EET acceptor (Fig. 10.6b). In this case, all the energy collected by the acceptor can be transferred in one step to the quencher. Here again, the profit is great. Instead of quenching the low-intensity signal of a single dye, an intensity existing on a much greater level can be quenched. In the case of existence of multi-step EET cascade (see Fig. 10.4), the quencher can be applied to any member of this cascade with equally dramatic superquenching (Demchenko 2013). Superquenching can be realized in all cases of collective behavior between interacting fluorophores, particularly in all types of nanocomposites connected by homoEET . We already discussed the issue of superquenching in the case of conjugated polymers (Chap. 8). This is a demonstrative example of excitonic coupling between fluorophores assembled into polymeric chains with high level of delocalization of excited-state electron density. The coupling of fluorescence emitters allows the excitation to migrate between different fluorophores in the system. Another type of superquenching is observed in J-aggregates of cyanine dyes. Such collective effects were observed for cyanine dyes covalently attached as side groups to watersoluble cationic polymer (Jones et al. 2001). The cyanine dye fluorophores strongly associate in a J-aggregate structure characterized by a sharp red-shifted absorption (compared to the monomer) and a similarly sharp red-shifted fluorescence. Collective effects in quenching demonstrate very large Stern−Volmer quenching constants. Superquenching was reported in lanthanide-polymeric conjugates incorporated into nanoparticles (Li et al. 2014). There is an unlimited range of possibilities to explore this phenomenon.

10.4 Optical Choice of EET Donors and Acceptors Many different types of reporters are applied as donors and acceptors in EET (Sławski and Grzyb 2019; Xu et al. 2020). With our knowledge of the properties of a variety of fluorescent materials, of their use as reporters and of new effects that appear on their assembling into nanoparticles and nanocomposites, we can discuss the construction of these assembles in the most optimal way. In the discussion that follows we make an attempt to provide systematic analysis of usage of different types of fluorophores as donors or acceptors in EET (Demchenko 2013) (Fig. 10.7). The donors should be the most efficient absorbers and the brightest emitters and, as a favorable property, possess long lifetime and high anisotropy of emission. In contrast, the acceptors should be the most ‘responsive’, changing the parameters of their emission in the

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FRET donors Conjugated polymers

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FRET acceptors Fig. 10.7 The scheme of preferences for different fluorescent (luminescent) emitters to be the donors or acceptors in energy transfer in EET-based nanocomposites. The arrows indicate the preferred directions of energy flow (Demchenko 2013)

broadest range. Each of them has its advantages and drawbacks. Here we follow the idea to use EET as the mechanism that could allow strengthening the strong and suppressing or eliminating the weak points in fluorescence reporting by coupling fluorescence emitters and allowing the excited-state energy transfer to occur between them.

10.4.1 Lanthanide Chelates and Other Metal-Chelating Luminophores Short-wavelength excitations and very long lifetimes put lanthanide chelates (see Sect. 6.3) to the first line of donors. Their good partner acceptors are both QDs (Charbonniere et al. 2006b, Geissler et al. 2014) and organic dyes (Selvin 2002). Combining the two optimization parameters (brightness and lifetime) one may infer that the optimal EET donors could be the nanoparticles doped in high density with

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luminescent lanthanide chelates (Zhao et al. 2018). This allows increasing the duration of transfer and enhancing in this way the EET efficiency. Due to convenient positions of lanthanide emission bands for overlapping the absorption spectra of many organic dyes, these dyes can be used as EET acceptors. Even more attractive as such acceptors are semiconductor quantum dots (QDs), in view of their much higher brightness (Tian et al. 2009). Such particles have been described in the literature (Casanova et al. 2006; Monat et al. 2007) and their efficient EET to organic dyes (Casanova et al. 2006) and quantum dots (Charbonniere et al. 2006a) were demonstrated. There are good examples in the literature on illustration and application of this principle to phosphorescent chelating complexes. EET from iridium as the donor to semiconductor QD as the acceptor resulted in increasing the lifetime from 40 to 400 ns (Anikeeva et al. 2006). Dramatic (by two or more orders of magnitude) increase of lifetimes of organic dyes can be also achieved by energy transfer from excited europium luminescent and ruthenium phosphorescent complexes.

10.4.2 Semiconductor Quantum Dots (QDs) Quantum dots (see Sect. 7.2) occupy one of central positions in nanocomposite design (Hildebrandt et al. 2017). In addition to already mentioned advantages as EET donors, such as the possibility to excite fluorescence in a very broad wavelength range, to display narrow and strongly size-dependent emission bands and high brightness, they possess other useful properties. They are reasonably resistant to photobleaching, and their high brightness is also observed in the conditions of two-photon excitation (Jares-Erijman and Jovin 2003). Owing to their very broad short-wavelength excitation range and high brightness (Algar and Krull 2008; Medintz and Mattoussi 2009), the quantum dots are ideal for transferring their energy to organic dyes (Snee et al. 2006) and fluorescent proteins (Algar et al. 2010). With QDs, the excitation wavelength can be selected within broad ranges avoiding direct excitation of acceptors, and their narrow size-dependent emission spectra can be easily adjusted to acceptor absorption. They also possess the lifetimes of ~10–8 s, which are typically longer than that of organic dyes. Different sensors were designed based on QD-dye pairs (Suzuki et al. 2008). The donor–acceptor pairs can be easily selected from QDs of different sizes and the series of dyes possessing also gradual shifts of their fluorescence spectra, such as Alexa dyes. EET between quantum dots emitting at 565, 605, and 655 nm as energy donors and these dyes with absorbance maxima at 594, 633, 647, and 680 nm as energy acceptors were tested (Nikiforov and Beechem 2006). As a first step, the covalent conjugates between all three types of QDs and each of the Alexa labels were prepared. The EET can be easily detected and the binding quantified in homogeneous system on formation of complexes avidin–biotin and antigen–antibody. Competitive binding assays can also be developed in such systems.

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The approach based on QD donors and dye acceptors has found many applications: in DNA and RNA hybridization assays, in immunoassays, in construction of biosensors based on ligand-binding proteins and enzymes. Usually the following configuration is used: QDs are conjugated with the receptor molecules, and organic dyes are used either for target labeling or for labeling the target analogs in competitor displacement assays. The same properties that make QDs so attractive as donors do not allow them to serve as efficient EET acceptors. Particularly, their absorbance extending to UV and occupying part of the visible range does not allow avoiding their direct excitation. Whereas the combination of quantum dots as EET donors and organic dyes as acceptors is very efficient, the reverse combination, of dyes as donors and QDs as acceptors is not efficient at all (Clapp et al. 2005a). Still, their role as acceptors can be efficiently realized with lanthanide chelates as the donors due to their long-lifetime emission (Charbonniere et al. 2006b) and also with upconversion nanoparticles, due to the absence of QDs absorption in the near-IR region (Hildebrandt et al. 2017). In the latter role they have found a broad range of applications, from immunoassays to multiplexed specific RNA detection.

10.4.3 Upconverting Nanomaterials The upconverting emitters (Sect. 7.5) have deserved their special role primarily as EET donors (Chen et al. 2015). Due to extremely sharp and narrow emission bands characteristic of luminescent lanthanide ions, no donor emission can be detected at the wavelengths of sensitized emission of the acceptor. In addition, no acceptor emitting in the visible range can be excited directly because the excitation is in the nearIR. The EET from upconverting phosphors to QDs (Bednarkiewicz et al. 2010) and carbon nanoparticles (Wang et al. 2011) was demonstrated to be highly efficient. Even more, the energy transfer to small-molecular dye was used for diagnostics directly in serum (Kuningas et al. 2006), showing the absence of external interference. An attractive sensor platform was demonstrated based on EET quenching by graphene oxide (Liu et al. 2011). Thus, organic dyes and other luminophores absorbing light in the visible range can be used with these nanoparticles also as EET acceptors (Dukhno et al. 2017; Muhr et al. 2017). This allows modulating emission wavelength and generate informative sensor signal. Of great importance here, as in any other sensor applications of upconverting nanoparticles, is the absence of any background emission. Meantime, these nanoparticles need themselves a substantial improvement, since their typically very weak and narrow absorption bands together with their size-dependent luminescence efficiency can limit their application potential. Their improvement can be achieved by creating a sophisticated core–shell particle architectures with organic dyes as antennas generating the near-IR emission that can be transferred for their excitation (Chen et al. 2015; Zou et al. 2012). By constructing a simple water-dispersible micellar system that transfers energy from the near-IR excitable

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Fig. 10.8 Light harvesting excitation of upconverting nanoparticles with near-IR dyes incorporated into micellar shell (Saleh et al. 2019). Oleate-capped NaYF4:20%Yb(III), 2%Er(III) upconversion nanoparticles were used and an enhancement of the green Er(III) emission compared to direct Er(III) excitation at 808 nm is observed. a Antenna dyes (violet) absorb near-IR light (808 nm) and transfer it to the particle core (blue), where the upconversion occurs. b The dye concentration dependence of fluorescence spectra in Pluronic F-127 micelle and c the same for Twin 80 micelles. Note that these functions are strongly nonlinear and after sharp rise demonstrate slow decrease

dye, we could demonstrate dramatic enhancement of luminescence of upconverting nanoparticles (Saleh et al. 2019). Interestingly, this effect of enhancement depends strongly on excitation power density and on dye loading (Fig. 10.8).

10.4.4 Conjugated Polymers Conjugated polymers (Sect. 8.5) are also in the line of optimal donors because of their very high brightness (Reppy 2010; Thomas et al. 2007), although their excitation is commonly spatially distributed over the entire polymer chain and they cannot be considered as point emitters. Their brightness is further increased reaching record values on formation of EET complexes with organic dyes (Wu et al. 2008) and QDs (Jiang et al. 2009). Their partner organic dyes are able to transmit EET into

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informative signal demonstrating modulation of their own response or displaying superquenching (Pu and Liu 2009; Thomas et al. 2007). Conjugated polymers allow realizing different signal transduction and reporting mechanisms, being active participants in different supramolecular constructs. They endow these structures with the ability to exhibit collective effect in PET and EET quenching. A designed combination of this polymer with another partner serving as a quencher or EET acceptor opens many interesting possibilities in sensing. One of them is a combination of conjugated polymer with organic dye. The dye can be either directly attached to the polymer chain or co-incorporated into nanostructure with efficient collective effect in EET. The latter is very efficient when it is directed from excited polyanionic conjugated polymer to a cationic cyanine dye (Tan et al. 2004). Interesting is the possibility of combining different conjugated polymers in one unit. Their chemistry is well developed and involves the formation of not only the polymer films or latexes but also of well-organized nanoparticles - dendritic conjugated polymers (Zhang and Feng 2007). Such encapsulation leads to new spectroscopic properties. A very small proportion of polymers that may serve as the EET acceptors allows observing only their emission with excitation of the major component excited as the donor (Wu et al. 2006). These experiments demonstrated that fluorescence brightness of the blended polymer nanoparticles could be much higher than that of inorganic quantum dots and dye-loaded silica particles of similar dimensions. Different nano-scale support media such as silica can be used for assembly of polymer-coated particles (Wosnick et al. 2005), in which different routes of reporter signal transduction can be activated. An exceptional ability of gold nanoparticles to quench cationic conjugated polymer was demonstrated (Fan et al. 2003). During fabrication, these particles can be stabilized with citrate, so they can contain the negative charge. In the bound form, they produce the quenching effect, so that one gold nanoparticle could ‘superquench’ several polymer chains. A promising field of application of conjugated polymers is the sensing technology based on thin films, especially in gas sensing (Volume 2, Chap. 8), and further development of artificial ‘noses’ and ‘tongs’ (Volume 2, Chap. 18). The proper polymer design allows avoiding the quenching in aggregates. For instance, poly(phenylene ethynylene)s with specially designed substitutions are highly emissive in aggregated forms due to formation of intermolecular excimers (Kim et al. 2005). A series of non-aggregating carboxylate-functionalized poly(phenylene ethynylene)s have been synthesized for immobilization via electrostatic adsorption onto Eu3+ -polystyrene microspheres (Liao and Swager 2007). This system is shown to constitute a ratiometric system that measures fluorescence quenching with high fidelity.

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10.4.5 Organic Dyes and Visible Fluorescent Proteins In the most known to date applications, organic dye molecules (Chap. 5) dominate as EET acceptors from various kind of donors. This is because of their two important properties: (a) they are small molecules that allow easy and versatile conjugation with nanoparticles and (b) they may be highly responsive as reporters by changing different parameters of their emission. Their excitation via EET increases the sensitivity of response but may not change the mechanism of this response based on its interactions and dynamics. Nanoparticles made of organic dyes or formed by their inclusion into polymer structures (Chap. 8) open new area in this research. They are efficient as EET donors and with proper functionalization as the acceptors and terminal emitters in sensor technologies. The energy transfer to them was described from conjugated polymers, AIE-luminogens (Liu et al. 2018), and many of such examples are mentioned in cited above publications (Sławski and Grzyb 2019; Xu et al. 2020). Fluorescent proteins are famous for their ability to be synthesized in living cells and as cellular products for providing their sensing ability (Sect. 5.5). We described their successful applications as sensor dimers responding by coupling-decoupling of EET. Their application is much broader when they are assembled with dyes or nanoparticles, thus, intracellular pH sensor can be constructed based on fluorescent protein coupled with quantum dot (Dennis et al. 2012).

10.4.6 Noble Metal Nanoparticles As we discussed in Sect. 6.1, the clusters of gold and silver, of several atoms in size, possess spectroscopic properties similar to that of organic molecules. Their behavior in EET is also quite similar, which was witnessed by recent data on EET from gold nanoclusters to J-aggregates of cyanine dyes (Banerjee et al. 2020). If we refer to Fig. 10.8, its bottom is represented by metal nanoparticles that if located at short distances to the donors (organic dyes, QDs, etc.) can serve as nonemissive EET acceptors, quenchers (Sen and Patra 2012), in addition to their known role as PET quenchers. Therefore, together with different nanocarbon structures, they can serve as strong ‘universal’ (wavelength-independent) fluorescence quenchers that can be used in combination with other emitters in different sensing technologies (Swierczewska et al. 2011).

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10.4.7 Fluorescent Carbon Nanoparticles The spectral range available to fluorescent nanodiamonds (Sect. 9.1) that was much narrower than that to organic dyes or quantum dots has increased in novel developments, but their low absorbance remains to be a serious drawback. Still, with their emission maxima in the red region they can serve as the EET donors for near-IR emitting dyes (Chen et al. 2011). The less common blue emitting nanodiamonds can transfer their energy to green emitting dye (Maitra et al. 2011). Carbon dots, graphene and graphene oxide nanoparticles (Chap. 9) are the newcomers in the family of efficient nanoscale fluorophores. Their efficiency in the excitation energy transfer systems is still not recognized in full, but their easy and cheap production together with good brightness and photostability and other attractive properties, has resulted in their high popularity among the researchers. At the time of writing, the general photophysical mechanism of their emission is not established and is actively debated. The present author suggested excitonic model that involves H-aggregates (Demchenko 2019), Initially, their excitation spectra were found at 350–400 nm and emission spectra in the blue-green wavelength region. At present, new materials and new methods were developed for their production, so their emission covers the whole range from violet-blue to near-IR. Their application as EET donors has been suggested by many authors, usually to appended organic dyes serving as acceptors (Li et al. 2015; Sun and Lei 2017). The λ-ratiometric signal can be obtained in sensing by switching between donor and acceptor emissions. Regarding their application as acceptors, one has to account their very high absorbance in the whole UV region extending to visible range exciting nonemissive states. Since it is uncoupled with the emissive state (Dekaliuk et al. 2014) this may provide optical screening. Similarly to QDs (Huang et al. 2006), carbon dots can nicely serve as EET acceptors from chemiluminescent donors. Thus, we clearly observe the ‘energy flow’ from blue to red emitters, from long to short lifetime emitters, from emitters of higher to lower brightness, sometimes down to efficient quenchers. The strongly responsive emitters, such as organic dyes and their nanoscale forms together with fluorescent proteins, are requested mostly at the acceptor side. In such compositions, the nanoparticles attain very useful properties that guarantee their numerous applications. In these properties and applications there is no sharp margin between inorganic, organic or inorganic–organic hybrid materials (Zhang et al. 2017; Zhao et al. 2018).

10.5 Wavelength Referencing, Multiplexing and Multicolor Coding Now we address the problem of creation of nanocomposites, the incorporated fluorophores in which perform the necessary functions to serve as references in λratiometric sensors or providing color diversity in multiplexing or multicolor coding.

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Referencing of fluorescence signal is required in quantitative intensity sensing (Sect. 3.2) and selective labeling of nano- or microparticles is needed for their identification, “barcoding”. The optical codes on nanoscale level are needed for their recognition in mixtures. Such recognition is required when they are applied in fluorescence sensing in homogeneous format (Sect. 1.1). Within a single nanocomposite, the functions of its specific recognition and of fluorescence reporting can be combined.

10.5.1 Wavelength Referencing and λ-Ratiometry Fluorescence intensity is the parameter that is rather simple for recording. Being expressed in arbitrary units, it is of limited quantitative value since it depends on fluorophore concentration, on its distribution in studied objects and also on instrumental parameters, temperature, quenchers, etc. Because of this reason, the analytical signal is difficult to scale and compare with the results obtained in different conditions of measurement. This creates many problems in sensing and imaging. Introduction of reference that can be excited together with the sensing fluorophore but emitting at a different wavelength (see Sect. 3.2) yields the ratiometric signal independent of these factors (Demchenko 2005, 2010). Combining two types of emitters in one nano-sized unit allows using the sensor as the instrument of quantitative analysis. As we observed in Sect. 3.2, there are two design strategies to generate the λratiometric signal operating with emission intensities at different wavelengths. One of them uses a combination of responsive and irresponsive fluorophores, so that irresponsive one provides the reference signal. The other strategy is based on generation of two target-responsive signals. Being coupled or uncoupled, their intensity should change responding to target binding in different manner or, better, in opposite directions. For example, the presence of target can induce the increase of intensity at one wavelength, accompanied with the decrease at the other, thereby producing a larger change in the ratio of intensities. Both of these strategies can be realized in nanocomposites (Bigdeli et al. 2019; Huang et al. 2018). One of the problems in realizing the first strategy is the requirement to exclude interactions between two or more types of fluorophores within one particle and make their emissions independent, so that the variation of reporter signal in its dynamic range should not influence the reference signal. The ideal case could be when different types of emitters are excited at the same wavelength and demonstrate very different emission spectra. This idea was illustrated by coupling the quantum dots, providing the reference emission signal, with responsive organic dyes. Since QDs demonstrate very small Stokes shifts, the problem is in selection of responsive dyes. For providing strong band separation, this shift for them should be very significant. In our studies (Peng et al. 2011), the ultra-small QD possessing a small Stokes shift was covalently bound to 3-hydroxychromone dye, in which the Stokes shift is extremely large. So, there is no EET between them and they emit independently. This allows the dye to exhibit its responsive fluorescence properties that, if necessary, can be translated

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Fig. 10.9 Nanocomposites of ultra-small CdSe quantum dots and HF-N-LA 3-hydroxychromone dye (Peng et al. 2011). a Absorption (left scale) and emission (right scale) spectra of CdSe quantum dots (solid line) and HF-N-LA molecules (dashed line). b Emission spectra of nanocomposites by combining different concentrations of HF-N-LA solutions with fixed amounts of QDs

into λ-ratiometric response. Moreover, the component emitters can be combined in different proportions, and the range of relative intensities can be variable on a very broad scale and even allow obtaining the composed ‘white light’ emission (Fig. 10.9). Lanthanide complexes (see Sect. 6.3) are ideal as the reference dyes in fluorescence sensors based on two-wavelength λ-ratiometric recording (Bunzli and Piguet 2005). Their broad excitation spectra allow exciting them at the same wavelength as the reporting dyes and providing stable signal that is well separated from that of the reporter. Low dependence of emission spectra on the temperature and on the presence of quenchers makes lanthanide chelates very attractive for this purpose. Regarding the strategy of the ratiometric analysis with the two bands changing in opposite directions, though it is quite popular with the use of two-band ratiometric organic dyes (see Sect. 3.6), its realization is not easy on nanoparticle level (Bigdeli et al. 2019). The advantage of nanoscale emitters is a great intensity enhancement, and the problem of its realization with all attractive features of λ-ratiometry remains a challenge. The examples of successful applications of polymer dots (Wang et al. 2019b), AIE nanoparticles (Wang et al. 2019a) and metal–organic frameworks (Chen et al. 2020) demonstrate promising prospects.

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10.5.2 Multiplex Assays and Suspension Arrays Multiplex analysis allows simultaneous detection of several analytical targets within a single, sample. This approach is definitely different from that involving simultaneous measurement of different samples for single analyte. It is needed in all cases when a rapid and accurate diagnostic of the studied system must be provided in many variables. It offers essential advantages regarding time, reagent cost, sample requirements and the amount of data that can be generated. It allows automation. Particularly, this approach is realized in multiplex immunoassays (Marquette et al. 2012). Commonly, multiplex immunoassays are taking place at the surface of a solid support and use fluorescence as a conventional detection technique. Growth of these analytical tools is fantastic. The development of DNA microarrays started from 256 probes per chip and now exceeded several million. Success in protein arrays is much more modest but no less important. In homogeneous-type assays (Sect. 1.1) there is a necessity of distinguishing several types of sensor-target interactions (and ideally, their great number). This can be realized if we are able to label every type of sensor molecule or nanoparticle with a certain ‘barcode’ that could be recognizable in chromatography or flow cytometry (Lim and Zhang 2007). The dyes (and nanoparticles) emitting at different wavelengths may serve for constructing such multicolor codes (Jun et al. 2012). This allows mixing the differently encoded sensor particles and detecting the binding of specific targets to each of their diverse types. The principle of operation of such sensors is illustrated in Fig. 10.10. The suspension arrays operating in solutions without any support may contain many thousand types of sensor particles. They must be different in target recognition units but display the same type of fluorescence response. To be recognized, the sensor containing the recognition and reporting units should contain also its own unique marker. In theory, six colors at six different levels of intensity should provide nearly 40 000 unique codes (Wilson et al. 2006). In practice it is hard to achieve such values in view of spectral overlaps between the dyes and variability of fluorescence intensities introduced both on the steps of sensor production and reading the output signal. Also, the cross-talk between different types of fluorophores that appears with the presence of EET should be considered and minimized. Step-by-step these essential problems tend to be resolved. Thus, it was reported on nanocomposites detecting simultaneously Cu2+ ions and pH (Han et al. 2015), pH and hydrogen peroxide (Liu et al. 2017) or hypochlorite and temperature (Chen et al. 2017).

10.5.3 Materials for Multicolor Coding There are tremendous possibilities for achieving the required diversity of emission patterns using nanoscale composite particles with incorporated fluorescence materials. The technically convenient starting conditions could be: (a) excitation of all

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Fluorescence intensity

Target 1 λ

Target 2 λ

Target 3 λ Barcoding signal

Response signal

Fig. 10.10 The barcodes for fluorescence sensors. As an example, three types of emitters (violet, blue and yellow) assembled in different proportions into nanocomposite make every nanocomposite recognizable by characteristic pattern of relative intensities of three bands. Fluorescence sensor included into nanocomposite can respond to the binding of its specific target (shown here as the dye changing the intensity of red light). The sensor particle interacts with particular target and its type is recognized by the barcode

members of such array by light of single wavelength, (b) narrow emission bands in dye composition forming the color code, and (c) chemical and photochemical stability that will not allow destroying the coding signal. Organic dyes satisfy these requirements only partially, but their performance can be improved substantially on incorporation into nanoparticles (Jun et al. 2012). The composing dyes can emit individually forming the composite spectrum, but since a strong variation in positions of fluorescence bands is required on excitation at single wavelength, this can be realized by wavelength-converting cascade EET (see Sect. 10.2). The dye molecules can be incorporated in different proportions into silica particles and coupled by EET cascade reaction (Ma et al. 2007). Still, their broad emission profiles are fundamental drawbacks reducing multiplexing capabilities. In this respect, quantum dots have better prospects than organic dyes because they possess much narrower fluorescence spectra covering the whole visible range, up to near-IR. They can be excited at a single wavelength and, in addition, they exhibit higher photostability (Eastman et al. 2006). Such attractive features of QDs were recognized (Medintz et al. 2005). Employing them as EET donors and organic dyes

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as acceptors allows realizing many new possibilities (Clapp et al. 2005b). Coating QDs with a silica layer can provide improved stability without affecting their optical properties. In addition to capping the single QDs with a silica monolayer, multiple QDs can be also encapsulated into silica nanobeads (Gao and Nie 2003). Their encapsulation into monodisperse polystyrene latex nanobeads has also been suggested (Han et al. 2001). The swelling method was used for tagging 1.2 μm beads with different combinations of QDs of various colors to create QD barcodes. It was demonstrated that the use of six colors and ten intensity levels can theoretically encode up to one million types of particles. Barcodes with application of upconverting nanoparticles deserve special attention. They can be designed on the basis of a single host material NaYF4 doped with variable amounts of Yb3+ , Tm3+ , and Er3+ ions. This allows obtaining a broad range of color output by single wavelength excitation using a cheap diode laser at 980 nm just by variation of dopant concentrations (Wang et al. 2010). Even broader assortment of colors can be achieved when these particles are decorated with EET-acceptor organic dyes (Gorris et al. 2011). In this way, a large library of emission spectra in the visible and near-IR spectral region can be created, which is particularly useful in multiplexed labeling. The range of applications implying the potential of such nanobeads to incorporate QDs of different size and, therefore, different fluorescence spectrum, is tremendous. Each particle can incorporate several different QDs that may compose very specific ‘color barcode’. Such coding is needed in microsphere-based array technology (Sect. 15.5). The DNA hybridization studies demonstrate that the coding and target signals can be simultaneously read at the single-bead level, which allows to apply this technique in high throughput screening, and in medical diagnostics. Thus, assembly of fluorescence emitters into nanocomposites allows achieving very important functions that are not observed in separate emitters alone. Since all types of fluorescent molecules and nanoparticles can be combined in nanocomposites, the new routes are opened to perfection of fluorescence reporters by their designing on nanoscale.

10.6 Sensing and Thinking: Multiple Functions of Small Nanoparticles, How to Achieve that? In this Chapter we were focused on different possibilities for endowing the molecules and nanoparticles with new functions by assembling them into functional nanocomposites. We achieved, together with dramatic increase of brightness, the possibilities of manipulation with positions of luminescence spectra and lifetimes in very broad ranges. We learned how to make the effects of superenhancement and superquenching useful for the increase of sensitivity in sensing and imaging. We observed that many of these achievements were based on the phenomenon of excitation energy transfer that is responsible for the migration of excitation energy within the dye ensembles and,

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if necessary, for the shifts to its lower values. Providing EET is easy, since according to Förster resonance energy transfer (FRET) mechanism, it proceeds at distances comparable with nanoparticle dimensions and does not require strict orientation of EET partners. However, in the domain of sensing with referencing, in optical barcoding and in multiplex sensing the requirements are quite the opposite. Multiple emissions should not converge to a single emission band. The luminophores constituting nanocomposite should emit independently, without the excited state energy exchange or flow between them. The solution here can be more difficult, and in general case was not found. What can be proposed is only the minimal overlap integrals between donors and acceptors, crossed orientation of their absorption and emission dipoles and their strongest separation within nanocomposite. In Sect. 10.3 above we discussed the optimal choice of donors and acceptors. Here we have to choose the least efficient of such pairs. Thus, the combination of different types of fluorophores offers a promising route for the fabrication of multi-chromophore systems that generate new properties and allow modulating the fluorescence response in very broad ranges (Table 10.1). Table 10.1 Summary of techniques used for modulation of fluorescence response in nanocomposites Effect achieved

Technique

Mechanism

Enhancement and modulation of intensity

Plasmonic enhancement

Interaction of different Brightness, quantum fluorescent emitters with yield noble metal NPs producing plasmonic effect

Modulated parameter

Enhancement and modulation of intensity

Doping into polymers Assembly of many emitters in a nanoscale volume

Molar absorbance

Enhancement and modulation of intensity

Light harvesting

Excitation of small number of emitters by energy transfer from large number of EET donors

Molar absorbance and EET efficiency

Shift of emission wavelength to a desired range

Wavelength conversion

Using cascade of Excitation and emission sequential EET reactions wavelengths

Extension of lifetime

Modulation of EET acceptor decay

EET from long lifetime donor to short lifetime acceptor

Repolarization of fluorescence emission

Red Edge effect

Suppression of EET on Fluorescence anisotropy excitation at the red edge

Switching of fluorescence color

Photochromism

Reversible light-induced Two-band ratiometry switching between two forms of EET acceptor

Fluorescence lifetime

10.6 Sensing and Thinking: Multiple Functions …

431

In order to check your acquired knowledge, respond to following questions: 1.

2. 3. 4. 5.

6. 7. 8. 9.

10.

11. 12. 13. 14.

15.

List all discussed above possibilities for fluorescence enhancement offered by constructing nanocomposites using molecular and nanoscale fluorophores and explain their background mechanisms. What are the excitons? How their effects are displayed in molecular aggregates, conjugated polymers and semiconductor nanocrystals’. What are J-aggregates and how their presence can be recognized in fluorescence spectra? Explain the new features that are observed when the fluorophores are assembled and exhibit homo-EET. Compare the parameters of EET acceptor excited via energy transfer and on direct excitation. What of these parameters are different and why? Estimate the scale of their variation on direct excitation and via EET. Explain the mechanism of light harvesting. What parameters of fluorescence emission change in this process? Wavelength conversion, in which direction it proceeds on the wavelength scale and why? What fluorescence parameters change in this process? Quantum dots of different sizes are assembled in nanocomposite. Which one will primarily absorb light and which will be the emitter? Imagine that you change the excitation wavelength to excite instead of D, either I1 or I2 (see Figs. 10.4 and 10.5). What will be the changes of spectra and lifetimes? Explain the role of each participant in a two-step cascade energy transfer. Analyze, what will be the result of changing the fluorescence parameters of each of them. What is the benefit of extending the emission lifetime? How to achieve that in nanocomposites? Why anisotropy is low in the systems of fluorophores coupled by homo-EET? What are the spectroscopic means to enhance it? Explain the mechanism of modulation by exposure to light the emission intensity switching between two colors. Explain your choice of EET donor in design of nanosensor with response of organic dye as the acceptor. Could it be quantum dot, lanthanide chelate, conjugated polymer or another dye? Explain, what are the advantages and disadvantages in all these cases. In what technologies and why the optical barcoding imprinted in nanoparticles becomes necessary? Do we need to activate or to suppress EET in this application?

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Stein IH, Steinhauer C, Tinnefeld P (2011) Single-molecule four-color FRET visualizes energytransfer paths on DNA origami. J Am Chem Soc 133:4193–4195 Stewart ME, Anderton CR, Thompson LB, Maria J, Gray SK et al (2008) Nanostructured plasmonic sensors. Chem Rev 108:494–521 Sun X, Lei Y (2017) Fluorescent carbon dots and their sensing applications. TrAC, Trends Anal Chem 89:163–180 Suzuki M, Husimi Y, Komatsu H, Suzuki K, Douglas KT (2008) Quantum dot FRET biosensors that respond to pH, to proteolytic or nucleolytic cleavage, to DNA synthesis, or to a multiplexing combination. J Am Chem Soc 130:5720–5725 Swierczewska M, Lee S, Chen X (2011) The design and application of fluorophore–gold nanoparticle activatable probes. Phys Chem Chem Phys 13:9929–9941 Tan CY, Alas E, Muller JG, Pinto MR, Kleiman VD, Schanze KS (2004) Amplified quenching of a conjugated polyelectrolyte by cyanine dyes. J Am Chem Soc 126:13685–13694 Tang F, Ma N, Wang X, He F, Li L (2011) Hybrid conjugated polymer-Ag@ PNIPAM fluorescent nanoparticles with metal-enhanced fluorescence. J Mater Chem 21:16943–16948 Thomas SW 3rd, Joly GD, Swager TM (2007) Chemical sensors based on amplifying fluorescent conjugated polymers. Chem Rev 107:1339–1386 Tian Z, Wu W, Li AD (2009) Photoswitchable fluorescent nanoparticles: preparation, properties and applications. Chem Phys Chem 10:2577–2591 Varnavski OP, Ostrowski JC, Sukhomlinova L, Twieg RJ, Bazan GC, Goodson T (2002) Coherent effects in energy transport in model dendritic structures investigated by ultrafast fluorescence anisotropy spectroscopy. J Am Chem Soc 124:1736–1743 Wang F, Tan WB, Zhang Y, Fan X, Wang M (2006a) Luminescent nanomaterials for biological labelling. Nanotechnology 17:R1–R13 Wang L, Wang KM, Santra S, Zhao XJ, Hilliard LR et al (2006b) Watching silica nanoparticles glow in the biological world. Anal Chem 78:646–654 Wang F, Banerjee D, Liu Y, Chen X, Liu X (2010) Upconversion nanoparticles in biological labeling, imaging, and therapy. Analyst 135:1839–1854 Wang Y, Bao L, Liu Z, Pang DW (2011) Aptamer biosensor based on fluorescence resonance energy transfer from upconverting phosphors to carbon nanoparticles for thrombin detection in human plasma. Anal Chem 83:8130–8137 Wang H, He Y, Li Y, Zhang C, Zhang P et al (2019a) Selective ratiometric fluorescence detection of hypochlorite by using aggregation-induced emission dots. Anal Bioanal Chem 411:1979–1988 Wang W, Zhang Y, Liu Y, He Y (2019b) Highly selective and sensitive ratiometric fluorescent polymer dots for detecting hypochlorite in 100% aqueous media. Spectrochim Acta Part A Mol Biomol Spectrosc 207:73–78 Watrob HM, Pan C-P, Barkley MD (2003) Two-step FRET as a structural tool. J Am Chem Soc 125:7336–7343 Wiederrecht GP, Wurtz GA, Hranisavljevic J (2004) Coherent coupling of molecular excitons to electronic polarizations of noble metal nanoparticles. Nano Lett 4:2121–2125 Wilson R, Cossins AR, Spiller DG (2006) Encoded microcarriers for high-throughput multiplexed detection. Angew Chem Int Ed Engl 45:6104–6117 Wosnick JH, Liao JH, Swager TM (2005) Layer-by-layer poly(phenylene ethynylene) films on silica microspheres for enhanced sensory amplification. Macromolecules 38:9287–9290 Würthner F, Kaiser TE, Saha-Möller CR (2011) J-Aggregates: from serendipitous discovery to supramolecular engineering of functional dye materials. Angew Chem Int Ed 50:3376–3410 Wu CF, Szymanski C, McNeill J (2006) Preparation and encapsulation of highly fluorescent conjugated polymer nanoparticles. Langmuir 22:2956–2960 Wu C, Zheng Y, Szymanski C, McNeill J (2008) Energy Transfer in a nanoscale multichromophoric system: fluorescent dye-doped conjugated polymer nanoparticles. J Phys Chem C Nanomater Interfaces 112:1772–1781 Wu C, Hansen SJ, Hou Q, Yu J, Zeigler M et al (2011) Design of highly emissive polymer dot bioconjugates for in vivo tumor targeting. Angew Chem Int Ed 50:3430–3434

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Chapter 11

Passive Support Materials for Fluorescence Sensors

In this Chapter we address the problem of controlling the spatial arrangement and orientation of the desired molecules and particles to generate highly-ordered nanostructures needed for sensing. They may be “passive” regarding sensing, but they carry the important function—the forming an integrity of structure. Both molecules and nanoparticles can be used for forming the self-assembled monolayers on plane surface using dip-coating and spin-coating techniques. More precisely controlled location and orientation of amphiphilic molecules can be achieved in LangmuirBlodgett films, and layer-by-layer technique allows obtaining multiple layers with polyelectrolyte polymers. Inorganic and organic nanoscale structures can serve as scaffolds. Of great importance are the scaffolds made of proteins, peptides and DNA. Due to important role of lipid bilayers, their properties are discussed at some length. Affinity coupling and self-assembly are the two principles of formation of functional constructions. Covalent conjugation is also used, particularly for introduction of fluorescence reporters. The conjugations involving fluorescence-generating click reaction are discussed. The Chapter terminates with the section “Sensing and thinking” with the questions to readers for checking their acquired knowledge. Synthetic strategies based on covalent linking, affinity coupling and self-assembly on flat and nanoparticle surface offer many advantages in designing efficient sensors. They include the minimization of synthetic work, the ease of modification and optimization of the sensor, the possibility to tune its properties by a simple adjustment of the number and quantity of components. The applications of these new concepts tend not only to combine the target binding and the response but also to improve this response by creating optimal supramolecular ensemble allowing activating the best communication and signal transduction between its structural elements. The recognition units can be formed by assembly of functional molecules with the appearance of new functions. Incorporation onto planar surfaces and nano-sized structures provides the support for functional materials that allows their stabilization and integration into selfassembled nanoscale recognition units. The use of nanoparticles can make these recognition units nano-sized and multivalent. In addition, the self-assembled systems © Springer Nature Switzerland AG 2020 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-030-60155-3_11

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allow great possibilities for introduction of fluorescence response functionality and thus for providing additional important properties. In the assembly, the receptor and the reporter units may not interact directly and the signal on target binding can be transmitted due to their spatial closeness ensured by the template and even indirectly via the change of integral property of nanoscale system. The problem of controlling the spatial arrangement and orientation of the desired molecules and nanoparticles to generate highly-ordered nanostructures needed for sensing is discussed here. Both molecules and nanoparticles of inorganic or organic origin can be used for forming the self-assembled monolayers on plane surface using dip-coating and spin-coating techniques. Providing precisely controlled location and orientation of amphiphilic molecules can be achieved by simple preparation of Langmuir-Blodgett films. Using the layer-by-layer technique, one can obtain the surface covered with multiple layers composed of polyelectrolyte polymers with alternating charges. Inorganic and organic nanoscale structures are considered as scaffolds for attaching functional elements to their surface. Well-organized and rigid can be the scaffolds made of proteins, peptides and DNA, which allows their precisely controlled assembly. Lipid bilayer structures occupy special role in biosensing, and their properties are discussed. On a general scale, affinity coupling and self-assembly are the two principles, following which the functional constructions can be formed. Chemical synthetic means allow covalent conjugation, particularly for introduction of fluorescence reporters. We will see that serving as support media and templates, DNA, proteins and lipid bilayers allow precise positioning of different functional units, and based on biospecific interaction, precise self-assembly can be realized. In assembled structures, carbon nanoparticles (graphenes, fullerenes, nanotubes and dots) can play additional role as fluorescence quenchers. Synthetic polymers being polyelectrolytes or possessing alternating hydrophylic-hydrophobic domains can form a variety of self-assembled structures ready for targeted functionalization by covalent linkage. Clearly, this relatively new field of development of fluorescence sensors is far from being mature. Whereas the advance in recognition units and fluorescence reporters, though promising many improvements, follows already well-established trends, the optimization of their performance based on the principles of integration and selfassembly promises new exciting discoveries that will result in new useful products.

11.1 Self-assembly on the Surface Self-organization of essential sensor components on a proper template is an important approach to the realization of functioning of self-assembled structures as sensors. In this case, the receptor and the reporter do not always need to interact directly (Arduini et al. 2007). The communication between them can be provided by spatial proximity of these two units on the template only, so that a signal transduction mechanism can be intermolecular but efficient. Thus, template can play important role both in formation of functional supramolecular structure and in maintenance of its integrity.

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11.1.1 Template-Assisted Assembly Different sensing technologies, such as heterogeneous immunoassays and heterogeneous DNA conjugation techniques, require functioning of sensors on solid-liquid interface. Plain sensor modules are also needed for gas sensing. This is the reason of high interest towards the surface-related technologies. The main advantage of template-assisted assembly is the possibility of choosing the template and its immobilization strategy, selecting from broad range of possibilities. Depending on the template, little or no synthetic modifications of the receptor are needed, and this allows an easy selection of their optimal configuration. The spatial proximity of a large number of sensor units in the assembly leads to collective effects and the properties that may contribute to the improvement of sensors performance. Different types of templates have been used to guide the self-organization of sensors, spanning from micellar aggregates to monolayers, to surfaces of inert materials (e.g. glass) and, finally, to nanoparticles. Each of these templates has its own peculiar features that are reflected in a characteristic performance of the resulting sensor. Two simplest methods for obtaining thin films on the solid surface are dip-coating and spin-coating (Fig. 11.1). Both methods use adhesion of molecular or nanoparticulate material to the surface. Dip-coating is convenient for formation of multilayers (Liu et al. 2018). In contrast to dip-coating, the spin-coating needs only flat surfaces, but the obtained film thickness of deposited sample is more homogeneous. Extra sample spins off the edges of the plate. Moreover, the speed of rotation may allow regulating the film thickness (Tyona 2013).

a

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Fig. 11.1 Comparison of dip-coating and spin-coating techniques. a Dip-coating involves immersing of a substrate into a tank containing coating material, removing the piece from the tank, and allowing it to drain. The coated piece can then be dried and prepared for a new step of layering. b Spin-coating uses the centrifugal force to deposit material on flat substrate. The slow speed rotation allows homogeneous distribution of this material on the surface

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Dip-coating and spin-coating are the proper methods for template-assistant assembly, since they allow formation of single-molecular layers. Additional benefit of template-assisted assembly is the possibility of micropatterning—the formation on the surface of molecular clusters with desirable pattern. The active areas for accommodating these clusters with the size of 100 nm or less can be pre-fabricated on the surface. This approach with the aid of surface chemistry merges fabrication and self-assembly and thus combines bottom-up and top-down approaches (Henzie et al. 2006). Compared to nanolithography (the technique of nanoscale etching, writing or printing on a material surface), the self-assembly on surface may allow obtaining highly ordered and even 2-D crystalline structures. The two techniques can be combined, offering the self-assembly in nanoscale patterned wells (Cui et al. 2004). Surfaces play very special roles in fluorescence sensing. Despite the fact that, unlike potentiometric or cantilever sensors, their presence is not so critical for reporting event, they can allow realizing many possibilities for modification and optimization of the sensor by simple adjustment of the proportion of surfaceattached components. With the combination of covalent binding and self-organizing methodologies, the more complicated multifunctional sensors can be constructed.

11.1.2 Formation of Supporting and Transducing Surfaces The role of surface as a passive support can be very important in many sensor constructions. When the sensor molecules, nanoparticles or supramolecular structures are immobilized on the surface, they cannot diffuse freely in solution and mix with other species. Many attractive analytical advantages can be derived from sensor attachment to the surface that can be realized in heterogeneous formats (see Sect. 1.1). We indicate several of such new possibilities. (a) The sensors composed in this way allow their use for many times with simple sample addition-washing protocols or providing continuous monitoring in the flow. (b) In such many-use systems, the reagent additions can be easily applied, for instance, the addition of fluorescent competitor. (c) Due to fixed spatial separation between sensor molecules and applied patterns, the sensing of a large number of analytes (multiplex sensing) can be made on the same plate simultaneously. For such multiplex sensing the microarrays (sometimes called biochips) can be developed for simultaneous sensing of hundreds or even thousands of targets (see Sect. 15.4). The choice of methods of surface immobilization depends on the chemical structures of sensor and the surface; these methods are well described in original literature and reviews (Sobek et al. 2006). They do not differ much from those that are used in surface modifications of semiconducting (see Sect. 7.2), magnetic (see Sect. 12.3) or other sensor technologies, where the surface immobilization is also needed, such as

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surface plasmon resonance. Meantime, each sensor and each application may demand its own solution; the demands on applied solid support are thereby as manifold as the applications themselves. Immobilization should satisfy many requirements, particularly, it should involve proper orientation of sensors and availability of receptor sites for target binding. The hydrogel layers allow high density of sensor immobilization. Hydrogels are the three-dimensional (3-D) polymer networks that swell, but do not dissolve in water. They can be made optically transparent and fluorescent-free, being formed of different hydrophilic polymers, such as polyacrylamide and polyethylene glycol (Svedhem et al. 2001). Their development into 3-D microarrays for DNA hybridization and protein detection (Rubina et al. 2003, 2004) allowed increasing substantially the density of sensor locations. One of proposed procedures (Burnham et al. 2006) looks in the following way. Disulfide-crosslinked derivative of hydrogel is deposited on the quartz or silicon surface. Then the application of reducing agent provides generation of reactive SH groups throughout the hydrogel leading to ‘activated hydrogel’. These SH groups can be readily modified with the attachment of proteins or other functional groups resulting in functional hydrogel. Different materials that can serve as supporting media, transducers and even imprinted recognition elements can be made in a sol-gel process. Both inorganic and organic/inorganic composite materials can be obtained by this procedure (Kamaruddin et al. 2011). The colloidal suspensions or ‘sols’ are formed via hydrolysis of alkoxy metal groups in the precursors with subsequent polycondensation. The obtained transparent glass-like structures can attain different shapes and may be obtained with high porosity. The pore size distribution can be controlled by chemical composition and by the reaction conditions. Importantly, fluorescent dyes can be incorporated into these structures in several ways: by adsorption within the pores, by incorporation into reaction mixture (due to mild conditions of synthesis there is no decomposition of dyes) and also by covalent binding to finally obtained material. Covalent attachment should be preferred for obtaining the sensors of high stability.

11.1.3 Self-assembled Monolayers Self -assembled monolayers (SAMs) are monomolecular ordered structures that can form spontaneously on a chemically active surface (Flink et al. 2000, Ruckenstein and Li 2005). They can be prepared simply by adding a solution of monolayerforming molecules onto the surface and washing off the excess. The monolayer is formed due to strong interaction energy of one of the groups (usually, terminal) of a monolayer former with an active surface (Sharpe et al. 2007). It can be additionally stabilized by side interactions in the monolayer. Gold and silver surfaces form very stable and ordered monolayers with thiolcontaining compounds because of strong binding energy between them (Fig. 11.2). In the case of alkane thiols deposited on gold, this energy can be as high as 100– 150 kJ/mol. SAM of long chain alkane thiol produces a highly packed and ordered

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Fig. 11.2 Formation of thiol surface-adsorbed monolayer (SAM) on gold surface. SAMs are frequently used for spontaneous modifications of gold and silver surfaces with the compounds containing sulfur. R denotes the active group needed for further modifications, often they are carboxylic groups

surface, which can provide a membrane-like microenvironment, useful for immobilizing biological molecules. In addition to the low molecular mass of thiol derivatives, the self-assembled monolayers on gold can be formed by SH-substituted peptides, proteins, carbohydrates (Revell et al. 1998), DNA and peptide nucleic acids (Briones and Martin-Gago 2006). It was reported that polythiophene monolayers on gold retain their fluorescent properties (Shinkai 2002), as well as some modified fluorescent dyes (Kriegisch and Lambert 2005). Meantime, a strong quenching of fluorescence of closely located dyes by PET and FRET mechanisms is a well-known property of gold surfaces. This property can be useful (Perez-Luna et al. 2002). One of such possibilities is the following. By immobilizing the analyte of interest (or its structural analogue) to a metal surface and exposing it to a labelled receptor (e.g. antibody), the fluorescence of the labelled receptor, being in close proximity to the metal, becomes quenched. If a free analyte is present, the labelled receptor dissociates from the metal surface with an increase of fluorescence intensity. In many cases, the quenching by metal surface has to be avoided, and another type of support has to be used. Alkyl silane molecules (e.g. octadecyltrichlorosilanes) form the self-assembled structures on silicon oxide surfaces, and also a number of different surfaces can be modified with alkyl carboxylates. More recent developments in the chemistry of SAMs on glass suggest new technologies for fluorescent sensor design. Trialkoxysilanes or halogenosilanes reacting with hydroxylated surfaces with the formation of SAMs offer such possibilities (Sullivan and Huck 2003). Alkyl siloxanes (Parikh et al. 1997) are formed on the surface of silicon or glass. The reactive siloxane groups can form on a surface a cross-linked network by condensing with hydroxyl groups on the surface, water and neighboring siloxanes. This binding is more stable than of alkane thiolates on gold, but there is a stronger limitation on the chemistry of second functional group that will extend from the formed surface and will be needed for further modifications. Designed polypeptides with variable repeat length containing N-terminal cysteine dimer were suggested as scaffolds for surface immobilization of quantum dots

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(Medintz et al. 2006). Many other methods have been developed for modification of the surfaces of these particles that attain increasing importance in sensing. Dense and highly regular two-dimensional distribution of exposed reactive functional groups of SAMs offers many possibilities for further attachment to solid support of the whole sensors or their assembly on the surface from receptor and reporter components. SAMs offer a simple and straightforward method to generate modified surfaces with transducing function. This property is of extreme value for making functional microfluidic devices (Sect. 15.6), in which due to a narrow size of produced channels a high surface-to-volume ratio can be generated. Even in these microscopic structures, the deposition of SAMs is simple. It requires only the flow of a solution or of a gas stream of adsorbate molecules through the channels. Thus, one of the actively used functions of SAMs is hosting of biosensor molecules and cells. This includes confining and aligning of biological macromolecules and the immobilization of cells in a way preventing their adhesion. The other important problem is the immobilization of fluorescence reporters and providing signal transduction function to these reporters. Thus, the general role of SAMs is to form an interface between the surface and the molecular sensors or nanosensors. Such possibility can be realized both on flat surfaces, in microfluidic cavities and on the particles of different size. Supramolecular interactions, such as of π-π type, have been recently exploited to make sensitive glass surfaces. It was reported on the functionalization of glass surfaces (quartz, glass slides and silica particles) with 2,7-diazapyrene derivatives (Fig. 11.3). Specifically, they were used for the detection of catecholamine neurotransmitters, such as dopamine (Cejas and Raymo 2005).

Support Fig. 11.3 Schematic structure of 2,7-diazapyrenium monolayer formed on silicon substrate

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Thus, the most attractive feature of self-assembled monolayers is their ability to change easily the surface properties of the layer, including its binding properties, by tuning the chemistry of terminal group that appears on its surface without significant influence on the formation of these layers.

11.1.4 Langmuir-Blodgett Films Formation of Langmuir-Blodgett (LB) films (Roberts 2013) is an alternative to selfassembly method of producing regular monolayers on solid support surface (Davis and Higson 2005, Matharu et al. 2012). This method is less demanding to the strength of interaction of monolayer-forming molecules with the support but requires them to be amphiphilic, i.e. to be composed of separated polar and apolar parts. The monolayer is primarily formed at the air-liquid interface, and then it is transferred to the surface of solid plate by immersing it to this liquid (Fig. 11.4a). By repeating the operation of immersion and emersion several times, several regular monolayers can be deposited. When primarily formed at the air-water interface and then transferred to hydrophobic support, these films expose polar groups. This is very useful for further modifications, such as attaching of various molecular and nano-composite sensors operating in aqueous media. Langmuir-Blodgett films with all their modifications made in solvent can be used in the air, which makes them applicable in the sensing of vapors. There are amphiphilic fluorescent dyes, such as Nile Red, that can form LangmuirBlodgett films by themselves (Dutta et al. 1996). Meantime, there are many possibilities of forming LB monolayers in mixed composition with the inclusion of fluorescent dyes (Hussain et al. 2006). In particular, amphiphilic analogs of polarity-sensitive dyes can be mixed with fatty acids to form stable monolayers (Alekseeva et al. 2005). Formation of stable films by luminescent complexes of europium have also

Support

a

b H2O

H2O Support Fig. 11.4 Different methods of functional modification of the surface. a Langmuir-Blodgett films. The films are first formed on a liquid surface by ordered amphiphilic molecules forming ordered layer and then transferred to solid-liquid interface. b Films obtained by layer-by-layer procedure, in which the layering on the solid surface is provided based on electrostatic interaction of deposited polyelectrolyte polymer layer with the previously formed layer

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been described (Xiang et al. 2006). Conjugated polymers can be used as monolayer formers (Mattu et al. 2006). All that suggests many attractive possibilities for fluorescence sensing. But, surprisingly, this methodology is used rarely. It should be indicated that Langmuir-Blodgett films when formed do not allow easy incorporation of compounds dissolved in external solution (to which the plate is exposed). Such incorporation can be better provided on the step of their formation; the incorporated molecules should be amphiphilic and possess some similarity with the major component of the film. There is also a possibility to make a second layer on top of the film. If this layer is formed by phospholipids, many possibilities appear for incorporation of a broad variety of different molecules, including biomembrane proteins. There are the results (Davis and Higson 2005) showing that the single-stranded DNA molecules can be incorporated into the films formed by octadecylamine and that they can be hybridized with complementary strands. In general, this old and simple method deserves much more attention. Its weak point is low stability of formed monolayers. It can be improved by several ways. One is the covalent cross-linking of molecules in monolayer. The other is overlaying by highly ordered 2-D structures, such as formed by S-layer protein (see Sect. 11.3).

11.1.5 Layer-by-Layer Approach Layer-by-layer technique is a version of self-assembly that is available to polyelectrolytes and uses the property of a molecular layer formed by charged polymer to absorb a molecular layer of a polymer with an opposite charge (Ariga et al. 2006, Chaki and Vijayamohanan 2002, El-khouri et al. 2012). Such layer-by-layer technique allows obtaining multilayers with precise thickness (Fig. 11.4b). Since many natural and synthetic polymers contain the charges, this technique is useful for the formation of surfaces with desired properties (Borges and Mano 2014, Paterno and Soler 2013). In addition, the distance from the surface monolayer to solid support can be precisely controlled. A smaller-size charged molecules including fluorescent dyes (Egawa et al. 2006) can participate in the formation of layers. The structure formations are not limited to flat surfaces; the layers can be assembled onto different charged particles and nanowires (Srivastava and Kotov 2008). It can be applied to any charged molecules, polymers and biological macromolecules (Ai et al. 2003). These films are highly stable, due to multiple interactions providing the charge neutralization. However, in comparison with Langmuir-Blodgett films, the formed layers do not show a very high degree of order. The order in polymer layers themselves cannot be very high, and this makes the structures rather amorphous. In aqueous solutions, the structure formation can be affected by pH and ionic strength, which is not always a favorable property. The layers formed by conjugated polymers (Ferreira and Rubner 1995) deserve special attention. Polyelectrolyte nature of many of them makes very good prospects for making ‘responsive’ layers with their inclusion (Li et al. 2013). It is known that their fluorescence superquenching effect (see Sect. 8.5) in thin films is much greater

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compared to that in isolated conjugated polymer molecules in solution (Chang et al. 2004). This is because the efficient exciton length is larger in two dimensions than in one-dimensional polymer chain. Precise deposition of quenchers that could be a part of reporting mechanism is possible in this system. The layer-by-layer approach can be extended to nanoparticles. The stepwise construction of a novel kind of self-assembled organic/inorganic multilayers was reported (Crespo-Biel et al. 2005). It is based on multivalent supramolecular interactions between the guest-functionalized dendrimers and host-modified gold nanoparticles. The interactions between optically responsive monolayers can be studied as a function of distance between them, which is regulated by a number of intermediate inert layers. It was found (Ianoul and Bergeron 2006) that in order to minimize the quenching of fluorescence signal, twenty polyelectrolyte monolayers are necessary to be deposited between the nanoparticles and the dye by a layer-by-layer deposition technique producing a 15–20 nm separation cushion. The combination of the layer-by-layer method of multilayer formation with other fabrication techniques such as spraying, spin-coating and photolithography offers many new possibilities for creation of sensor arrays (Ariga et al. 2007).

11.1.6 Prospects for Designing Smart Surfaces Design of fluorescence sensors using functionalized surfaces has many technical advantages. It also extends the possibilities of sensing, particularly to the gas phase. With surface immobilization, it is much easier to perform the sensors that allow multiple functioning, continuous targets monitoring in tested medium and different manipulations, such as washing-off the non-target species or reagent additions. The surface deposition allows spatial separation of sensors oriented to tested medium and, therefore to provide multiplex sensing. Surfaces can play an active role by participating in signal transduction, and such participation is versatile. The gold and silver surfaces are very good as SAM formers and also as fluorescence quenchers. This favorable combination can be used in the design of many sensors exploring the variation of distance between the surface and fluorescent dye (or nanoparticle). This and other, more inert, surfaces can be modified with the introduction of SAM made of fluorescent molecules or of a layer formed by fluorescent conjugated polymer. This offers many additional possibilities. A variety of amphiphilic, ‘fatty-acid-type’ molecules can form Langmuir-Blodgett monolayers and multilayers with incorporation of fluorescent dyes, and this possibility should not be overlooked. In addition, the last point. Surface can be extensively used as a platform for solid-phase synthesis of peptides and oligonucleotides with the receptor functions for analyte. Affinity coupling and self-assembly as important steps for making the sensors of high complexity, can be efficiently provided on the surface. The thin film technology is also one of the most powerful strategies for immobilization of sensor units on artificial devices such as optodes.

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11.2 Operating with Building Blocks for Nanoscale Sensors It could be ideal to develop one or several ‘standard’ reporting units that could be compatible to self-assemble with the library of receptors that are able to detect a huge variety of targets. Then it may be possible to use unified technique for their detection. The scientists indicate that despite the fact that there are interesting examples of large self-assembled structures, there is still a general lack of understanding how to introduce the standardized procedures for the step-by-step synthesis of nanosized composites. This direction, meantime, is the only way to fill the gap between miniaturization of macroscopic approaches (known as ‘top–down’ principle) and chemical synthesis that has a limit on increasing complexity of molecular structures.

11.2.1 Inorganic Colloidal Scaffolds In Chapter 7, the properties of different nanoscale inorganic materials were overviewed. Being made of conducting, insulating or semiconducting materials and possessing different optical properties, they have something in common: they cannot be used ‘naked’ and they have to be ‘decorated’. This means that their surface should attain similar properties as functionalized flat surfaces that were discussed above. The nanoparticles made of different materials commonly attain the spherical form. It is the form with the lowest or smallest surface-to-volume ratio that is thermodynamically stable in the conditions of their synthesis. Due to the same reasons, the versatile multifunctional core-shell nanoparticles are produced in relatively easy way (Chen et al. 2015, Kalambate et al. 2019). The steps in such decoration are illustrated in Fig. 11.5. In such already modified state they can be functionalized or assembled into nanocomposites. For performing such manipulations with them, they have to be present in dispersed forms and stabilized against aggregation. Details of such stabilization reactions can be found in the literature (Sperling and Parak 2010). Different reagents can be used for this purpose, but the general principle is the following. The stabilizing agent should be bi-functional. It has to bind strongly to the particle surface and, simultaneously, prevent the aggregation. Usually the stabilizing agent is present in the medium of nanoparticle synthesis, but the surface layer that it forms is not optimal for controlled assembly, the introduction of receptors, fluorescence reporters or other functionalities. Therefore, the ligand molecules on the surface can be exchanged by others. Usually the incoming ligand molecule binds stronger to the inorganic nanoparticle surface and allows new possibilities for inducing new properties. A common example of ligand exchange is the treatment of Au nanoparticles that in aqueous solutions are synthesized by citrate reduction. They have negatively charged citrate ions adsorbed on their surface and are thus stabilized by electrostatic repulsion. On subsequent steps they can be replaced by sulfonated phosphines or mercaptocarboxylic acids (Sperling and Parak 2010).

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Fig. 11.5 A nanoparticle stabilized with different hydrophilic ligands (Sperling and Parak 2010). The space-filling models and chemical formulas presented together were shown for those molecules (left to right): mercaptoacetic acid (MAA), mercaptopropionic acid (MPA), mercaptoundecanoic acid (MUA), mercaptosuccinic acid (MSA), dihydrolipid acid (DHLA), bis-sulphonated triphenylphosphine, mPEG5-SH, mPEG45-SH (2000 g mol−1 ) and a short peptide of the sequence CALNN

In common cases, the partners that are selected for self-assembly are dissolved in non-mixing solvents of different polarities. Then the phase transfer steps are needed. Three strategies can be selected for that: ligand exchange, ligand modification and additional layers of molecules that stabilize the particles in the desired phase (Sperling and Parak 2010). Being a part of these approaches, silanization is used for surface modification and phase transfer. Organic polymers are often applied for formation of coating layers. Further steps of functionalization may involve covalent attachment of proteins, nucleic acids, etc. Supramolecular structures with the function of fluorescence sensing are seen as self-organized complexes between or with designed partners, in which recognition and reporting can be coupled in a most efficient way. In an effort for developing better sensors, an important role is given to nanoscale support materials. They include different types of molecules and particles representing inorganic, organic and biological worlds (Oshovsky et al. 2007).

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11.2.2 Graphene and Graphene Oxide Enormous enthusiasm of researchers involved in molecular electronics that predict leading role of carbon to be the basis of technological revolution, is supported by scientists working in the field of sensing technologies. Nanoscale carbon materials can be very strong light absorbers, light emitters and light quenchers. Their light emitting properties were discussed in Chapter 9. They require quantum confinement effects and therefore they can be observed only on nanoparticles of a very small size. Meantime, the role of carbon materials as the building blocks is much broader, especially when it is coupled with quenching that can give an output signal in sensing. The family of π-conjugated inorganic carbons involves graphene and its oxidized derivatives, carbon nanotubes and fullerenes (see Fig. 9.1). Graphene is a 2-D atomthick lattice of carbon atoms, so that graphite is a stack of graphene layers. Carbon nanotubes are the rolled-up cylinders of graphene, and fullerenes (C60 is shown) are the balls consisting of wrapped graphene through the introduction of pentagons into the hexagonal lattice. The basic platform of nanoscale carbon is graphene. Its carbon atoms form a densely packed regular two-dimensional pattern of atomic width. The sp2 bonding is the same as in polycyclic aromatic hydrocarbons. It allows delocalization of electrons that are responsible for unique electron conduction. Graphene sheet is an ideal absorber of different types of molecules, especially of aromatic nature, of biological macromolecules and nanoparticles. Different aromatic compounds demonstrate increased affinity to graphene being stabilized by π-π stacking interactions. Figure 11.6 illustrates examples of these interactions for the compounds discussed in ref. (Mann and Dichtel 2013). Those molecules that being bound to graphene sheet lose their fluorescence (e.g. pyrene derivatives), can regain it after dissociation, If the target recognition is coupled with this event, such fluorescence enhancement can be used as the reporter response. Other possibilities arise if the reporting is based on association-dissociation of fluorescently bound receptor. This, DNA molecule can be bound if it is single-stranded and it dissociates if it acquires the double-helix form, and several reported methods of studying DNA hybridization are based on that (Deng et al. 2014). The sticky ends of graphene sheets are amenable to different covalent modifications. The familiar graphite can be viewed as the structure composed of many layers of such graphene sheets with strong interaction between the atoms within the sheets and week between the sheets. Hiding sticky ends, graphene sheet can be rolled up into cylinder forming carbon nanotube, and with the inclusion of pentagon elements it can form fullerenes, the structures in the form of hollow spheres. Graphene oxide (GO) and reduced graphene oxide (rGO) were previously considered only as graphene derivatives and predecessors in its synthesis, but now their special role is broadly recognized. In such graphene sheets, the polar groups are present. They break up the regular graphene structure into connected islands. In addition to increasing the solubility, there appear additional sites of covalent binding and self-assembly. Graphene and GO interact strongly with polymers (Salavagione

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Fig. 11.6 Molecules containing exposed aromatic groups interacting with graphene sheet. The interaction results in fluorescence quenching in electron-transfer process

et al. 2011), proteins (Zhang et al. 2013) and, notably, with single-stranded nucleic acids, but not with double helices. Based on this feature, the methods of the DNA hybridization were developed (Lu et al. 2010). These techniques are simple: a conjugate with sensing DNA sequence and fluorescence label is exposed to the test system. The emission is quenched due to attachment to GO support. If target DNA is present in this system, it forms double helix with the sensing sequence, which after dissociation from carbon surface starts to fluoresce. Highly efficient quencher ability for not only nucleic acids but also for various organic dyes and quantum dots is a characteristic feature of graphene materials due to very efficient electron transfer (see Sect. 4.1) from the excited dye to graphene sheet. In the case of organic dyes and polymers with heterocyclic side groups including single-stranded nucleic acids there is a strong interaction together with strong quenching through π-π stacking between the ring structures in correspondent partners that makes electron transfer quenching extremely efficient. In the case of DNA, the double strand conformation makes these interactions not possible. Many possibilities can be realized exploring the quenching-enhancing effects on association-dissociation with graphene or GO surface. They are used as scaffolds to which the assembled recognition and reporting functionalities can be attached. In close proximity to the surface the emission of fluorescent dye or nanoparticle is quenched and the target binding results in disruption of this interaction leading to fluorescence enhancement.

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11.2.3 Carbon Nanotubes Tubular structures are probably the most rigid mechanically and versatile functionally modules that can be used both as nanoscale support materials and as transducers. Those are carbon nanotubes that are very promising materials as molecular wires in electronics and also as the building blocks of various nanocomposites (see Fig. 9.1). Their inherent well-defined structural features, such as cylindrical dimensionality forming precisely established inner and outer volumes, are very useful for that. They possess their own optical properties useful in sensing. These tubes have a tunable near-infrared emission (at 1200–1400 nm) that responds to changes in the local dielectric function (Choi and Strano 2007), but remains stable to permanent photobleaching. This emission can be quenched by appropriate redox-active dyes, and with proper construction this effect can be used in sensing (Satishkumar et al. 2007). This emission in the near-IR, which is already used in sensing (Barone et al. 2005). Meantime their most requested role in sensing technologies is to serve as platform for nanodevice assembly and also as a very potent fluorescence quencher. The techniques of integration of nanotubes into various devices and designing the integrated sensing elements were developed (Mahar et al. 2007). This makes good prospect for the creation of smart sensors, in which the signal transduction is provided by fluorescence quenching and charge transfer. The first attempts along this line were successful (Barone et al. 2005).

11.2.4 Fullerenes Fullerenes (see Fig. 9.1) are known as very potent electron acceptors, and this property is used in different optoelectronic devices (Ito and D’Souza 2012, Wróbel and Graja 2011). Though they look as point light absorbers, their own absorbance in the visible is very low (for C60 it is ~760 M−1 cm−1 at the band maximum ~455 nm).This suggests for these particles a convenient role of electron acceptors in nanocomposites together with different types of fluorophores providing quenching-enhancement response. Though, there are practical difficulties due to low solubility of fullerenes and their tendency to aggregate. The examples presented above show that carbonic nanostructures can play a very important role in fluorescence sensing technologies. These nanomaterials can serve not only as scaffolds; they can be efficient quenchers. Typically, most of these nanomaterials, despite the position of their absorption peak, are capable of quenching the fluorescence from different light emitters with varying emission wavelengths. An important role in this process is played by electron transfer and also by non-resonant energy transfer operating by electron-exchange mechanism, both resulting in quenching. Because of their advantage as universal quenchers, the

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carbonic fluorescence-quenching nanomaterials eliminate the difficulty in selecting a wavelength-matching fluorophore and a quencher that may be a problem with conventional fluorescence-activated biosensors.

11.3 Organic and Biological Structures as Scaffolds It is commonly thought that if we deal with biological macromolecules in relation to sensors, the research will be always oriented to studies of biological target. In reality, their application can be much broader. This is due to the fact of their enormous possibility of variation their primary chemical structures and enormous possibility of these structures to adopt the three-dimensional conformation. Meantime, the adopted conformations can be very rigid.

11.3.1 Two-Dimensional Self-assembling of S-Layer Proteins Peptides and proteins provide a variety of opportunities for controlled and specific self-assembly. Mechanistically, peptide self-assembly can result from amphiphilic properties and from complementarity of partner structures. This complementarity can pre-exist in components, but can also appear in the course of interaction, the process that is known as ‘induced folding’ (Demchenko 2001). Designed peptides forming the dimers can be used for assembling the nanoparticles (Aili et al. 2007). The proteins that can assemble into two-dimensional crystals deserve special attention (Sleytr et al. 2007). Such proteins form the surface layers (S-layers) characteristic for prokaryotic organisms (archaea and bacteria) (Schuster 2018). They vary in molecular mass of subunits (40-200 kDa) and the thickness of the formed layer (from 5–20 up to 70 nm). As S-layers are the periodic structures, they exhibit identical physicochemical properties for each molecular unit down to subnanometer level and, when assembled, possess pores of identical size and morphology. Protein engineering allows obtaining their truncated forms and also covalently coupled hybrids with other proteins, of which the most interesting is the coupling with streptavidin. The latter allows obtaining complicated constructions based on two-dimensional S-layers as the building blocks (they will be illustrated in Fig. 11.14). Many applications of S-layers in biotechnology (Sharma and Singhal 2019) depend on the ability of isolated subunits to re-crystallize into monomolecular layers in suspension or on suitable surfaces and interfaces, such as liposomes (see Sect. 11.4). Because of these properties and using the possibility of modifications with various protein reagents, S-layer lattices can be exploited as scaffolding and patterning elements for generating more complex supramolecular assemblies and structures. One of such possibilities is illustrated in Fig. 11.7.

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Fig. 11.7 Schematic representation of two-dimensional structures formed of engineered S-layer proteins fused to functional or structure-forming protein (e.g. streptavidin). The latter can form supramolecular structures by affinity coupling (such as streptavidin with biotin) or by self-assembly. Different possibilities can be realized in these constructions, such as biotinilated nanoparticles or antibodies. This ensures the binding of receptor elements in defined spacing and orientation

11.3.2 Peptide Scaffolds The ability of peptides and proteins to form three-dimensional structures of various complexities and of various functional uses will stimulate the researchers for many years ahead. Innumerable possibilities for structure formation based on variation in a sequence formed by 20 amino acids can be used not only for the construction of sensor units (as will be seen in Volume 2, Chapter 4) but also for making the scaffolds for supramolecular sensors. Here we present the example of formation of scaffolds for such sensors by association of peptides. Peptides with the properties of forming the self -assembled tubular structures were first suggested as anti-microbial agents. Later, it was realized that such peptide structures can serve as ideal scaffolds for supramolecular sensors (Gao and Matsui 2005). Surprisingly, it was found that even the peptides as short as Phe-Phe dipeptide can assemble into stable nanotubes. Via their molecular-recognition functions, the self-assembled peptide nanostructures can be further organized to form nanowires, nanoparticles and even ‘nanoforests’ (on solid support). It has been demonstrated that cyclic peptides arranged into planar rings by alternating L- and D- amino acids self-assemble via hydrogen bonding to tubular open-ended and hollow structures (Brea et al. 2007). The application of such molecular self-assemblies as the sensor building blocks is well predictable; it is robust, practical and affordable. It is also beneficial, that smart functionalities can be added at desired positions in peptide nanotubes through well-established synthetic techniques. They may include both recognition and reporting units with exploration of fluorescence-based technologies. The other types of peptide-based scaffolds are their associates based on the principle of amphiphilicity (the separation of polar and unpolar sites). Their polar sites can be represented by 1–2 charged amino acids, and hydrophobic part—by four or more sequential hydrophobic amino acids. They form ordered nanostructures similar

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to that formed by lipids (Jiang et al. 2007). There are many opportunities for hierarchical formation of supramolecular assemblies of larger complexity based on these peptide assemblies. The principle of formation of the third type of these scaffolds is borrowed from the known examples of appearance in vivo of amyloid fibrils that are associated with a large number of human diseases. Application of peptides forming amyloid fibrils as scaffolds was suggested based on the facts of formation by them of very rigid β-sheeted structures (del Mercato et al. 2007). The peptides assembled on the basis of noncovalent intermolecular interactions allow realizing three most important types of these interactions: (a) electrostatic interactions based on complementarity of charged groups, (b) hydrogen bonding that involves complementarity of proton donor and acceptor groups and (c) hydrophobic interactions that provide stabilization of contacts between low-polar groups in polar media. Individually, these bonds are weak, but their collective action provides significant effects of stabilization of intermolecular complexes. The top achievement of today is the computer design and technical realization of structures formed of coiled peptide fragments of desired, even chimeric, configuration. These structures resemble DNA origamis, discussed below, and can be called protein origamis (Ljubetiˇc et al. 2017). It was demonstrated that such structures can be assembled not only in vitro but also in vivo (Lapenta et al. 2018). We can admire such pieces of art realized on molecular level due to unique ability to locate strands and kinks of the structure in a well précised manner (Fig. 11.8). We can think also on the possibilities to use these extremely versatile materials with programmable three-dimensional structures. Fig. 11.8 Design of the four-sided pyramid and triangular prism coiled-coil protein origami folds (Ljubetiˇc et al 2017). a A topology is shown for each polyhedral shape. b Representative all-atom models. The scale bar denotes 5 nm

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11.3.3 DNA Templates. Origamis Let us for the moment forget about a very important functional role of DNA and RNA and consider oligo- and polynucleotides of different lengths as scaffolds for the assembly of nanocomposite sensors. Their role is not limited to nucleic acid hybridization and may allow extremely broad range of applications in sensors operating on supramolecular level (Wilner and Willner 2012). Highly selective binding of complementary DNA strands with any desired sequence of bases makes them very efficient supports for molecular sensors and the sensors themselves. Their great advantage is the possibility of designed location in the sequence of nucleic acid bases of different building blocks that can provide programmable arrangement of rigid structure-forming strands. They can serve as scaffolds for further modifications. DNA is a polymer made of building blocks, nucleotides, which are the purines: adenine (A) and guanine (G), and the pyrimidines: thymine (T) and cytosine (C). The nucleotides bind to each other to form a linear chain through the phosphodiester bonds. This represents the so-called single-stranded DNA molecule. Single strands can wrap around each other to form the well-known helical structure (double helix). The challenge in creating DNA nanostructures is to specify the appropriate DNA sequences in a way that the desired structure (geometry) forms have to be thermodynamically stable. The stability of double stranded DNA structure is provided by pairing of A with T and G with C coupled with hydrogen bonds. Constructing the DNA-based templates can be modulated by temperature (the ‘thermal annealing’ uses controlled high-temperature DNA melting) and also by attached macromolecules and ions. Strong advantages of nucleic acids are seen in construction of aptamers. Aptamers are the single-stranded DNA or RNA oligonucleotide sequences that possess the ability of recognizing various molecular targets, including peptides and proteins (Hamula et al. 2006, Tombelli et al. 2005). The design and application of aptamers as the sensors will be extendedly discussed in Chapter 5 of Volume 2. Here I stress that innumerable number of possibilities exists for formation of structures stabilized by intramolecular formation of short double-helical segments composed of complementary nucleic acid bases. Such structures may exhibit an ideal combination of rigidity and flexibility in intermolecular interactions. Moreover, selection of these structures can be made by repeated increase of their desired properties using the selection-amplification (SELEX) techniques that can create a large pool of similar compounds, a combinatorial library, and to screen this library. Likewise with synthetic peptides, the manipulation and incorporation into sensor constructs can be made. The main difference between them is the selection step. The advantage of nucleic acid aptamer libraries over protein or peptide libraries is their large size (up to 1015 –1016 different sequences) and the easy identification of selected binders by enzymatic amplification with polymerases. Automated selection procedures allow rapid identification of DNA and RNA sequences that can target a broad range of extra- and intracellular proteins with nanomolar affinities and high specificities.

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Design of specific topology based on sequence design with active use of these modulating factors have led to DNA origamis—the DNA chains forming very complex structure and topology (Fan et al. 2019, Halley et al. 2019). There are different possibilities for constructing origamis (Kuzyk et al. 2018). One uses already assembled DNA origami as a prefabricated template and then different materials can be attached. In another approach, assembly of the DNA origami structure can be used to drive the assembly of materials, i.e. both assemblies can take place simultaneously. Very strict precision of formation of designed structures can be achieved, as illustrated in Fig. 11.9. A prefabricated template can be made from DNA origami for precise streptavidin assembly (Kuzyk et al. 2009). Utilization of DNA origami can advance the assembly of nanoscale materials by allowing programmable fabrication of non-periodic complex structures with ~6 nm resolution. Thus, it was shown that smartly assembled origamis can entrap in desired positions the gold nanoparticles, for instance, providing their linear 1-D arrangement (Julin et al. 2018). Being arranged by origami, 2-D and 3-D configurations of metal nanoparticles can also be realized (Mathur et al. 2020).

Fig. 11.9 Self-assembly of DNA polyhedron origami (He et al 2008). a Three different types of DNA single strands stepwise assemble into symmetric three-point-star motifs (tiles) and then into polyhedra in a one-pot process. There are three single-stranded loops (colored red) in the center of the complex. The final structures (polyhedra) are determined by the loop length (3 or 5 bases long) and the DNA concentration. b Three views of the reconstructed DNA tetrahedron structure. The scale bar denotes 5 nm

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Conjugation with peptide nucleic acids (see Volume 2, Chapter 11) can be explored on this new level of structure formation. The examples of the studies in right direction are the formations of porphyrin arrays on DNA template (Fendt et al. 2007). The designed pattern and precise spacing of their location adds unlimited possibilities in a bottom-up approach toward self-assembly. Fluorescent analogs of DNA bases as well as intercalating dyes can serve as reporters (Barthes et al. 2015, Michel et al. 2020), see Fig. 5.26. Short DNA-like oligomers with fluorophores replacing DNA bases can be synthesized either as the sequences with precise location of selected dyes or as the libraries with their random location (Kwon et al. 2015). Being attached to polymer beads, they generate different fluorescence colors due to multiple interactions between closely spaced fluorophores. The color changes on interaction with different analytes.

11.4 Functional Lipid and Polymer Bilayers The membrane of a living cell is probably one of the brightest examples of how Nature uses self-assembly of relatively simple building blocks to create highly organized structures. Membrane can be viewed as a structure formed of lipids with different integral proteins and glycolipids incorporated into it. Biotechnology uses different structures formed of lipids (Fig. 11.10). Supported monolayer

Support

Hydrophobic

Polar

Arrangement of phospholipids

Fig. 11.10 Popular schematic view of the structures formed by lipid molecules. Stability of these structures is determined by amphiphilic properties of constituting molecules and their arrangement in monolayer. Polar heads tend to be exposed to aqueous solvent, whereas hydrophobic tails tend to be screened from it, attaching to hydrophobic support or forming the central part of bilayer

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Hydrophobic surfaces can be covered with phospholipid monolayers, which can be formed spontaneously, using, for instance, the Langmuir-Blodgett technology (Czolkos et al. 2011). The stability of such structures can be increased by thermal treatment (Stine et al. 2005). Such arrangement may help solubilizing the hydrophobic particles in polar media and also incorporating into them of targetbinding functionalities. In contrast, formation of flat lipid bilayers occurs on polar surfaces by vesicle fusion (Hardy et al. 2013) and by other means (Jackman and Cho 2020). Lipids alone can form artificial self-assembled structures that in many properties resemble the cell membranes, the basic element of which is the lipid bilayer. Lipid bilayers (see Fig. 11.10) can assemble into the structures of different curvatures, from planar membranes to small nanoparticles (of the smallest size of ~50 nm), forming a closed volume. The latter are called the lipid vesicles or liposomes. Common phospholipids forming these structures are the molecules with polar heads and two long (16–18 carbon atoms) hydrocarbon tails. The bilayer formed in aqueous media consists of two monolayers, in which the apolar tails assemble forming hydrophobic layer. The polar heads stretch out to form two outer surfaces extending to aqueous solvent. When a vesicle is formed, some of the heads appear facing the inner volume, and the others extend to the outer surface. Their stability allows to fill their inner volume with water-soluble materials and to use them as drug carriers. Bilayers themselves allow incorporation of membrane proteins, making them convenient models of biological membranes. The small low-polar molecules can be dissolved in the membrane between hydrophobic tails of bilayer (between the heads).

11.4.1 Liposomes as Integrated Sensors Bilayer membranes, especially in the form of vesicles (liposomes), offer very interesting possibilities for fluorescence sensing. Liposomes can be spontaneously formed in water from suspension of hydrated lipids (phospholipids or sphingolipids) by sonication, injection through porous membrane or just by gentle agitation. They possess very important collective properties. If they are made of a single type of lipid, the remarkable cooperativity in temperature-dependent structural transitions is observed. They change the structural arrangement in the bilayer at fixed temperature within the transition range as narrow as 0.6° C. These transitions influence the mobility of lipids in bilayer, but membrane integrity is intact. They are extremely sensitive to incorporation of some other molecules, such as cholesterol. The structural and dynamic properties of the bilayers can change in a specific manner on incorporation of many different compounds (and the general anesthetics are the well-known examples). Important to note, all these changes involve only arrangements and dynamics of lipids and do not produce the change of overall structure and integrity of liposome. Being very thin (about 4 nm in width) the bilayers possess tremendous depth-dependent gradients of polarity, hydration and electric fields. They are practically impermeable to ions, even to protons.

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Fig. 11.11 The first-generation 3-hydroxychromone dyes for biomembrane studies (Klymchenko et al 2002b). Dye F does not occupy a well-determined position (its motion is shown by arrows). Dependent on its position, it may or may not form hydrogen bond with water. Location of Dye F2N8 is at the polar interface. Dye F4N1 in vertical orientation goes deeper into the bilayer, on the level of the lipid sn1 carbonyls. Dye PPZ is in inverted orientation, it goes deeper than the carbonyl groups. Such location is supported by the results on quenching by nitroxide paramagnetic quenchers that were covalently attached to the lipids (shown in the left part of the figure, marked as TC, 5 and 12)

The latter properties make the bilayers the ideal transducers in sensor technologies. From the time of discovery, liposomes are the objects of active research with various fluorescent dyes serving as the ‘probes’ for their structure and dynamics. The targeted synthesis of smart derivatives of wavelength-ratiometric two-color fluorescent dyes made possible their spontaneous incorporation into the bilayer in desired depth and orientation (Klymchenko et al. 2002a) (Fig. 11.11). This allowed determining for the first time the polarity and hydration in the bilayer as easily distinguishable parameters (Klymchenko et al. 2004a, b), and characterization of produced electrostatic potential (Klymchenko et al. 2003). Thus, we know that liposome can interact with different molecules, and this interaction changes its collective structural and dynamic properties, without changing its integrity. We know also that these interactions can depend strongly on lipid composition of the membrane, and in this sense can be very specific. We possess novel fluorescence dyes as the tools to provide the most sensitive response to these interactions. What remains, is to make the sensors. It was demonstrated that the lipid bilayer itself can serve as the sensor element that allows for the efficient detection and characterization of molecules that interact with the membrane and changes its structure. Possessing high cooperativity, the whole bilayer structure can serve as a composed nanosensor combining the binding and reporting functions, so the transduction occurs due to cooperative properties of the bilayer. Located in the bilayer, fluorescent dyes provide the reporting signal. This idea was explored for the development of prototype sensor for cholesterol. The changes in lipid membranes on incorporation of cholesterol are well-known. They are especially great for bilayers made of lipid sphingomyelin (SM) that are able to form with cholesterol the very rigid structures, “rafts”. We formed the vesicles made of SM, incorporating functional 3HC dyes and observed dramatic

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Without cholesterol Lipid bilayer in disordered state

With cholesterol Lipid membrane in highly ordered state

Fig. 11.12 The prototype for two-band ratiometric fluorescence sensor for cholesterol that uses sphingomyelin (SM) bilayer vesicles with functionalized 3HF dye (Turkmen et al. 2005). Thin solid line represents the spectrum in SM bilayer. Upon addition of cholesterol, the relative intensity of the short-wavelength band dramatically (by ~80%) decreases (thick solid line). This does not happen when cholesterol is added to phosphatidylcholine (DOPC) vesicles (compare dash and dot-dash lines). The arrow shows dynamic range of this effect.

changes of florescence color upon incorporation of cholesterol (Turkmen et al. 2005), Fig. 11.12. We observe that upon addition of cholesterol, the relative intensity of the short-wavelength band dramatically (by ~80%) decreases in relative intensity. This dramatic change of color can be easily detected even visually and, of course, with the application of a simple fluorescence detection technique.

11.4.2 Stabilized Phospholipid Bilayers The weak points of liposomes are well known to those who work with them. It is not easy to make them homogeneous in size, and their long-term stability is bad. Therefore, the idea appeared to stabilize them by forming them around micro- or nanoparticles, to form core-shell liposomes. The cores of these particles may be of noble metals, polymeric or composed of quantum dots with the exploration in full of their signal transduction and fluorescence possibilities. In such construction, the particle serves as the ‘core’, it is surrounded by lipid bilayer as the ‘shell’ (Mornet et al. 2005, Troutier and Ladavière 2007, Weingart et al. 2013). The core determines the particle size and shape and protects it from spontaneous decomposition.

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Different types of nanoparticles can play the role of a core in such nanocomposites (Weingart et al. 2013). It can be selected in order to obtain additional valuable properties. For instance, it can be made magnetic (Shinkai 2002), which will facilitate separation of sensor particles from tested media and their repeated use (see Sect. 12.3). Alternatively, if the core is composed of a polymer or silica bead doped with fluorescent dye (as described in Chapter 8) or quantum dot (see Chapter 7), it can provide fluorescent emission. The procedures of making these composites are simple due to spontaneous formation of bilayers (Mornet et al. 2005). The nanocomposites based on silica core and phospholipid bilayer shell are quite popular in fluorescence sensing technologies (Chemburu et al. 2010). The other popular system is a hydrogel core enclosed within a lipid bilayer (Kazakov 2017). The stabilizing core effect on phospholipid bilayers is frequently observed. The membrane-inserted sensor functionalities can be engineered in bilayers formed on the surface of polymer latex beads (Ng et al. 2001). These composites called lipobeads (Fig. 11.13) can be transformed into fluorescent sensors by incorporation into the bilayer of various fluorescent dyes that possess sensing properties or are conjugated with recognition units. Such lipobead nanosensors were suggested for intracellular measurements of pH, Ca2+ , and O2 (Gao et al. 2011, Ma and Rosenzweig 2005). There are large possibilities in the modification of the surfaces of these particles with bioactive molecules for the development of biosensors. The membrane shell can possess both receptor and reporter properties and can be modified for that in many ways. Lipid self-assembly on particles allows receptor insertion and amplification of receptor-target recognition. One of such incorporated receptors can be monosialoganglioside GM1, which can specifically bind cholera toxin (Carmona-Ribeiro 2001). Utility of self-assembled vesicles, bilayers or monolayers at interfaces is limited only by our own imagination. The planar surfaces of optical waveguides and microfluidic devices can also be modified by depositing the lipid bilayers resulting in their stabilization and allowing Hydrogel

Phospholipid bilayer

Lipid anchor

Phospholipids

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Lipobead

Fig. 11.13 Schematic illustration of the self-assembly of a lipid membrane around a hydrogelphospholipid conjugate

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incorporation of different types of recognition units. Among interesting developments is the formation of bilayer membrane that recognizes cholera toxin directly on the surface of a planar optical waveguide (Kelly et al. 2006). This approach can be extended to the development of sensor arrays on planar support (Yamazaki et al. 2005). Incorporation of integral membrane proteins into the arrayed membranes enables the study of ligand/receptor binding, as well as the interactions with intact living cells.

11.4.3 Polymersomes Polymersomes are synthetic analogs of liposomes made of amphiphilic vesicleforming block copolymers (Rideau et al. 2018). The advantage of polymersomes over liposomes is their increased stability, which contributes to the increased lifetimes of these structures. In contrast to common liposomes that have high tendency to collapse with time, polymersomes can be made to be stable eternally. This can be achieved by their chemical cross-linking (Discher et al. 2002). For the formation of bilayers, the polymersomes have to be assembled according to the same principle as the lipid vesicles. In this case they consist of two blocks— polar heads and hydrophobic tails. The vast amount of available monomers and the ability to vary the ratio of the two blocks make it possible to tune the properties of the resulting vesicles, for example, their size, polarity, stability, etc. In general, the membranes of block copolymer vesicles possess higher thickness and less fluidity compared to liposomes (Antonietti and Forster 2003). These properties can vary in broad ranges depending on the polymer composition (Bermudez et al. 2002). Formation of polymersomes and subsequent manipulations with them can be provided with the aid of click reaction (Rein et al. 2016). This allows obtaining coreshell nanocomposites of various structures and properties (Balasubramanian et al. 2016, Li and Binder 2011), which makes an interesting platform for sensing and imaging (Ghoroghchian et al. 2005, Leong et al. 2018).

11.4.4 Formation of Protein Layers Over Lipid Bilayers The layers formed by two-dimensional crystallization of S-layer proteins (see Fig. 11.7) usually need some support for their formation. With this support, different novel nanocompositions can be created, which could allow making a 2-D crystalline surface layer amendable to further modifications (Fig. 11.14). It was demonstrated that they can be readily formed over liposomes (Moll et al. 2002). This allows activating an important line of development in combining the S-layer and lipid membrane technologies (Huber et al. 2006). Although, the development of S-layer technologies was focused primarily on liposomal drug delivery systems, an important field of future applications emerges in sensor technologies (Pum et al. 2013).

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Fig. 11.14 Schematic drawing showing the reassembly of S-layer proteins in solution, on solid supports, at the air-water interface, at lipid films, and at liposomes and nanocapsules (Pum et al 2013)

The interaction of S-layer proteins with lipid molecules is noncovalent. Electrostatic interaction between exposed carboxy groups on the inner face of the Slayer lattice and the zwitterionic lipid head groups at the outer face of liposomes is primarily responsible for the binding and defined orientation of the S-layer subunits. As a result, they can form a closed shell around liposome and this allows obtaining the composites with essentially increased stability. However, these interactions do not reduce substantially the lipid mobility in the plane of bilayer. A formed closed lattice structure is amendable to different modifications. Moreover, since S-layer proteins can be genetically modified and fused with other structure-forming proteins, the supramolecular functional constructs can be assembled. Concluding, there are many very positive features offered by lipid bilayers and their analogs. These structures can be formed spontaneously and they allow spontaneous incorporation into them of different functional molecules, both receptors and reporters. They can respond to target binding by the changes of their integral and easily measurable properties, such as the lipid order and dynamics. When they form the particles with closed inner volumes (liposomes), these particles can retain

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in this volume many different molecules or particles with diverse functional possibilities. The composites containing single particles surrounded by the bilayer are of special importance because they allow achieving a strongly increased stability of the bilayer towards aggregation or decomposition and also because of possibility to provide additional useful properties to the core particles. Synthetic lipid analogs and bilayer-forming polymers expand these possibilities.

11.5 The Principles of Formation of Assembled Structures New rapidly developing chemistry based on the principles of self -assembly and self organization tends to complement, and to a significant extent, to substitute common organic synthetic chemistry in an attempt to produce supramolecular systems of greater complexity and higher level of functionality. The new strategy requires the design and synthesis of a limited number of relatively simple building blocks that are then allowed to self-organize into complex structures. Following it, without strong synthetic effort, one can assemble the recognition unit together with the signal transduction and reporting unit into one block with superior properties. In this case, there may be no need for covalent links between these units; they only have to be designed according to a productive strategy, and noncovalent interactions between them are quite sufficient for functioning. Supramolecular chemistry has developed into a strong interdisciplinary field of research (Lehn 1995). One of its rules is the requirements for topological saturation. Saturation by non-covalent interactions should provide collective effect that has to bring to the system a necessary structural stability. Additional stability may be achieved with covalent cross-links formed after the assembly. A ‘hot spot’ should remain only for recognition site and, if necessary, for providing the binding to a surface or location in heterogeneous structure, such as the living cell.

11.5.1 Affinity Coupling In general, there are two principles of formation of supramolecular structures: affinity coupling and self-assembly. The examples of affinity coupling are the interactions antigen-antibody and biotin-avidin (or streptavidin) that are the most frequently used for creation of supramolecular structures. The advantage of antibodies is their versatility, since antigenic determinants can be inherently present or artificially incorporated into any structure of protein, nucleic acid or carbohydrate origin. They can be obtained as binding sites of many artificial molecules including organic dyes. The possibilities of manipulating with them have grown with introduction of monovalent single-chain antibody fragments (Piervincenzi et al. 1998). Thus, for efficient highly selective high-affinity coupling, one of assembling units should contain antibody or its fragment and the other—antigenic determinant recognized by it. With a broad

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variation of affinities (K d ≈ 10−8− −10−13 M), the antigen-antibody interactions may not in all cases be sufficiently strong for the formation of stable supramolecular structures. Binding of avidin or streptavidin with biotin is different. Protein avidin can be found in the white of chicken eggs. It binds a rather small molecule biotin of 244.3 Da (also known as vitamin B7) with a uniquely high affinity (K d ≈ 10−14 −10−15 M). Streptavidin is a bacterial analog of avidin, it has similar properties as avidin but can be produced in bacteria. It is a globular 60 kDa protein with a high (compared to other proteins) thermal and pH-stability. (Figure 11.15). Since each avidin molecule can bind four biotins or biotin-labeled units, the complex, precisely arranged and very stable supramolecular constructions can be designed. Therefore, if streptavidin is a part of one component of the structure and biotin is attached to its another component, these components find each other in solution and associate irreversibly with the formation of a series of noncovalent bonds between complementary groups of interacting surfaces that are formed together and are, therefore, as strong as covalent bond (Fig. 11.16).

Biotin

Biotin

Streptavidin Fig. 11.15 The streptavidin-biotin system. a Streptavidin tetramer (PDB code 1SWE) in complex with biotin. The protein is shown in cartoon representation. The biotin molecules are in spacefilling representation and their location is circled and marked by arrows. Streptavidin monomers are colored red, green, blue and yellow respectively. The parts of the protein, which are in front of the biotin molecules are made semi-transparent for clarity

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b

a

Streptavidin

Support

Biotin

Fig. 11.16 Typical supramolecular complexes that can be assembled using tetrameric streptavidinbiotin junction. a The complex obtained with tetrameric streptavidin covalently attached to the support surface. It interacts with biotin-labeled fluorescent structure. b The complex combines two nanoscale structures. One of them is shown to contain fluorescence reporter

For providing efficient affinity coupling with the use of biotin-streptavidin pair, the SH-reactive biotin derivative is applied. It yields biotinilated hydrogel, which is ready for binding streptavidin (Burnham et al. 2006). The latter one is particularly useful for immobilization of biotinylated aptamers (Schaferling et al. 2003). Since biotinylated proteins are deposited from solution, such system is advantageous for deposition of proteins that need delicate conditions of treatment. Generally, in addition to increased binding capacity, immobilization within three-dimensional hydrogels offers many advantages over binding to flat two-dimensional surfaces, such as ‘wet’ and friendly polar environment with stabilizing effect of the gel matrix. A disadvantage is a slow rate of establishing the target-binding equilibrium that is limited by target diffusion in the gel. Streptavidin-biotin pair has now become a standard tool for providing strong and specific assembling of supramolecular structures. In contrast to antibodies, their complementary surfaces do not allow much structural variation, but there are many possibilities of reacting or substituting the groups that are outside their contact areas. In biological world, a number of additional partner pairs can be found that are characterized by high affinities. Such are the pairs: lectin—carbohydrate or enzyme— suicide inhibitor. They possess subnanomolar dissociation constants. The binding properties of biotin but not of avidin or streptavidin can be simulated with designed peptides. Trivalent vancomycin/D-Ala-D-Ala—complex may probably be suggested as a pair with relatively tight binding of smaller molecular weight units (Rao et al 1998). High affinity in interaction can be achieved involving cucurbit[7]uril (Shetty et al. 2015) and other molecules with host-guest coordination properties (Liu et al. 2017). However, they did not find extensive practical use. It is surprising that by now organic synthetic chemistry could not suggest a proper substitute to streptavidinbiotin pair that could compete in simplicity, stability and prize. So, streptavidin— biotin pair remains the most requested in generating molecular junctions. Affinity coupling is an extremely valuable tool in intracellular research. For many of these studies it is needed that the fluorescently labeled partner should be introduced from outside into the cell, where it should find its second partner out of thousands

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of types of other molecules. Unfortunately, neither streptavidin-biotin nor antigenantibody pair can be efficiently used for this task.

11.5.2 Self-assembly Self -assembly involves the use of topological complementarity and saturation of intermolecular interactions between the partners on a broader scale with possible adaptation of conformation of partners for achieving this saturation (Demchenko 2001, Lehn 1995). Self-assembling and self-organizing methodologies are the powerful tools for the “bottom-up” approach for the realization of complex structures with functional properties. Recently, this concept has been extended to the design of fluorescent chemosensors providing new exciting potentialities for the development of innovative sensing systems (Mancin et al. 2006). The idea of this approach is, again, borrowed from Nature. Living cells do not appear as a result of ‘nanofabrication’. All the processes leading to formation of supramolecular structures, such as chromatin, membranes, microfilaments or viruses, start from synthesis of individual molecules and their subsequent assembly. Being the building blocks for such assembly, biomolecules are adapted for assembly by their inner structures forming the contact surfaces. The same is required from artificial systems. In such self-assembling systems, the principle of molecular recognition (Volume 2, Chapter 1) is actively explored. In addition to steric complementarity, there should be maximal intermolecular contact with maximal saturation by noncovalent bonds. Thus, amphiphilic structures favorable for hydrophobic interactions are essential for self-assembly by incorporation into micelles and lipid bilayers, but they are ‘lubricants’ that lack specificity that may be often necessary. Electrostatic interactions can provide the necessary recognition pattern, especially when they are assembled to clusters. They govern the layer-by-layer formation of multi-layer scaffolds. Hydrogen bonds are individually weak, but ensemble of these interactions is responsible for stability of DNA and of α-helices and β-sheets in proteins. Experimental evidence exists that such multivalent interactions can increase the affinity compared to univalent interactions by many orders of magnitude (Baldini et al. 2007). Many different hydrogen-bonded supramolecular structures comprising tens hydrogen bonds have been suggested (Vriezema et al. 2005) to ensure strong binding. These structures can be less stable in water and other protic solvents, in view that these solvents themselves can be good hydrogen bonding partners. The full advantage of hydrogen-bonding complementarity can be explored in precise and programmable self-assembly using the principle of formation of DNA double helix (Seeman 2003). The partners that contain complementary sequences can assemble in a thermally reversible manner (as do the double-helical DNA strands) over a range of length scales. DNA can be conjugated to other materials (molecules and nanoparticles) or attached to solid support. The organizational capabilities of structural DNA

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nanotechnology are just at the beginning to be explored. It is expected that they will find broad application. An interesting perspective is to make the self -assembly responsive, i.e. controllable by different external stimuli (Grzelczak et al. 2019, Yang et al. 2014). Such stimuli-responsiveness can originate either from the organic ligands adsorbed on nanoparticle surfaces or from their inorganic cores. The factors that control the selfassembly can be chemical stimuli—the solvents, acid/base and redox signals, metal ions, gases, biological macromolecules. Among the physical stimuli are the temperature, magnetic fields, and light. Modulating the self-assembly by removable ligands is the possibility to make this reaction under control. Thus, with the increase of size and complexity, the preparation of molecular conjugates by covalent synthesis becomes a very difficult, time-consuming and even unrealizable choice. In contrast, supramolecular chemistry based on self -assembly may offer the creation of nanoscale systems with a broad range of sensor applications. Self-assembly of composite sensor units is based on the same principles as the sensing itself—on complementarity between interacting partners and formation of non-covalent interactions between them, such as hydrogen bonds, salt bridges, solvation forces, π-π stacking and coordination of metal ions. Due to multiplicity of these bonds, the interactions between the partners can compete in strength with covalent bonds.

11.6 Conjugation, Labeling and Cross-Linking Integration of nanoparticles and their conjugation with biomolecules leads to novel hybrid systems, which may couple the recognition, cell penetration or catalytic properties of biomaterials with the attractive multifunctional characteristics of nanoparticles. In the development of supramolecular sensors, synthetic chemistry plays a very important role, but often on a high level of modification and integration of nanostructures. The is a need for modifying the molecular and supramolecular structural blocks by covalent attachment of recognition and reporting units and of additional stabilization of self-assembled structures with covalent bonds. Several key approaches on this pathway will be outlined below. With different types of particles, a multipoint conjugation can be achieved (Aubin-Tam and Hamad-Schifferli 2008). The techniques have been developed for specific binding of nanoparticles with different small molecules (Algar et al. 2011), and also with DNA (Boeneman et al. 2010), peptides and proteins (Aubin-Tam and Hamad-Schifferli 2008, Prasuhn et al. 2010). Binding with whole bacterial cells (Moyano and Rotello 2011) becomes possible. Formation of supramolecular structures based on biospecific molecular recognition will be of special concern in our discussion.

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11.6.1 Nano-Bio Conjugation The nanomeric size of fluorescent particles allows many different possibilities of chemical manipulations with them: formation of single biomolecule-particle conjugates (Katz and Willner 2004), attachment to nanoparticles of multiple molecules in a well-controlled manner (Peng et al. 2011) and also the formation of their hetero-composites of high complexity (Algar et al. 2011). Meantime, each type of nanoparticle structure requires its own conjugation chemistry. Functional groups on polymer surfaces are the easy targets for bioconjugation, and exposed amino groups of PAMAM dendrimers are especially convenient for such modifications (Bergamini et al. 2010). Many possibilities exist for modifications of fluorescent conjugated polymers that are discussed in Sect. 8.5. The literature examples include their coupling with lipids with potential for gene delivery (Feng et al. 2010) and with short peptides that can be functionalized for cellular research (Pu et al. 2010). In the latter case, variation of peptide coverage changes the polymer emission color. Regarding semiconductor quantum dots (Sect. 7.2), their bioconjugation is more difficult, and several strategies for that have been developed (Algar et al. 2011, Biju et al. 2010, Hötzer et al. 2012, Jin and Hildebrandt 2012). Their further functionalization with the attachment of ligands to cellular receptors can be achieved via interaction of streptavidin-QD with biotinilated ligand (Lidke et al. 2007). As we observed above, the biotin-avidin (and also neutravidin and streptavidin) interaction is a popular approach for connecting components in functional nanocomposites.

11.6.2 Techniques of Conjugation and Labeling Covalent linkage was and remains the most frequently used means in self-assembly of inorganic, organic and organic-inorganic structures for their stabilization, functionalization and labeling. The most frequently used reactions for covalent modification and conjugation of inorganic nanoparticles can be found in ref. (Erathodiyil and Ying 2011) and the publications cited therein. Amino groups and SH-groups are the usual targets for chemical modifications, and their presence at the sites of desired modification with the absence in other sites may guarantee the necessary precision (Brinkley 1992). SH-targeting is more actively used with peptides and proteins because these groups, present as Cys residues, are less abundant than the amino groups (N-terminal and that of Lys residues). Solid phase synthesis of peptides and site-directed mutagenesis of large proteins are used for incorporation of SH- groups into desired sites of the sequence for subsequent attachment of fluorescent dyes according to well-known chemistry. Usually the fluorescence dyes are synthesized possessing reactive group for labeling.

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The three-dimensional structures of biological macromolecules, if they are known, are actively used for determination of the optimal labeling sites. Comparative studies of sensor proteins, in which the engineered Cys locations were rationally designed based on the strongest differences of environments of these sites in unbound and bound forms derived from X-ray structures (Marvin et al. 1997) witness for usefulness of this approach. The retention of native protein structure during SH labeling is especially important for proteins that contain significant amount of S-S bonds that are necessary for supporting this structure, and, particularly, for antibodies. Thus, the skill is to select the location of introduced SH- group in such a way that could allow correct protein folding with correct formation of S-S bonds that are essential for structure and, simultaneously, to keep the introduced SH-group free and available for chemical modification. It was found (Renard et al. 2002) that a mild reducing treatment is necessary to reactivate mutant Cys residues before coupling with SHreactive dye. The reaction of coupling in these conditions allows preserving the essential disulfide bonds of the antibody variable domains. In the same study, the structural and energetic criteria were applied to locate the sites of introduction of reactive Cys residues by mutagenesis. The authors selected the target residues as that belonging to topological neighborhood of the antigen in the structure of complex between antibody and antigen, its absence of functional importance for binding the antigen and its solvent accessibility. The feasibility of this rule was shown in experiment.

11.6.3 Co-synthetic Modifications Co-synthetic incorporation of fluorescent dyes into polymer and biopolymer structures is less frequently used, though such procedures may be of choice for sensing peptides and also of oligonucleotides as aptamers. In Chapter 4 of Volume 2 we will observe that in some sensing technologies the large globular protein structures are not required, and the necessary effect of target recognition can be achieved by smaller peptides. They are more flexible in application and more tolerant to environment conditions with better performance regarding reproducibility and multi-use operation. In this case, peptides can be obtained by chemical solid-state synthesis and any possible variation of amino acid sequence can be easily achieved. They may include co-synthetic incorporation of fluorescent reporter groups at any position in the sequence, which makes unnecessary the site-specific modification of SH- or NH2 groups (Link et al. 2003). An alternative possibility, the incorporation of non-natural amino acids in a cellfree peptide and protein biosynthesis on ribosome, is also possible. In this case, a non-natural aminoacyl-tRNA is constructed and incorporated into synthetic process (Katzen et al. 2005). Regarding covalent labeling of nucleic acids, both 5’ and 3’ terminals are available for that. There are also many new possibilities to synthesize artificial fluorescent nucleic acid bases that can be incorporated co-synthetically (Michel et al. 2020).

11.6 Conjugation, Labeling and Cross-Linking

Click reaction Alkyne

Azide

473

Triazole conjugate N N N

Cycloaddiction

Fig. 11.17 Schematic representation of a typical fluorogenic click reaction. Non-fluorescent alkyne- and azide-containing starting materials are covalently coupled together in one step to afford a highly fluorescent product

11.6.4 Chemical and Photochemical Cross-Linking Self-assembly, if necessary, can be combined with chemical cross-linking and photopolymerization (Mancin et al. 2006). This will provide higher stability to the formed structures and allow integrating molecules with different functions. Such applications are immense, and the reader is advised to consult classical monographs (Hermanson 1995) and reviews (Brinkley 1992, Brunner 1993), in which these modifications are described in sufficient detail. These reactions, however, were not sufficient to satisfy the demands of bioconjugation chemistry, macromolecular science and, particularly, the design of supramolecular ensembles. All that stimulated the development of click chemistry that rapidly became a general tool for achieving modular and highly specific cross-linking occurring with high chemical yields and with a product being stable in physiological conditions. The azide alkyne cycloaddition reaction catalyzed by copper and occurring in mild conditions at room temperature fully satisfies the click chemistry concept. Moreover, this reaction can be fluorogenic (Le Droumaguet et al 2010, Plass and Schultz 2011), i.e. it can generate fluorescent product from non-fluorescent predecessors (Fig. 11.17). Essentially, this reaction is not limited to small molecules and organic dyes containing the complementary groups. It can provide the linkage between inorganic and organic macromolecular species. An acetylene-functionalized lipase has been attached to azide-functionalized water-soluble gold nanoparticles. The product containing about six enzyme molecules per nanoparticles retained in full its catalytic activity (Brennan et al. 2006). Another example was the demonstration that azidefunctionalized gold nanoparticles can be specifically coupled to an alkyne-modified DNA duplex, resulting in a chain-like assembly of nanoparticles on the DNA template (Fischler et al. 2008). Here azide terminate nanoparticles reacted with alkynemodified DNA duplex, resulting in regular chain of gold nanoparticles formed on DNA scaffold (Fig. 11.18). Silica beads, quantum dots and other nanomaterials can be assembled with this reaction and decorated with different types of chemical and biological receptor and reporter units expanding the possibilities of fluorescence-based technologies (He

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Azide-terminated nanopar cles

Alkyne-modified DNA

DNA with a ached nanopar cles

Fig. 11.18 Regular chain of gold nanoparticles formed on DNA scaffold with the aid of click reaction. Azide terminate nanoparticles reacted with alkyne-modified DNA duplex

and Gao 2011, Le Droumaguet et al. 2010). The click reaction shown in Fig. 11.17 and a number of other cycloaddiction reactions can be realized within living cells (Plass and Schultz 2011). More information about that will be presented in Volume 2, Chapter 15.

11.7 Sensing and Thinking. Extending Sensing Possibilities with Smart Decorated Surfaces and Nano-Ensembles Reading this Chapter, one can imagine a myriad of opportunities for manipulating with interactions of molecules, nanoparticles and surfaces. Since there is no limit regarding the sensor construct, the choice of optimal solution can be made on a very broad scale. This may involve not only molecular complexes and responding nanoparticles, but also the self-assembled supramolecular structures, composite nanoparticles (that combine recognition and response properties) and various types of surfaces and interfaces. In many technologies, target and sensor are in different phases, and in order for them to approach, at least one of these partners should possess sufficient mobility residing in gas or liquid phase. The other partner can be in solid state, and here follows the important role attributable to interfaces. For flat surfaces and nanoparticles, the creation of supportive structures and interfaces follows quite different experimental procedures. But then, for further functionalization, the applied principles and techniques can be similar. The principle of such self-assembly is also the same as for the sensor-target interaction. It is the molecular recognition that involves energetic and steric complementarity. The covalent cross-linking can also be explored, if necessary. Improving the functionality is always increasing the complexity. For overcoming the observed difficulties, the libraries of sensor building blocks have to be created. The well-established streptavidin-biotin pair is not optimal for many applications;

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therefore new pairs for molecular assembly, especially of synthetic origin, should appear offering new possibilities. Their libraries are expected to be made available and then easily combined to produce the most suitable system for the desired use. The reader is requested to answer the following questions: 1.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Why the support becomes needed for fluorescence sensors? In which sensor technologies it must be used in the form of plain surfaces or as the cores of nanoparticles? Suggest several examples for using dip-coating and spin-coating. Which of them you would prefer in your suggested examples? Can you use lithographic printing to make surface-selective (e.g. mosaic) pattern of binding using dip-coating or spin-coating? What are the conditions for making the self-assembled monolayers? What forces operate between layered molecules and with the support on formation of Langmuir-Blodgett films? How the layer-by-layer technique operates? What are the requirements for layered molecules? What is the ligand exchange? How does it operate with metal nanoparticles? The graphene support, what can be its functional role in sensing? Why and how the S-layer proteins can be used as 2-D crystals? What are the DNA origamis and protein origamis? Explain the principle of formation of the lipid bilayer. Which part of liposome can accommodate polar or nonpolar compounds, and why. What about large nanoparticles? What about fluorescence probes? Explain the principle of self-assembly. What is the topological saturation? Why biotin and streptavidin are so popular? The click reaction may lead to fluorescent product but may not. Explain how you will use this reaction to incorporate the desired fluorophore.

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Chapter 12

Fluorescent Composites Combining Multiple Sensing and Imaging Modalities

Smart nanoscale composites can be designed not only for performing functions associated with absorption and emission of light. They can comprise other modalities that are required for obtaining and analysis of information using quite different chemical structures and different instrumentation. This information can be complementary to that obtained by optical methods and can be efficiently used in sensing, but mostly in imaging. The combination of fluorescence probing with X-ray and NMR methods and also based on magnetic properties will be discussed below. Regarding imaging, the methods based on X-rays and NMR allow deep penetration into human tissues, which is not possible with optical methods. We demonstrate the possibilities to realize this coupling by designing the composites that on supramolecular or nanoscale level combine the probing and contrasting efficiencies for different modalities. In the last Section “Sensing and thinking” I encourage the reader to think on possibilities of application of such multimodality-based approach. In previous Chapters of this book we analyzed many possibilities to design optimal fluorescence reporters for different scientific and practical needs. We observed that with smart nanocomposites often incorporating organic and inorganic components, one can achieve dramatic improvement. Many important developments are observed. These nanocomposites demonstrate dramatically increased brightness, the ability to concentrate emission on particular luminophores (antenna effect), to shifting the emission spectrum to a desired spectral position (wavelength conversion). The emission anisotropy and lifetime can be modulated in broad ranges. New sensing technologies were devised on this basis, which include multiplexing and multicolor coding in suspension arrays. In parallel, other methods for research and practical application, such as based on X-rays and NMR, develop. They require quite different instrumentation and data analysis. The atomic and molecular structures that are the source of information are quite different. What is the need to combine them with fluorescence sensing and imaging? The answer is very simple. Each of these methods brings often complementary information, and systematic analysis based on its assembling and systematic analysis brings new knowledge. Such need of complementary information is especially © Springer Nature Switzerland AG 2020 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-030-60155-3_12

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X-ray CT

NMR imaging

+

+

Luminescence

MulƟmodal agents Fig. 12.1 Illustration of multimodal approach combining luminescence imaging with the Xray computer tomography and NMR imaging. The applied multimodal contrast agents serve for obtaining images that use the combination of these methods

seen in imaging of living cells and tissues. Fluorescence is not the only method for studying the living cells. It is suitable for multi-scale imaging, extending to the level of whole-body animals, but in this application it is hindered by a very limited imaging depth, below 1 cm. One has also to account the limited resolution in imaging, the need for application of external fluorescent reagents and the commonly present background emission. Other methods have their own serious limitations in terms of spatial, temporal and energy resolution. How to combine different seemingly incompatible methods based on different physical principles and using quite different instrumentation and data analysis for focusing on the same object of study? This can be done with the application of probes operating on nanoscale and satisfying the requirements of these methods together (Zhao et al. 2018). Such probes should be so designed that they have to serve as the agents for optical, X-ray and NMR contrasting, as well as magnetic, studies simultaneously. This task is not simple. Below, we will discuss different possibilities and successful results of its realization. More specifically, we focus on optical luminescent (mainly fluorescent) imaging probes adapted for multimodality imaging that integrate luminescent reporting groups (luminophores) with other contrast agents used in X-ray computed tomography (CT) or nuclear magnetic resonance (NMR) imaging (Fig. 12.1). Outside our discussion in this Chapter will be X-ray excited and radioactivity-induced visible fluorescence probes. They will be overviewed in Sect. 14.3. Photoacoustic spectroscopy is also very rapidly developing domain offering high-contrast imaging. Amazingly, this method does not require a multi-composite design. The light used for excitation can produce in one material two effects, the generation of the emission and production of the acoustic signal. In Sect. 14.6, we will provide more information on this interesting issue.

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In this Chapter, the sensing and imaging technologies are described that are based on smart composition materials and allow simultaneous application of several detection/imaging modalities. The emphasis is made on design strategies of optical fluorescence imaging probes that are currently implemented and are prospective to afford physically, chemically and biologically compatible multimodality.

12.1 Multimodal Approach in Sensing and Imaging Let us first specify some terminology, in order to avoid confusion. Under multifunctionality we mean a combination of essential properties of reporter that may include the property of selective binding to a particular target (macromolecule, living cell or its organelle), of guiding to a particular target by physical means (e.g. by being magnetic), or using specificity of molecular recognition (e.g. by attached antibodies). Thus, control on location of the reporter and on physical or chemical action at the site of location (e.g. singlet oxygen generation or photothermal action) can be coupled in one nanocomposite. Performing these functions, the reporter elements can vary in broad ranges. They can be organic dyes (Chap. 5), complexes and compositions of inorganic atoms (Chap. 6), nanoparticles of inorganic nature (Chap. 7), conjugates of organic fluorophores (Chap. 8), carbonic nanostructures (Chap. 9) and also three-dimensional structures forming interfaces of different nature (Chap. 11). They provide different types of response but this response is combined by common mechanism, which is the generation and deactivation of electronic excited states. We will use the term multimodality (Louie 2010), in contrast to multifunctionality (Jarzyna et al. 2010), if these mechanisms are different and based on different physical principles. Even if they are addressed to the study of the same object and use (as we will see below) the same nanocomposite tools, they can be multimodal. Thus, in early cancer diagnosis the probes can be used that combine fluorescence, Xray computed tomography and nuclear magnetic resonance imaging that together may give three dimensional (3-D) details of tissues and cells of high resolution and sensitivity (Xing et al. 2012). The multimodal imaging probes are aimed to overcome intrinsic drawbacks of each imaging modality with complementary information from different modalities for improved sensitivity and accuracy. A request for multimodality becomes very important in various applications, particularly for the whole body imaging and clinical diagnostics together with treatment. However, the route to achieving such multimodal probing is not easy. Operating with multifunctional nanostructures (Chap. 10), we learned how to combine in a nanoscale structure the species possessing different properties. This experience is actively used for composing multimodal structures, with the account that here their basic elements are mechanistically different. One of the technical problems is related to the fact that many of these elements can interact with each other. For instance, heavy atoms used as contrast agents in CT or making the nanoparticles magnetic can strongly quench fluorescence. We have to deal with design strategies that could be implemented to create physicochemically

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and biologically compatible multimodal fluorescence imaging probes to overcome intrinsic drawbacks of each imaging techniques. No new drawbacks should appear on this route. The other problem is the nanocomposite size that may be important if we combine molecular structures. The smaller these particles are the better. Whereas in homogeneous assays they can be in the form of microscale beads, for the studies of living cells the size of particles less than 50 nm is highly desirable (Xing et al. 2012). Also, the particles of the size much smaller than the wavelength of light can be considered as zero-dimensional (0-D) quantum emitters, “dots”, which simplifies the analysis of their spectroscopic behavior. Of course, it could be easy to use in imaging a “cocktail approach”, i.e. to employ a mixture of various contrast agents together in a single dose (James & Gambhir 2012). However, it is almost impossible to achieve spatiotemporal consistency for all imaging techniques. One has to take into account that being different species, these agents will diffuse in different rates and will be present in the studied location sites in different concentrations. They may behave differently even if they incorporate the same additional functional moieties such as targeting groups or therapeutic agents. They can be differently modified or destroyed within the cells or tissues. On the level of living bodies, their pharmacokinetics, most probably, will be different. Therefore, a single probe, which integrates dual or multiple imaging contrasting agents, should be definitely preferred for dual- or multimodality imaging applications. Thus, the materials developed for multimodal technologies should be used as single composed units, they should be physically compatible within these units, and if they are used in biological systems, they should be compatible with these systems (James and Gambhir 2012).

12.2 Design and Functioning of Multimodal Supramolecular Structures and Nanoparticles 12.2.1 The Principle of Formation of Materials with Multimodal Properties There are several basic principles of formation of multimodal structures (Fig. 12.2). One relies on assembling into supramolecular structure of molecular moieties. They can be, for instance, chelation complexes of Gd3+ ion coupled with fluorescent organic dye, luminescent lanthanide chelate or other molecular construction of relatively small size. One example of this type shown in Fig. 12.2c is represented by fluorescent probe GdDO3A-ethylthiourea-fluorescein (Mishra et al. 2006). The other principle of formation also shown in Fig. 12.2 is the assembly based on nanoparticle core. For instance, superparamagnetic nanocrystal Fe3 O4 can serve as such core and fluorescent dyes or small-size luminophores can be incorporated into the shell. If quantum dots, upconverting nanoparticles or organic polymer structures

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a

b Dye

Gd3+

Gd3+

QD

Gd3+ Gd3+

c

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Gd3+

d

Fig. 12.2 Schematic presentation (a, b) and examples of structural realization (c, d) of bimodal small-molecular structure containing (c) fluorescent moiety (fluorescein dye) and d quantum dot (QD) coupled with gadolinium ion in a chelating complex. In both cases the two moieties are covalently coupled through a spacer. Graphical elements are not to scale

are used as such cores, displaying fluorescence modality, then multiple binding of Gd3+ complexes can impose the MRT modality. Thus, the composite structure assembling the near-IR-emitting CdSeTe/CdS quantum dots together with chelating groups hosting Gd3+ ions was constructed (Jin et al. 2008). In this design, reduced glutathione was used as a surface coating agent and then the coated surface was functionalized with Gd3+ -DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) complexes (Fig. 12.2d). In similar manner, fluorescent carbon dots were loaded with Gd3+ ions (Bourlinos et al. 2012). The multimodal structures can be also performed by incorporating smaller units (molecules or nanoparticles into hybrid formed by silica or organic polymer. Different magnetic nanoparticles can be coupled with fluorescent molecular and nanoscale structures to make such hybrids. As an example, bi-functional magnetic fluorescent silica nanocapsules were synthesized by employing conjugated polymers as the fluorescent emitters and iron oxide nanocrystals as the magnetic constituent, both of which were encapsulated inside the nanocore of a thin silica shell (Tan et al. 2011). There is also a possibility of linking covalently the nanostructures with different modalities. Thus, a nickel-containing nanoparticles were first synthesized based on SiO2 , and then fluorescent carbon dots were linked to its surface. Both fluorescence and magnetism are retained in synthesized nanocomposites (Wang et al. 2014). The methods for assembling components of multimodal structures into composites have been developed (Canovas et al. 2019).

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12.2.2 Fluorescence Combined with X-Ray Computed Tomography X-rays are a form of electromagnetic radiation with wavelengths roughly between 0.01 nm and 10 nm. They demonstrate unlimited penetration into tissues and fast acquisition of 3-D anatomical image. The X-ray computed tomography (CT) is a technique based on formation of contrast images recorded as the difference in the absorbance of X-rays in various analyzed media, such as the tissues of living beings (Lusic & Grinstaff 2013). The term tomography (Greek: tomos = slice, graphein = draw) now means the obtaining of three-dimensional image by rotating a radiation source and detector around an object. In experiments with small animals, the object can also be rotated. A series of scans with small angular displacements are then processed computationally to create a 3-D image of the scanned object. Resulting three-dimensional image can be used for detecting different pathologies, including medical diagnosis of tumors, brain injury, lung pathology and other disease conditions (Liu et al. 2012). However, overexposure to X-rays can be harmful. Therefore, the use of potentially harmful radiation is a shortcoming of CT scans. While CT scans allow obtaining detailed skeletal structures of the human body, the differentiation of morphological structures between normal and pathological tissues is limited without CT contrast agents. The general requirement for contrast agent here is the presence of heavy atoms that should be good X-ray scatterers (Hsu et al. 2020). Traditionally, iodinated low molecular weight compounds were used as contrast agents (Lee et al. 2013). They demonstrate very rapid clearance from the body. Therefore, for increase the circulation time and decrease the adverse effects, the nano-sized iodinated CT contrast agents have been developed. In addition to iodine, nanoparticles based on heavy atoms such as gold, lanthanides, and tantalum are used as efficient CT contrast agents. Gold nanoparticles are thought to be the most prospective. Still, they demonstrate a rather low sensitivity to contrast the soft tissues. Spatial resolution of 0.5 mm is considered to be good when using this method with application of contrast agents. Still, it is considered to be one of the most powerful imaging modalities. The harmful effects of ionizing radiation and the need for improvement of imaging sensitivity are still the limitations. It might be improved by the development of short acquisition-time techniques. Beside large difference in optical resolution as compared with luminescence methods, there are several points of inconsistency. One is, of course, the penetration depth. The reader may address Sect. 14.6 to see how shallow is the penetration into the living tissues of even of the most efficient near-IR light. Thus, Illumination of the same structures located at different depths is not possible, except for very small surface regions. The other problem is the lack of correspondence in sensitivity. In order to equalize it, a much higher concentration of contrast agent should be applied.

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Still, several successful results can be cited. In one research (Qin et al. 2015) the luminescent Ag2 S nanoparticles emitting light at 1170 nm were used. This wavelength belongs to the second NIR window. In this near-IR II range the electronic transitions are very rare, so the contribution of tissue autofluorescence is negligible. The contributions of water vibrations in this range are also low, which allows the light to penetrate deeper than usual (this issue will be extensively discussed in Volume 2, Chap. 16). In this work, the introduced CT contrasting component was iodine oil. Stabilized by polymer and lipid, such nanocomposite contained folate group on its surface, which allowed selective accumulation in tumor tissue. In vivo experiments revealed that the probe has a rather long circulation time (blood half-life of 5.7 h), When injected into nude mice by the tail vein, the probe elicited intensely enhanced fluorescence as well as X-ray CT signals in the tumors after 24 h, and the structure, the size and position of tumor tissue were shown clearly. The near-IR emitting Ag2 Te nanoparticles were suggested to use with the same purpose (Yu et al. 2020). In a second example (Hu et al. 2017), a simple method for inducing high-intensity upconversion luminescence in a core–shell-structured NaLuF4 :Yb, Tm system was developed. The applied doping of this structure by Li+ ion dramatically increased its near-IR luminescence. In vitro and in vivo experiments showed that the high-intensity luminescence of nanoparticles exhibited depth penetration ability for biological tissue. The nanoparticles, which had a size of 23 nm, showed good CT imaging performance when compared with a clinical contrast agent, in addition to allowing for deep tissue imaging. The excellent optical and CT imaging properties of the Li+ -doped highly luminescent core–shell upconversion nanoparticles suggest their potential use in both optical imaging and CT imaging for multimodal diagnosis. The high X-ray contrasting properties of gold nanoparticles were used as a core of double-modality composite (Fokkema et al. 2018). The rhodamine dye was located in a shell and in this way it was isolated from the core by a thick silica layer. In general, for efficient functioning of such composites, the heavy atoms and the structures formed of them are used. It should be accounted that, being good for CT, these species are strong fluorescence quenchers.

12.2.3 Combining Fluorescence with NMR Imaging Magnetic resonance imaging (MRI) is a versatile imaging modality based on nuclear magnetic resonance (NMR) (Prasad 2006). The main advantage of NMR imaging over optical imaging is the possibility of three-dimensional tomography of deep tissues. Therefore, the dual-functional imaging probes integrating the NMR tomography with fluorescent imaging capabilities may become increasingly important (Marcelo et al. 2020). Compared with CT, MRI uses non-ionizing radiation that is much safer in applications. However, it requires strong magnetic field to polarize the magnetic moments of certain atomic nuclei and also radiofrequency waves to study their resonance behavior.

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Fig. 12.3 Fluorescence imaging vs. magnetic resonance imaging (MRI). Comparison between both imaging techniques showing their complementarity (Lartigue et al. 2020)

In sensing and imaging, the fluorescence and magnetic resonance technologies are really complementary (Lartigue et al. 2020), as it can be seen in Fig. 12.3. Poor penetration into living tissues of visible and near-UV light is counterbalanced by unlimited penetration of magnetic field. However, much higher spatial resolution power, higher sensitivity by many orders of magnitude and innumerable possibilities of functional response makes fluorescence-based technology a good partner. Fluorescence is the best to fit to intermolecular interaction, visualization of organelles, intracellular dynamics, metabolic features on cellular level, and MRI is the most valuable for in vivo tracking. Direct overlapping of two images is possible only in the studies of small animals. However, the valuable information on localization, dynamics and toxicity of MRI agents can be studied by fluorescence. Contrast in MRI images is produced by the relaxation processes of hydrogen protons in different microenvironments. When the hydrogen protons are exposed in an external magnetic field (B0 ), some portion of the nuclei aligns in this direction, and the nuclei rotate with a frequency proportional to the applied field. When a second magnetic field oscillating with a radio frequency is perpendicularly applied to B0 in a resonance condition, the energy is absorbed due to the net magnetic precession to flip on the perpendicular plane. Removal of the resonant field causes the relaxation of the net magnetic moment with the release of the absorbed energy. The relaxation processes can be measured by z-axis and xy-plane in 3-D directions, which are defined by longitudinal T1 relaxation and transverse T2 relaxation, respectively. The images are generated when specially designed radiofrequency pulse sequences are applied from the differences in longitudinal relaxation time (T 1 ) and/or transverse relaxation time (T 2 ). This process requires the presence in a studied system of a specific NMR-active nuclide. In biological and clinical applications, the most common nuclide used in MRI is 1 H due to its ubiquitous existence in water molecules and its high NMR sensitivity

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among all nuclei. With increasing the instrument frequency in recent years, MRI on 19 F nuclei becomes used, due to their low background and relatively high sensitivity in biological samples. Applying MRI with 1 H and 19 F nuclei, one can provide 3-D tomography generating anatomical as well as physiological information depending on the specialized MRI technique used. A drawback of this reagent-free type of MRI application is its low sensitivity and resolution. Depending on the power of the magnetic field, high spatial resolution of 10–100 µm can, in principle, be acquired. However, the limitation exists for using in humans the magnetic field strengths no stronger than 3 T (Tesla units), and common medical inspections proceed on 1.5 T instruments. They provide resolution less than 1 mm. This may be enough for obtaining detailed anatomical information, but due to low sensitivity, the images for molecular information are very limited. In order to improve such low MRI sensitivity, various special imaging contrast agents have been developed. There are two types of these agents (nuclides), one influencing T 1 and the other T 2 relaxation. Among the MRI contrast agents that are able to exert distinctive proton relaxation effects by increasing the T 2 relaxations, the superparamagnetic iron oxide nanoparticles (SPIONs) are frequently used (Duli´nska-Litewka et al. 2019). Their core radius ranges from 5 to 15 nm, and the hydrodynamic radius (core with shell and water coat) is between 20–150 nm Fig. 12.4a). Superparamagnets have the property that unless there is a magnetic field, their magnetization us absent (Lee and Hyeon 2012). They can be used in different technologies, beside multimodal operation (Sect. 12.3). When applied in MRI imaging, they produce darker spots, so these agents are also called negative contrast agents. Iron oxide (Fe3 O4 ) in the form of nanoparticles is the most popular T2 agent. Here the size is very important. Only very small (smaller than ~15 nm) iron oxide nanoparticles demonstrate superparamagnethism (Thorek et al. 2006), because in this case they have the property of a single magnetization domain. In this size range, their induced magnetization is observed on a strong level,

a

b

Fig. 12.4 The properties of fluorescent silica nanoshell-coated iron oxide nanoparticles (Jang et al. 2016). a Representative TEM images. Carbon oxide core is black and thick silica coat is grey. b Magnetization versus applied magnetic fields changing its sign (measured at T = 300 K)

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but only in an external magnetic field. When this field is removed, any magnetization is lost (see Fig. 12.4b). Different NMR contrasting agents were produced for clinical application. Such SPIONs differ mainly by multitude of their surface modifications that allow providing their targeting functions in vivo (Avasthi et al. 2020; Duli´nska-Litewka et al. 2019). In construction of multimodal nanoparticles, one has to account the luminescence quenching effect of iron; therefore the direct contact with fluorescence moiety should be avoided. Using SPIONs, the strategy was developed to modify the particle surface first and then attach the dyes. In one of the recent reports (Yan et al. 2017) the MRI/optical dual-modality probe was constructed by coupling polyethylene glycol (PEG)-modified nano-Fe3 O4 with specific targeting cyclopeptide GX1 and nearinfrared fluorescent dyes Cy5.5. MRI/optical imaging effects of the probe were observed, and the feasibility of application of these nanocomposites for in vivo double-modality imaging was discussed. It was shown, that these nanocomposites are selectively absorbed by gastric cancer cells and provide bimodal imaging of location of cancer tissues. The gadolinium (Gd3+ ) metal ion has its own magnetic moment, which makes it the most suitable for providing strong dipolar interactions with water nuclei protons. Its paramagnetic complexes decrease T 1 , which results in the appearance of brighter areas in obtained images. The Gd ion forms kinetically and thermodynamically stable complexes with different specially designed ligands. These ligands may occupy eight of the nine available coordination sites. The ninth coordination site remains available for fast exchange of coordinated water molecules with those surrounding the complex to transmit the paramagnetic relaxation effect to bulk solvent. Possessing seven unpaired electrons, gadolinium ions can efficiently alter the relaxation time of surrounding water protons and improve in this way the contrast in NMR measurements. They are able to recognize specific target molecules and to induce an enhancement of water proton relaxation rate on interaction with the target. The gadolinium ions are extensively used for 3-D NMR imaging diagnosis in routine clinical practice. Wide ranges of nonspecific Gd3+ complexes are being used in these applications, mainly as contrast agents for evaluation of physiological parameters. However, the introduction of paramagnetic ions directly in vivo is limited by their inherent toxicity. Therefore, acyclic and macrocyclic polyaminocarboxylates are used as strong ligands to sequester the material and prevent toxicity (Mishra et al. 2006). Different constructions combining Gd3+ chelators and molecular or nanoscale fluorophores have been suggested (Yang et al. 2020), as illustrated in Fig. 12.2. Modifying the ligands, high level of targeting and functionalization can be achieved. The Gd3+ complexes with quantum dots have received much attention (McAdams et al. 2017; Pereira et al. 2019). The composites allowed secure implanting of several hundred magnetic centers per quantum dot unit. This allowed proportional increase of NMR signal per nanoparticle. The composites containing these ions can be attached to peptides, proteins they can be incorporated into liposomes in order to facilitate their penetration into the cells, which was monitored by taking fluorescent cell images. However, no coupled tomographic images were demonstrated.

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Fig. 12.5 An example of dual-modality fluorescence/MRI imaging probe DO3A-Gd-CA developed for detection of fluorine ion based on dissociating Gd3+ complex (Wang et al. 2016). This ion is embedded in a mononuclear nine-coordinate complex. Upon the addition of fluoride ions to its aqueous solution, the replacement of the coordination aromatic carboxylic acid (CA) led to an increase in longitudinal relaxivity (r 1 ) and an enhancement in fluorescence intensity, realizing the switch-on dual-modal responses towards fluoride ions

Many different bimodal sensors were obtained by incorporating Gd3+ ions into polymer nanoparticles, including those incorporating organic dyes (Xia et al. 2020). However, the coordination complexes themselves can serve as the units that can be coupled with organic dyes and the coupling-decoupling can be of value for sensing. Figure 12.5 serves as an example of such performance. The organic dye is attached to Gd3+ coordination complex. When the fluorine atoms are present, they substitute the binding site, which allows the dye to dissociate and indicate the presence of F− ion by changing the parameters of its emission. Magnetic resonance imaging in vivo in mice also indicates these changes. This bimodal sensor can be potentially used as a powerful tool for the detection of fluoride ions in live systems. Polymer with incorporated Gd-chelating sites together with naphthalimide-based fluorescence pH sensors as the side groups were suggested for cellular pH probing with some prospect for in vivo studies (Su et al. 2018). Fluorescent gold nanoclusters were not forgotten, and the composites based on their coupling with Gd3+ -containing complexes was described (Liang and Xiao 2017). Core-shell upconversion nanoparticles were also used for designing the dualmode materials. It was shown that these particles were enriched with paramagnetic Mn2+ ions (Lv et al. 2018). This dual-modal switching feature of the optical emission and MRI signals provides a platform for stimuli-responsive biosensing of reactive sulfhydryls and H2 O2 . The sensor was tested for intracellular testing of these compounds. For functioning as upconverting fluorescent and magnetic resonance dual-modality imaging contrast agents, the upconverting nanocrystals were coated with shells formed of paramagnetic lanthanide complexes (Wang et al. 2013). They show excellent enhancement of both fluorescent and NMR signals in experiments in vivo.

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Core: CT or iron oxide based contrast agent Core

Layer of Gd-based contrast agent Layer of organic dyes (or quantum dots) serving for luminescence-based contrast agent 1, 3 – thin polymer shells

5 – surface layer needed for solubility and biological funcƟonalizaƟon

Fig. 12.6 Design of an “ideal” nanocomposite as contrast agent for multimodal imaging. The multilayer structure consists of CT contrasting agent (e.g. gold) or carbon oxide nanocrystal that can be also used in MRT. Next layer can be made of Gd3+ ions for producing the MRT contrast. The other layer should be presented by luminescence (fluorescence) contrast reagents. The intermediate layers of silica or organic polymer are needed to avoid interaction between functional layers. The surface layer serves for solubility, chemical modifications and biological functionalization. It can be used for attaching a targeting group the desired sites for their selective contrast

12.2.4 Nanocomposites Demonstrating Triple-Modal Imaging It is quite possible to develop materials for three-modal and multimodal imaging (Rieffel et al. 2015; Tian et al. 2014). This can be achieved by assembly of multiple components in one structure. Figure 12.5 demonstrates the principle of formation of an “ideal” or “all-in-one” multimodal composite. The realizable procedure is the step-by-step addition of desired structures in a layer-by-layer manner. With proper surface modifications, the conjugation of a large number of targeting groups (e.g., antibodies) is possible. There is a danger however that such airplane will not be able to fly. It will be too expensive and of too large size for practical use (Fig. 12.6). Different authors report on achieving three or more modalities in their designed nanocomposites (Zhao et al. 2018). Meantime, even simple contrast agents themselves can be used for multimodal functioning. Thus, fluorescent nanoclusters of gold and silver (Sect. 6.1), being composed of heavy atoms, can increase the CT contrast. Likewise, upconversion nanoparticles (UCNPs) assembling several types of lanthanide atoms (Sect. 7.5) have to be good CT contrast agents (Hu et al. 2017). It was shown that luminescent NaErF4 @NaLuF4 UCNPs can provide triple response, also in CT and MRT (Li et al. 2018). Core@shell structures of Fe3 O4 @Mn2+ -doped NaYF4 :Yb/Tm nanoparticles demonstrate multiple merits (Luo et al. 2017). The near-IR luminescence is ascribed to the Tm3+ ions, T1 -weighted MRI attributed to the Mn2+ ions, and T2 -weighted MRI ascribed to the Fe3 O4 core of the hydrophilic Fe3 O4 @Mn2+ -doped NaYF4 :Yb/Tm nanoparticles. Incorporation of both Gd3+ and Mn2+ ions increases both T1 and T2 resonance sensitivity. It is known also that along with high atomic numbers, lanthanide atoms possess higher k absorption edge values than iodine, which allows using them for increasing

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the CT instrument sensitivity. Therefore, Gd3+ ions are well suited also for X-ray computer tomography. The Gd3+ ion can be incorporated into luminescent UCNP nanocrystal (Choi et al. 2019). Also, the iron oxide nanoparticles, together with MRT contrasting, can provide the CT contrast. Therefore, adding one more modality to such smart materials becomes easier. The structure of NaLuF4 :Yb,Tm@NaGdF4 (153 Sm) composite suggested as four-modal imaging probe contrasting the tumor (Sun et al. 2013). Whereas it is hard to couple experiments with probes displaying different modalities on an instrument level, one benefit is obviously seen. The sample is injected into animal once and its distribution and accumulation at particular sites in the body should be seen to be the same when studied by different methods. Each imaging modality has its own merits and disadvantages, and a single technique does not possess all the required capabilities for comprehensive imaging. Therefore, multimodal imaging methods are quickly becoming important tools for state-of-the-art biomedical research and clinical diagnostics and therapeutics.

12.3 Magnetic-Fluorescent Nanoparticles with Different Functionalities From discussion above one may infer that it is hard to combine fluorescence-magnetic nanocomposites in a single sensing or imaging experiment. Still, the range of use of these smart materials can be very broad (Fig. 12.7). The fluorescent property of the composite permits fine visualization, whereas the magnetic property enables imaging, magnetic separation, local heat production leading to higher chemical reactivity and destruction of target cells (Mahajan et al. 2013). There are many applications of these materials in imaging, separations, and theranostics (see Volume 2, Chap. 17). As their properties continue to improve, they have the potential to greatly impact biological imaging, diagnostics, and treatment. Different compositions of these nanoscale materials were tested and suggested to community (Cabrera et al. 2017; Lartigue et al. 2020), The superparamagnetic nanocrystals and fluorescent quantum dots are commonly used (Demillo et al. 2015), but there are bright possibilities to use plenty of choices existing from both sides. When magnetic nanocrystals are of very small sizes, 5–15 nm in core radius and 20–50 nm in hydrodynamic radius (Duli´nska-Litewka et al. 2019), they are single-domain superparamagnets. Therefore, they can be easily manipulated by the application of an external magnetic field, and this ‘action at a distance’ provides tremendous advantages for many applications. The range of these applications can be substantially extended by making their various surface modifications (Fig. 12.8). These well-developed procedures change dramatically their properties, making them functional. In biomedical field this allows their use for providing hypherthermia, guided magnetic drug delivery, cell sorting, protein immobilization, in addition of being the contrast agents in MRI.

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1 Fig. 12.7 Illustration of a broad range of possibilities for using combination of luminescence (fluorescence) and magnetism

Hydrodynamic diameter Zeta potenƟal Cellular uptake Toxicity Hydrophobicity

Fig. 12.8 The substances that are the most frequently used as a coating for SPIONs (Duli´nskaLitewka et al. 2019): (1) PEG (Poly(Ethylene Glycol)), (2) PVA (Poly(Vinyl Alcohol)), (3) PVP (Poly(Vinyl Pyrrolidine)), (4) chitosan, and (5) dextran. The figure also sums up the most important parameters that are affected by coating

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Glutaraldehyde

+ ImmunoreacƟon

+ +

EDC NHS

MagneƟc separaƟon

MagneƟc fluorescent composite nanoparƟcles CdTe quantum dots

Magnet

AVAV anƟgen or NDV anƟgen AVAV anƟbody or NDV anƟbody

Fig. 12.9 Schematic diagram of the whole process of the analysis method based on the magnetic fluorescent composite nanoparticles (Wang et al. 2010). The antibodies attached to fluorescence nanoparticles (quantum dots) react with antigens located on the surface of magnetic nanobeads. After immune reaction, the formed complexes are isolated by a simple application of magnet

Fluorescent composites of magnetic nanoparticles is a story that is not limited by only imaging (Lu et al. 2015; Serrano García et al. 2018). Biological functionalization of fluorescence-MRI composites involves protein binding (Cabral Filho et al. 2018; Marcelo et al. 2020), including antigens and correspondent antibodies. This opens the ways to realize new version of immunoassays, which is similar to immunoprecipitation method, but the antigen-antibody complexes formed on the surface of magnetic nanoparticle can be isolated from the reaction mixture by just applying a magnet (Fig. 12.9). One more area of research, development and application is the modification of living cells making them magnetic (Safarik et al. 2016). Such modifications are needed for many purposes, including the tracking of selected and modified cells within the living body and making different manipulations with them. Fluorescence response allows making the introduced magnetic nanoparticles visible within the cell, determine their location and interactions. Many different biotechnological procedures can be developed on this basis. Targeted capture of required (usually malignant) tissues in vivo becomes possible after the attachment of specific ligands. The iron oxide nanoparticles decorated with CdTe quantum dots were then functionalized with anti-epidermal growth factor receptor (EGFR) antibody to specifically target the cells recognizing the correspondent receptor that is expressed on the surface of tumor cells. This composite nanoscale system is shown to perform excellently for specific binding, magnetic separation and fluorescent detection of EGFR positive Molt-4 cells from a mixed population (Kale et al. 2011). Summarizing the story with multifunctional and multimodal nanocomposites, it can be stated that the range of their functional possibilities are much broader and some of the results are much brighter than that described in several chapters of this book. Within supramolecular structures and nanocomposites there opens an immense

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Increased brightness

InducƟon of biocompaƟbility and bioconjugaƟon

OpƟmized emission in wavelength, decay rate and anisotropy

Molecular recogniƟon and bar-coding on singleparƟcle level

Increased chemical stability and photostability

MulƟparametric and mulƟplex sensing

Broad-range modulaƟon of solubility

MulƟfuncƟonality (magneƟc, x-ray dense, NMR-contrasƟng, etc.)

Fluorescent nanocomposite

Fig. 12.10 Illustration of different functionalities that can be achieved in the design of fluorescent nanomaterials from molecular and nanoparticle components

number of possibilities to induce new functionalities and modify them for optimal performance, as illustrated below (Fig. 12.10). On nanoscale, there appears the possibility of obtaining versatile organicinorganic hybrid materials (e.g. up-converting nanocrystals decorated with organic dyes), which can be realized in very smart designs and attain unique features. Such nanostructures are often constructed based on a core-shell principle. This allows combining maximum of functionality with facile production. The core part commonly carries the supporting and reporting functions (typically they are electronically dense or magnetic), whereas the shell is responsible for target recognition and signal transduction together with such necessary properties as solubility or adhesion to particular surface. We observe also that there has been a great deal of enthusiasm in designing and exploiting the different multifunctional and multimodal nanoscale composites. It has injected new vigor into development. Significant achievements comes as an award for overcoming many difficulties.

12.4 Sensing and Thinking. Composites Assembling Several Modalities in Sensing and Imaging. What for? Above, we provided a brief discussion on design and applications of multimodal probes for sensing and imaging. We came to a vision that making such probes is difficult, but possible. More difficult and almost impossible is to combine these modalities on instrumentation level. It is hard to comprehend how to incorporate

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even the smallest and smartest fluorescence detectors into large and complicated CT and MRT devices. How to protect these detectors from damage by X-rays, even if they are not direct but scattered? What happens with the probe at such illumination? We know also about the harmful effect of high-energy electrons generated at this illumination (Sect. 14.3). The problems with combining in one measurement luminescence and MRT are no less essential. Strong magnetic fields may influence dramatically the behavior of such nanocomposites. Consistent analysis of the data may also be difficult, since these methods are based on different conceptual and computational basis. Thus, trying to combine these methods in a single multimodal experiment with a necessity to provide simultaneous analysis using different input and output channels, we return to the question posed at the beginning of this section. What is the need for that? Thinking about this question, an interesting analogy with amphibian car (Boerman and Oyen 2008) can be recollected. These vehicles can move on land and on water. By putting both features in the same vehicle, one can get something that is not as fast as a car on the road or a speedboat on the water, but somewhat acceptable being able to both drive on roads and float. Can we evaluate in the same way the attempts to implement multimodality measurements? In order to be convincing on this route, we have to address and resolve one of fundamental questions that cannot be resolved the other way. Respond to the following questions. 1. In what sense the methods of X-ray tomography, NMR tomography and luminescence imaging are complementary? Compare the parameters that are the most important for practical use. 2. Compare the terms multifunctionality and multimodality and the range of their use regarding sensing and contrasting materials. 3. What problems do you see in combining the three analyzed methods for particular tasks? Are these problems mostly in addressing proper tasks or in design of instrumentation? 4. What probes are required for improving contrast in X-ray computer tomography? How do they correlate with probes used in fluorescence imaging? 5. Explain the difference. The X-ray tomography and MRT are actively used without contrast agents, and these agents are used for improving the contrast, whereas fluorescence imaging heavily depends on the introduction of probes. 6. In several papers cited above, the cellular behavior (cell entry, distribution, toxicity) of bimodal nanocomposites was studied on cultural cell level and then they were used for in vivo tomography in CT or MRT studies. Explain the value of these experiments. 7. List and analyze the properties and applications of fluorescent-magnetic nanocomposites beside the imaging technologies.

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Wang G, Xie P, Xiao C, Yuan P, Su X (2010) Magnetic fluorescent composite nanoparticles for the fluoroimmunoassays of newcastle disease virus and avian virus arthritis virus. J Fluoresc 20:499–506 Wang Y, Ji L, Zhang B, Yin P, Qiu Y et al (2013) Upconverting rare-earth nanoparticles with a paramagnetic lanthanide complex shell for upconversion fluorescent and magnetic resonance dual-modality imaging. Nanotechnology 24:175101 Wang D, Guo Y, Liu W, Qin W (2014) Preparation and photoluminescent properties of magnetic Ni@SiO2 –CDs fluorescent nanocomposites. RSC Adv 4:7435–7439 Wang Y, Song R, Feng H, Guo K, Meng Q et al (2016) Visualization of fluoride ions in vivo using a gadolinium (III)-coumarin complex-based fluorescence/MRI dual-modal probe. Sensors 16:2165 Xia B, Yan X, Fang W-W, Chen S, Jiang Z et al (2020) Activatable cell-penetrating peptide conjugated polymeric nanoparticles with Gd-chelation and aggregation-induced emission for bimodal mr and fluorescence imaging of tumors. ACS Appl Bio Mater 3:1394–1405 Xing H, Bu W, Zhang S, Zheng X, Li M et al (2012) Multifunctional nanoprobes for upconversion fluorescence, MR and CT trimodal imaging. Biomaterials 33:1079–1089 Yan X, Song X, Wang Z (2017) Construction of specific magnetic resonance imaging/optical dualmodality molecular probe used for imaging angiogenesis of gastric cancer. Artif Cells, Nanomed Biotechnol 45:399–403 Yang C-T, Hattiholi A, Selvan ST, Yan SX, Fang W-W, et al (2020) Bimodality probes of Gd enhanced T1 -weighted magnetic resonance/optical imaging. Acta Biomaterialia 110:15–36 Yu M-X, Ma J-J, Wang J-M, Cai W-G, Zhang Z, et al. (2020) Ag2 Te quantum dots as contrast agents for near-infrared fluorescence and computed tomography imaging. ACS Appl Nano Mater 3:6071–6077 Zhao J, Chen J, Ma S, Liu Q, Huang L et al (2018) Recent developments in multimodality fluorescence imaging probes. Acta Pharmaceutica Sinica B 8:320–338

Chapter 13

Evanescent Field Effects and Plasmonic Enhancement of Luminescence in Sensing Technologies

An important and highly attractive property of light is the ability to provide optical excitation at distances shorter than its wavelength that can be realized at solid-liquid interface due to evanescent field generation. This allows separating by optical means the species bound to the surface and those that are free in solutions. By locating a thin layer of silver or gold at this interface, the surface plasmon resonance conditions can be achieved, which results in enhancement of emission of closely located fluorophores. If this layer is composed of plasmonic noble metal nanoparticles, this enhancement becomes much stronger. In solutions, the aggregation of plasmonic nanoparticles can be observed as the change of color even with naked eye. In all these conditions, the sensor-target formation can be accelerated by microwave radiation. By exploring such unique possibilities, one can achieve avoiding the amplification steps on the target/indicator level in sensing and imaging technologies. In the development of sensing and imaging techniques, the researcher is looking for possibilities to decrease the detection volume, to selectively excite the species within this volume and in these conditions, instead of losing, to gain the fluorescence intensity. This possibility exists due to the property of light to generate evanescent field at an interface in a layer that is thinner than the wavelength of light. The electromagnetic wave propagates into liquid phase decaying exponentially with the distance. This allows selectively excite those fluorophores that are immobilized at the surface (possibly, due to target-sensor interaction) leaving in the dark those that remain in the solution volume. Such spatial selectivity allows achieving the multiplexing in sensing and, particularly, in imaging. When a very thin (~50 nm) layer of silver or gold is deposited at this interface, a new phenomenon appears. The propagating plasmons in this layer produce the conditions of surface plasmon resonance, which may greatly, by several orders of magnitude, increase the emission intensity of fluorophores located within close proximity to a metal surface. This effect becomes even greater if this layer is composed of noble metal nanoparticles, due to the effect of localized plasmons. In addition, plasmon-plasmon interactions can report on association of gold nanoparticles by observing the change of color even with naked eye. All these observations allow © Springer Nature Switzerland AG 2020 A. P. Demchenko, Introduction to Fluorescence Sensing, https://doi.org/10.1007/978-3-030-60155-3_13

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Fig. 13.1 The local field effects on fluorescent dyes generated at interfaces that can influence dramatically on their spectroscopic properties. In the conditions of total internal reflection, an evanescent wave excites only those dyes that are located close to the surface. If a very thin gold or silver film is located at the interface, the generated propagating plasmons provide a strong emission enhancement. When the noble metal nanoparticles are located at the interface, the localized plasmons generate much stronger effects. Additional application of microwave radiation strongly accelerates the formation of informative complex with the target

deeper understanding of the phenomenon of plasmon-enhanced fluorescence and identify its two contributions. One is the increase of efficient light-absorption crosssection in strong local electromagnetic field, and the other—an increase of emission rate. The dramatic enhancement of fluorophore brightness is not the problem in itself. It is required for achieving the new steps in technology development. The amplification steps in DNA analysis and the enzyme amplification in ELISA have to be replaced by fast, cheap and efficient methods that must be able to detect several target molecules. The speed of formation of target-sensor complexes is critical here. Microwave treatment offers the solution for its acceleration for reaching these complexes in the conditions of equilibrium. Together with achievements in sensor design discussed in other parts of this book, a great progress is expected in the future, which is depicted in Fig. 13.1.

13.1 Evanescent-Wave Fluorescence Sensing When light is reflected at the interface between the optically highly dense medium and the low-dense medium, there is a finite decaying electric field across the interface that penetrates for some distance into the optically low-dense medium. This field is referred to as the evanescent field. Propagating in the media, from which it is totally reflected, such evanescent field behaves as the true light intensity rapidly decaying with distance on a scale of 100–200 nm. It can excite fluorophores located within this layer. Such excitation is explored to achieve spatial selection in optical excitation,

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since it allows exciting those species that are located close to interface between the two media (such as water and glass). The power of this approach is in its ability to be combined with different sensor technologies offering their improvement and, often, their dramatic transformation.

13.1.1 Excitation of Dyes by Evanescent Field The phenomenon of refraction of light at an interface between the media of different refractive indices is known from textbooks. The refraction strongly depends on the angle of incident beam, so with the increase of this angle there appears the condition when the light is totally reflected (Fig. 13.2a). This occurs when the angle, at which the light propagating in a medium of higher refractive index n2 encounters a medium of lower refractive index n1 , is higher than a certain critical angle θc . The latter is determined by equation θc = sin−1 (n1 /n2 ). It is interesting to note that the so attractive sparkling properties of finely processed diamonds are due exactly to this phenomenon, originating from extremely high optical density of diamond. However, the total internal reflection is not truly total and the geometrical principle is not totally valid. A small amount of electromagnetic field propagating with the frequency of light in the medium with n2 still penetrates at a short distance into the medium of lower n1 in the form of standing wave known as evanescent wave (Sapsford 2010; Taitt et al. 2005). This evanescent wave is identical in frequency to the incident light and generates electric field that sharply decays with distance from the interface (Fig. 13.2b). Evanescent wave decays exponentially as a function of distance. In practically realizable situations, it can propagate to a distance of 100–200 nm, which depends on refractive indices, incidence angle and the wavelength. Its energy is sufficient to excite fluorescence of species located within the range of action of this field.

a

b

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EVANESCENT FIELD y

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Fig. 13.2 The total internal reflection and generation of evanescent field. a Transmission and reflection of light entering from a medium with higher refractive index n2 to a medium with lower refractive index n1 . The rays incident at angles higher than a critical angle θc = sin−1 (n1 /n2 ) are totally reflected. b The rays internally reflected into the medium with n2 produce the evanescent field in the medium with n1 . This wave decays exponentially as a function of distance from the surface on a nanometer scale

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When special materials and special configuration of excitation beam were developed, this phenomenon found important application in sensing. The technique used for that is called the evanescent field excitation (by the physical principle) and total internal reflection spectroscopy or microscopy (by the method of its realization). This principle can be realized on optical waveguides (see Sect. 15.3) and in total internal reflection fluorescence (TIRF) microscopy (Volume 2, Chap. 15). The basic mode of operation of evanescent-wave sensors is easy to understand. The light propagates along optical waveguide without leaving it, exhibiting total internal reflection from its walls. The sensors (that may be composed of receptor units only) are attached to the waveguide surface and extend to the solvent. The difference between refractive indices of solvent water medium (n1 = 1.33 − 1.37) and fused silica from which the optical waveguides are usually made (n2 = 1.46) is quite significant for providing this optical trapping. The labeled targets that are bound to the sensor can be detected by their fluorescence emission. If the labeled species are not bound and are not under the influence of evanescent field, they are not excited and remain in the dark. Since only the bound species demonstrate the fluorescence response, being excited “from outside” and those remaining in solution do not respond, for providing or modulating the reporting signal there appears the possibility to avoid washing-out the unbound species. Being at a sufficient distance they remain in the dark. With the exploration of this effect, many different sensor configurations can be realized. For instance, the labeled species can be not necessarily the target but the competitor. Another possibility is to make fluorescent the sensors attached to waveguides, and the target or its competitor in this case can be the quencher. Moreover, under specific conditions of illumination, this effect can be used in microscopy. In all these cases, only the area of studied object that is attached to a glass support will be selectively illuminated and produce the response.

13.1.2 Evanescent Wave Sensing Technology Evanescent wave sensing technology cannot be considered separately from other developments in fluorescence sensing. Moreover, it can be used as a platform for adaptation of different already developed approaches and allows their substantial improvement (Ekgasit et al. 2004). Since all these sensors can measure surfacespecific binding events by collecting the emission selectively from the surface, many heterogeneous assays can be transformed into direct sensors. Versatility of this technology is seen from its application using both fiber optic and planar array fluorescence sensors (Sect. 15.3). The most extensive application this technology has found in immunoassays. Evanescent wave biosensors utilizing antibody-based sandwich fluorescence assays

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Fig. 13.3 Principle schematic of evanescent wave planar optical waveguide chip (Xiao-Hong et al. 2014). Evanescent field penetrates into the test liquid and is able to excite fluorophores that are located close to the waveguide surface providing the necessary selectivity

have been described for detection of gram-negative and gram-positive bacteria, viruses, and different proteins including toxins. In fact, any compounds, to which immunoassays are applicable, can be determined by this method. Competitive and displacement assays can be established for the detection of small molecular weight compounds, such as hormones and neurotransmitters. The reader can find many references in reviews addressing this field (Taitt et al. 2005, 2016). The recent developments include miniaturization of this technique, transforming it into format of re-usable chips (Xiao-Hong et al. 2014), see Fig. 13.3. One of the most interesting application areas of this approach is the hybridizationbased detection of target DNA/RNA molecules. It was shown that molecular beacon technique (Sect. 4.4.4) is quite applicable also in this case (Liu and Tan 1999). Biotinylated molecular beacons were designed and immobilized on an optical fiber core surface via biotin-avidin or biotin-streptavidin interactions. They can be used to directly detect, in real-time, the target DNA/RNA molecules without using competitive assays. The sensor demonstrates rapid, stable, highly selective, and reproducible performance. Spatial resolution offered by this technology is extremely attractive for the assays, in which the binding of labeled target or competitor can be measured with the excitation confined to narrow pre-surface layer. The other application is in cellular research with the development of specialized evanescent field microscope. In general, evanescent-field sensing can be considered as universal platform that may encompass different sensing technologies targeted to many analytes and provide very sharp images with spatially selective excitation. To conclude, the excitation via evanescent field effect is a powerful tool to introduce spatial resolution into the sensor system; it can be combined with different sensing technologies. Being introduced into heterogeneous assays, it allows providing a direct response to the target binding.

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13.2 Plasmonic Effects in Fluorescence A strong modulation of fluorescence emission intensity is observed when fluorescent molecules or nanoparticles appear close to noble metal nanoparticles (usually of gold and silver), even without their direct interaction. Such effects are also observed in the case of flat thin metal surfaces that are commonly used in surface plasmon resonance (SPR) technique. But if this surface is composed of nanoparticles, the effects are much stronger and the fluorophore emission attains new features (Merlen et al. 2010). The resonance conditions leading to plasmonic enhancement can be observed without increase and even with some decrease of emission lifetime, indicating the suppression of nonradiative channel of excited-state decay. Presently, the plasmonic effects have become a hot topic both in research and in sensor development, promising many new possibilities (Bauch et al. 2014; Emam et al. 2020; Li et al. 2015a). They extend into methods that provide an enhanced sensitivity and a wider dynamic range for chemical analysis, medical diagnostics, and in vivo molecular imaging.

13.2.1 General Character of Plasmonic Effects Plasmonic effects are described in many publications (Darvill et al. 2013; Stewart et al. 2008) and the books focused on this topic (Geddes 2010; Novotny and Hecht 2012). In optimal conditions, the increase in fluorescence emission intensity reaches very significant values, up to two-three orders of magnitude (Aslan et al. 2005). Even much stronger effects were obtained with gold nanoparticles of special design (Kinkhabwala et al. 2009). It is interesting to see that with weak fluorescence emitters the effect is much stronger, so that they can be converted into good ones (Aslan et al. 2005; Lakowicz 2005). Important also is the generality of this phenomenon. The emission enhancement is observed not only for organic dyes, but also for semiconductor quantum dots (Gryczynski et al. 2005; Lee et al. 2009), conjugated polymers (Park et al. 2007), lanthanide-chelating luminophores (Zhang et al. 2006b), etc. Up to five orders enhancement was reported for the two-photon excited dyes (Wenseleers et al. 2002). This effect is characteristic for not only fluorescence but also for phosphorescence (Zhang et al. 2006a), chemiluminescence (Aslan and Geddes 2009) and electrochemiluminescence (Feng et al. 2019), i.e. for different possibilities to achieve the electronic excited states without optical excitation, as discussed in Chap. 14. This indicates that the surface plasmons can be directly excited from chemically induced electronic excited states and excludes the possibility that they are created by incident excitation light. Different other photophysical processes and photochemical transformations are dramatically influenced by plasmonic effects. Thus, if the FRET donor-acceptor system is composed of organic dyes adsorbed on silver or gold nano-island films, this reaction is strongly accelerated (Giorgetti et al. 2009). Moreover, different chemical

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reactions can be accelerated in the field of plasmons (Xiao et al. 2013), which opened the area of plasmon-enhanced photocatalysis (Linic et al. 2015; Wang and Astruc 2014). In all these cases the major effect is attributed to the dramatically enhanced absorbance together with the increase of emission rate. Such increase of dye absorbance increases greatly the efficiency of two-photon microscopy (Gryczynski et al. 2002) and suggests the application for excitation of two-photonic dyes in sensing with the use of less expensive pulsed diodes. The fact that phosphorescence emission can also be enhanced on interaction with the metal (Zhang et al. 2007), and this effect is clearly observed with metal nanoparticles (Zhang et al. 2006b), suggests the use of plasmonic enhancement for triplet-states dependent generation of reactive oxygen species for phototherapy of cancer (Zhang et al. 2007). On the other side, the shorter lifetime of the metal-enhanced fluorescence results in less time for the excited state photoreactions, and thus more excitationemission cycles can occur prior to photobleaching. Thus, the increased photostability of fluorescent dyes is an important additional benefit.

13.2.2 Fluorescence Enhancement in Surface Plasmon Resonance Conditions The excitation of fluorescence emission by evanescent wave was discussed above (Sect. 13.1). This wave appears at an interface between the media of different refractive indices and extends into the medium with lower refractive index when the incident light beam is directed at angle higher than critical, in the conditions of all reflected light. It is known also that the evanescent field is augmented dramatically when a thin (about 50 nm) layer of noble metal (gold or silver) is deposited on the solid glass surface (Neumann et al. 2002). Such enhancement appears due to the presence of plasmons, which are the propagating in two dimensions oscillations of charge on the surface of thin metal films. Interacting with photons, they become propagating plasmons-polaritons (Sarkar et al. 2017). This effect depends on the refraction index in contacting media and is sensed at a rather long length (up to ~200 nm, a fraction of wavelength of light) decaying exponentially with the distance. The resonance conditions are observed only at a certain angle of incident light, and this angle changes with refraction index. The target binding to surface changes the refraction index, changing this angle. Detection of this change is used in surface plasmon resonance (SPR) sensing technique (Homola 2008). The enhancement of evanescent field occurs due to resonant coupling between the incident radiation and the surface plasmon wave, and this increase influences the photophysical and light-emissive properties. The well-known resonant enhancement of Raman scattering bands is also related to this phenomenon (Bell et al. 2020). With the knowledge of these findings, the reader must come with the idea of combining the benefits offered by surface-selective evanescent-wave excitation with the possibility of plasmonic enhancement of this emission. Technical realization of

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ReflecƟon Cone angle Fig. 13.4 Behavior of dyes located in the test medium of dielectric function εt (commonly, water) excited near thin metal surface of εm deposited on prism of εp . The excitation is provided through the prism at optimal angle. a The dye is located very close to the surface. The dominant decay channel is the energy transfer to metal, resulting in quenching. b The distance to metal is large. The dye produces free-space emission of photons. c The dye location is optimal. A strongly enhanced highly directional fluorescence emission is observed. It propagates through the prism in a cone of angles. d Graph showing the dependence of fluorescence intensity on distance from the metal layer

this possibility has led to establishment of surface plasmon field-enhanced fluorescence spectroscopy (SPEFS). This method exploits the strong SPR-generated evanescent field for selective excitation of surface-confined fluorescent dye (Ekgasit et al. 2004). The resonant excitation of an evanescent surface plasmon mode is used here to excite the dyes that can be chemically attached to the target or sensor molecules in different assay formats. When the reporting dyes are located close to the metal/solution interface, they become strong emitters. In this narrow pre-surface area, the dye is exposed to the strong optical fields giving rise to dramatically enhanced emission (Fig. 13.4). The distance dependence here is very important, since if the dye appears at very short distances to the surface, its emission will be quenched due to the energy transfer to metal plasmons. The enhanced local field decays steeply as the exponential function of distance, so if the distance is very large, the dye spontaneous emission is unaffected by the field. There is an optimal distance at which fluorescence is strongly enhanced, propagating within a cone of angles within the prism. If the labeled target is located within this area, it can be detected by an increased number of emitted quanta. An interesting property of surface-plasmon fluorescence spectroscopy is its ability to generate the angle-resolved emission (Ming et al. 2012). It was shown (Gryczynski et al. 2005), that upon excitation of quantum dots (QDs), the surface plasmons emitted a hollow cone of radiation (due to symmetry conditions) into an attached hemispherical glass prism located at a narrow angle. This directional radiation preserves the spectral properties of QD emission and is highly polarized irrespective of the excitation polarization. The development of these new techniques in combination with the

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use of highly photostable quantum dots addresses the issue of bioassay sensitivity and offers substantial improvement.

13.2.3 Modulation of Light Emission by Plasmonic Nanoparticles If instead of solid layer, the dye interacts with noble metal nanoparticles, additional important factors can modulate the emission in this system. This is the case of localized plasmons. Particularly, the scattering of incident radiation on nanoparticles and plasmon confinement effect induce new unusual features to emitted radiation. These effects are described in many publications (Emam et al. 2020; Li et al. 2015a). Figure 13.5 illustrates the fact that strong plasmonic enhancement can be obtained only with the use of small metal nanoparticles possessing high density of conduction electrons. The very small clusters of several atoms cannot produce this effect. They demonstrate, instead of plasmon effect, their own emission (Sect. 6.1). The larger particles are also different. They reveal the metallic large-scale electron conductance. In contrast to these cases, in small nanoparticles the oscillation of conduction electrons under the influence of electromagnetic field is restricted in space. This is important for generating the surface plasmon oscillations (Eustis and El-Sayed 2006). It turned out that the most efficient are the effects generated by nanoparticles smaller than the wavelength of incident light (Li et al. 2015a; Novotny and Hecht

Noble metals Fluorescent

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Fig. 13.5 The size-dependent optical properties of noble metal materials. In the particles of nanometer size, the oscillations of conduction electron density in the electromagnetic field of the incident light generate localized plasmons producing strong local electromagnetic field effects that can modulate optical properties of nearby molecules and nanoparticles

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2012). In this case, the collective oscillations of electrons occur in phase in subwavelength volume, and very high values of local electromagnetic field intensity are created around the metal nanostructures. This results in large absorption and scattering cross-section, as well as in amplified emission of fluorophores influenced by this field. This condition is called localized surface plasmon resonance (LSPR). Much effort has been provided to establish the optimum distance between the dye and the surface of metal particle for the maximal amplification effect. The fluorescence from a molecule directly adsorbed onto the surface of a metallic nanoparticle is strongly quenched (as will be discussed below). Meantime, at a distance of few nanometers from the nanoparticles it can be strongly enhanced. However, if the distance is too large, the enhancement effect has again to vanish, as well as for thin metal films (see also Fig. 13.4). The experiments focused on such distance optimization were performed with the metal particles covered with inert material of variable thickness with the dye adsorbed or covalently attached to its surface. Silica shells (Tovmachenko et al. 2006) and inert stearic acid spacer layers (Ray et al. 2007) were used to evaluate this distance dependence. The results presented in different publications exhibit rather broad variation in estimation of such optimal distance, from 5–10 to more than 20 nm. It is probably because this distance depends on orientation and spectral fitting of the dye and plasmon particle, the plasmon system, the excitation and emitted wavelengths. Elegant experiments were provided on a single molecular level (Anger et al. 2006). A gold nanosphere was attached to the end of a pointed optical fiber and scanned over a single fluorophore (Nile Blue) molecule dispersed in poly(methylmethacrylate) on a glass slide. Variation of fluorescence Intensity was recorded as a function of the spacing between the Au nanosphere and a single dye molecule. The maximum of enhancement effect was found to be on the level of ~8 nm. The intensity enhancement on interaction with localized surface plasmons depends also on the wavelength of incident light. The single-molecular studies using individual gold and silver particles excited at different laser frequencies have shown that the maximal enhancement should be observed when the emission frequency is red-shifted from the surface plasmon resonance band maximum of the particle (Bharadwaj and Novotny 2007). The plasmonic properties depend strongly on the type of material (gold or silver) and the shape of particles. For silver spherical particles, the resonance conditions are in blue-green and for gold particles in orange-red region (Fig. 13.6). If the particles are not spherical, their resonance regions are much broader extending to red and near-IR range of spectrum. In particles with sharp edges the local fields are strongly enhanced at these edges (Höppener and Novotny 2012). The basic effect of enhancement of emission intensity by neighboring metal material is very different from that commonly observed in fluorescence, where the freespace quantum yield, Φ o , and lifetime, τ0 , are observed to change in the same direction (Karolin and Geddes 2012). As described in Chap. 3, the following equations relate Φ o and τ0 to radiative and non-radiative rate constants: Φ0 = kr /(kr + Σknr ) = 1/[1 + (Σknr /kr )]

(13.1)

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Fig. 13.6 Spectral dependence of the plasmon resonance of differently shaped nanoparticles. Note that for Ag and Au nanospheres the resonance ranges are different and that for Au nanostructures of different shape the range of resonance energies is broader and mostly shifted to the red and near-IR region

τ0 = 1/(kr + Σknr )

(13.2)

Thus, in a common free-space case the ratio of radiative (k r ) and the sum of all non-radiative (k nr ) rate constants of excited-state decay change in such a way that for being highly fluorescent the dye should emit much faster than all other deactivation processes (with the rates k nr ) occur. In contrast, the plasmonic enhancement modulates the intrinsic decay rates. It was found, that in proximity to metal structures the changes of Φ and τ (marked with index m) are quite different, and the correspondent equation for Φ m can be written as: Φm = (kr + km )/(kr + km + Σknr )

(13.3)

Here k r is the unmodified radiative decay rate, k m is the metal-modified radiative decay rate, and k nr are, as above, the nonradiative rates combining all dynamic quenching effects. Similarly, the fluorophore metal-modified lifetime, τm , is decreased by an increased radiative decay rate: τm = 1/(kr + km + Σknr )

(13.4)

The unusual predictions follow from the theory of plasmon enhancement. It can be seen that as the value of k m increases, the quantum yield, Φ m also increases, instead of commonly expected decrease. Nevertheless, this increase results in decrease of

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emission lifetime, τm . These predictions were confirmed in experiment (Aslan et al. 2005). It also follows that the quantum yield Φ can be increased for molecules having a low value of the quantum yield Φ o , and the interaction with plasmons can transform bad emitters into good emitters. Examples of such transformation are numerous and demonstrate the generality of this effect. In plasmonic systems, it is often very difficult to separate the effects of enhancement and quenching, but the general rules for obtaining enhancement can be formulated. (a) The fluorophores should optimally fit by their absorption and emission spectra to the wavelength range of resonance scattering of plasmon nanoparticles. (b) They should be located at an optimal distance and in optimal orientation to nanoparticle of their dipoles. (c) The plasmon particle should be of such size and shape that it should be more scatterer than the absorber. (d) Its size, shape and interactions with other plasmon particles should better fit to resonance conditions. Hence nanostars, nanorods, and nanoshells and their associates generally have increased enhancement effect compared to nanospheres (Ming et al. 2012). Thus, the plasmons transform incident light into very strong electromagnetic fields within the sub-wavelength regions (near-fields). The high near-field intensities affect the electronic dynamics of fluorophore molecule, resulting in modifications in the decay rates and quantum yields.

13.2.4 Exciton-Plasmon Interactions Combination of semiconductor quantum dots and π-conjugated organic materials with noble metal nanoparticles represents an interesting case of exciton-plasmon interactions that go beyond plasmon enhancement that was discussed in the previous section. Despite many publications in this field, called ‘plasmonics’, the basic mechanisms that govern the response of excitons in QDs to the presence of metal nanoparticles remain to be established. In addition to plasmonic enhancement of emission, there are other mechanisms influencing the absorption and emission of different types of fluorophores. Particularly these are the photoinduced electron and energy transfers that are favored by small sizes of plasmonic particles and short distances to fluorescence emitters. Both effects lead to efficient fluorescence quenching. Interestingly, the metal nanoparticle surface can act as both donor and acceptor of electrons. Of particular interest are the interactions of metal nanoparticles and semiconductor QDs, for which both plasmonic enhancement (Gryczynski et al. 2005, Lee et al. 2009) and efficient quenching (Pons et al. 2007) are observed. Regarding the energy transfer quenching, it extends beyond the classical Förster range (Yun et al. 2005), showing invalidity of applying to the metal nanoparticles of point dipole approximation. The description by other parameters becomes needed, such as Fermi frequency and wave vector of the metal. Using the DNA template, the plasmonic and excitonic nanoparticles can be assembled into one unit. In such nanocomposites the distance between Au particles and QD,

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the number of Au atoms around the central QD, and the size of Au and QD components can be adjusted (Fu et al. 2004). Different types of binary superlattices of CdSe QDs and gold nanoparticles can be obtained by self-assembling (Shevchenko et al. 2008). There are experiments that show great potential for exploration of plasmonexciton interactions in sensing technologies. In semiconductor nanowires the exciton lifetime determines the emission wavelength because on exciton diffusion along the nanowire it becomes trapped in the regions of lower band gap, so its emission is shifted to the red. Accordingly, because of shortening the exciton lifetime the energy transfer quenching by nearby metal nanoparticle will be observed as the shift of nanowire emission to the blue (Lee et al. 2007). This suggests a new method to study intermolecular interactions. Coupling of molecular resonances with plasmonic resonances has been actively studied with organic dyes adsorbed on the surface of metal nanoparticles (Ni et al. 2010). Such systems exhibit plasmonic resonance shifts that can be modulated by changing the dye properties (isomerizations, pH sensitivity, photochromism etc.) (Ming et al. 2010). This opens many new routes for designing plasmonic nanocomposites with desired photonic properties. Of particular interest are the systems based on plasmon interactions with excitons generated in dye J-aggregates (Zheng et al. 2010) that are known to generate very sharp bands in electronic spectra (Bricks et al. 2017). Highly mobile excitons are also present in conjugated polymers that can be viewed as strongly coupled multi-chromophore systems (see Sect. 8.5). Their emission can be strongly enhanced on interaction with plasmonic nanoparticles, and different hybrids were suggested that display this effect (Tang et al. 2011). It has found application in DNA sensing (Geng et al. 2011). Assembling into nanocomposite of molecules and nanoparticles with different mechanisms of light absorption and emission is expected to open new pathways to create functional materials with optical and electronic properties not observed in any of the source materials. Accordingly, QDs and conjugated polymers generate different types of excitons (Wannier-Mott excitons and Frenkel excitons, correspondingly) and the properties of their hybrids deserve close attention. This area is open to active research and soon we expect many interesting results and important applications.

13.3 Light Absorption and Scattering by Plasmonic Nanoparticles The plasmonic modification of fluorescence intensity combines two contributing mechanisms. One of them is from the absorption side, and it occurs due to strongly enhanced local (on the scale of nanometers) electromagnetic field augmenting the probability of fluorophore excitation. In order to better understand the origin of this

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component of plasmonic enhancement one can use the term ‘optical extinction cross section’, which is the sum of the absorption and scattering cross sections for photons. This term is frequently used by physicists for describing the interaction strength between optical species and light. The strong color that can be detected visually on illumination of nanoparticles is due to a combination of two optical phenomena: light absorption and scattering (Yguerabide and Yguerabide 1998). Due to this fact, the correct term could be the ‘extinction’ instead of ‘absorption’, since it describes both absorption and scattering components to the observed colors. For better understanding the phenomenon of plasmonic enhancement, it can be imagined that a special hypothetical area surrounding a plasmonic particle exists, and this much larger area than its geometrical dimension absorbs and scatters light with dramatically higher probability. Whereas organic fluorophores and semiconductor QDs possess quite similar ratios of extinction cross sections versus their physical cross sections (which means that their extinction cross sections do not go beyond their actual sizes), for plasmonic nanocrystals this ratio is larger by 2–3 orders of magnitude (Ming et al. 2012). This means that very strong electromagnetic field is created around plasmonic particle on its interaction with light, exceeding substantially its size. For fluorophore located within this area the effect will be similar to that occurring on a manifold increase of incident light intensity. Therefore plasmonic nanoparticles are often called nanoantennas.

13.3.1 Going Deeper into the Properties of Localized Plasmons The plasmonic nanoparticles (especially of silver, gold and copper) are very strong light absorbers and scatterers. Under resonance conditions, surface plasmons in metal nanoparticles lead to light scattering that can be several orders of magnitude more intense than the fluorescence from efficient quantum emitters (Jain et al. 2006). The light scattering was found to be angle-dependent. The silver particles are several-fold better scatterers than gold (Yguerabide and Yguerabide 1998). Subsequently, light scattering by silver and gold nanoparticles can be detected at concentrations as low as 10−16 M. The scattered light by metallic nanoparticles is the highest at observation angles of 0 and 1800 with respect to the incident light, i.e. in the backward and forward directions. The angular dependence of the scattered light depends on the size, shape and composition of the nanoparticles. It is essential that enhanced fluorescence emission reproduces exactly this angular dependence (Aslan et al. 2007b). Consider the plasmonic particles demonstrating localized surface plasmon resonance (LSPR) that are smaller than the wavelength of light. Then, their electrons oscillate in phase (Li et al. 2015a, b). The relative contributions of these absorption and scattering effects is size-dependent and dramatically stronger than that of organic dyes or nanoparticles made of them (Fig. 13.7). The collective oscillations lead to

13.3 Light Absorption and Scattering …

a

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b

c Absorber ScaƩerer

R < 15 nm

R > 15 nm

Fig. 13.7 Light absorption and scattering. a Common organic molecules or nanoparticles. Absorption cross-section corresponds to geometric size and light scattering is very low. b Plasmonic nanoparticle of small size (15 nm) size but smaller than the incident light wavelength. The absorbance is also large, but much larger becomes the light scattering contribution. Resonance wavelengths are marked by vertical dashed lines

large absorption and scattering cross-sections, as well as to amplified local electromagnetic fields, but to a different extent. For smallest particles, less than ~15 nm, the effect of light absorption dominates over scattering. In contrast, for greater nanoparticles the scattering cross-section becomes much stronger, especially at wavelengths longer than at the resonance band. Since the optical cross section of the nanoparticle at the resonance energies is strongly enhanced relative to its off-resonance value, the light scattering, being increased with nanoparticle size, should also depend upon the energy of plasmon excitation. It is maximal at resonance wavelength.

13.3.1.1

Association and Aggregation of Plasmonic Nanoparticles

Variation of size and dimensionality of different types of plasmonic nanoparticles results in different spectroscopic effects, including the change of their color that can be detected by naked eye. It can detect their association due to plasmon-plasmon interactions between adjacent particles (Jans and Huo 2012; Saha et al. 2012; Valentini and Pompa 2013). The change in their aggregation that can be coupled with the target binding with the easily detectable change in optical properties of gold and

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Blue

Red

Target

Fig. 13.8 The change of visually detected reflected/scattered color of gold nanoparticles on their association caused by their binding polyvalent target

silver nanoparticles (Zhang et al. 2018). The characteristic visually detected red color of gold colloid changes to bluish-purple color upon colloid aggregation (Fig. 13.8). It was stated (Yguerabide and Yguerabide 2001) that the advantages of gold particles as detection labels are that (a) their light producing power is equivalent to more than 500,000 fluorescein molecules, (b) they can be detected by eye and a simple illuminator at concentrations as low as 10−15 M in suspension, (c) they do not photobleach, (d) individual particles can be seen in a simple student microscope with dark field illumination, (e) color of scattered light can be changed by changing particle size or composition for multicolor multiplexing, and (f) they can be conjugated with antibodies, DNA probes, ligands, and protein receptors for specific analyte detection. Due to these properties, this detection method has found application in polynucleotide analysis (Elghanian et al. 1997). On introduction into the test system of target polynucleotide, the gold nanoparticles labeled with nucleotides possessing attached complementary sequences start to associate. This induces a dramatic change of visually detected color (Yguerabide and Yguerabide 2001). Unlike in molecular fluorescence, the light-scattering signal from metal nanoparticles is quenchingresistant. Because of extremely strong light absorption of gold nanoparticles, the straightforward application of this colorimetric method in DNA assays allows achieving a detection limit of ~10 femtomoles (10−15 M) of oligonucleotide. This is already 50 times more sensitive than the sandwich hybridization detection methods based on fluorescence. In order to further increase this sensitivity, the intensity and color of scattered light was suggested to be recorded (Storhoff et al. 2004). This scatter-based method is so sensitive that it allows detection of zeptomole quantities of nucleic acid targets. There are many other detection methods suggested with the use of color change of noble metal nanoparticles (Jans and Huo 2012; Saha et al. 2012). The color change

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Fig. 13.9 Schematic design of sequence-selective single mRNA detection based on coupling plasmonic nanoparticles (Lee et al. 2014). Spectral characteristics of different nanoparticle structures formed by DNA hybridization for sequence-specific detection are shown above. Nanoparticle probes targeting different exons in the mRNA are highlighted by different colors. Monomers (light blue line) and dimers (purple line) can be differentiated from one another due to their unique spectral characteristics

based on analyte-induced aggregation/deaggregation test was suggested for the detection of low concentrations of heavy metal ions (Pb2+ , Cd2+ , Hg2+ ) in aqueous solutions. The ensemble in this case consists of gold nanoparticles functionalized with alkanethiol chains carrying carboxylate groups at the distal terminal ends. Aggregation of the particles upon the addition of metal ions occurs due to ion coordination by these groups belonging to two neighbor particles. Dextran-coated gold nanoparticles aggregated with concanavalin A were used for competitive glucose sensing in an extended millimolar-micromolar range (Aslan et al. 2004). Design of experiment on using DNA probes targeting specific sequences within the mRNA, conjugated to 40 nm gold nanoparticles (Lee et al. 2014) is depicted in Fig. 13.9. Hybridization of these probes to a single target mRNA resulted in the formation of nanoparticle dimers with small inter-particle distances producing spectral peak shift and several-fold increase of intensity due to strong plasmonic coupling.

13.3.2 The Effects of Particle Shape and Hetero-Composition Noble metal plasmonic nanoparticles can be synthesized with different shapes, and their shape together with size determines the spectroscopic range of plasmon efficiency (Fig. 13.6). A significant local electrical field enhancement around metallic nanoparticles contributes to fluorescent response, and this field becomes strongly anisotropic around the particles of different shapes (Li et al. 2015b; Zhao et al.

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2011). It was shown that the strongest plasmon field is concentrated at the sharp edges (tips, corners, etc.), which is the factor that must be accounted and actively used in sensor design. Preventing the possibility of direct quenching at too close distances should be also accounted. Gold nanobipyramids, as well as gold nanorods, deserve special attention. Both of them are the elongated plasmonic nanoparticles with their longitudinal dipolar plasmon wavelengths synthetically tunable from the visible region to the nearinfrared region (Chow et al. 2019). Because of their anisotropic geometries, they have highly polarization-dependent absorption and scattering cross-sections. Au nanobipyramids possess two sharp end tips, and, therefore, exhibit larger local electric field enhancements, larger optical cross sections, narrower line widths, better shape and size uniformity, and higher refractive index sensitivity than the rods. The other important and interesting features of plasmonic nanostructures is the possibility to provide broad variations of their properties by making heterocomposites between gold and silver (Ha et al. 2019), silver-palladium (Zhu et al. 2017) and other (Ha et al. 2019) noble metals in different proportions. Compared to homogeneous plasmonic nanostructures, the multicomponent-based heterogeneous structures exhibit more diverse or improved properties and thus can offer more opportunities in controlling, improving, and widening the structures, properties, and functions of nanocomposites. The possibility was shown for combining several components within a single structure with controlled interfaces, high precision, quantitative, and stable surface modification (Ha et al. 2019). Combining plasmonic and non-plasmonic materials is also possible, which further increases their diversity.

13.4 Broad-Scale Applications of Plasmonic Sensors The application of plasmonic biosensors has become very popular (Bauch et al. 2014; Jeong et al. 2018; Li et al. 2015a), mostly in DNA hybridization, immunoassays and pathogen detection. Plasmonic enhancement of fluorescence can be coupled with all assay formats. Heterogeneous assays may benefit from application of flat films that can be formed of both continuous and nanostructured noble metal layers. For homogeneous assays, the composite nanoparticles incorporating noble metal component can be fabricated. Combination with technologies based on other sensing principles can be also realized. The most characteristic examples are overviewed below. A straightforward use of metal enhancement is for providing an increased sensitivity compared to traditional detection techniques. An example is the work (Aslan et al. 2006), in which the application of this approach resulted in detection of only several femtomoles (10−15 mol) of RNA rapidly and without the use of time- and reagent-consuming PCR procedure. This approach has a strong potential to be developed into different single-molecular detection methodologies. In a recent development (Wei et al. 2017), the sensitivity sufficient for detecting less than 100 dye

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Fig. 13.10 Illustration and photograph of smartphone surface-enhanced fluorescence microscopic device. a 3-D illustration of the smartphone attachment with the cutaway view of the inner sample stage. b Schematic of the configuration implemented in the smartphone attachment

molecules was achieved using a simple device containing thin plasmonic film with detection on smartphone (Fig. 13.10). The other important advantage in sensing applications is the localized nature of the enhancement effect. The enhanced evanescent field mode of excitation localizes the excitation volume in close proximity to the metal nanostructures. As in other evanescent-field excitation techniques, this allows exciting only the bound species and eliminates the need for any washing steps. In contrast to plain surfaces, the nanoparticles possess dramatically larger surface areas. Together with their effect as scatterers, this offers a broad flexibility in the assay development. Plasmonic nanoparticles can be incorporated into fiber-optics biosensors (Chang et al. 2009) and microfluidic chips (Li et al. 2015a). Since it is technically possible to monitor simultaneously the reflected light (for obtaining the SPR signal) and surface plasmon enhanced emitted light from the same surface, this approach can provide two complementary channels of information. Many benefits can be obtained from such marriage. For instance, the unlabeled targets of large mass, producing a strong change of refractive index in the interfacial region, are active in SPR. In contrast, the small-size fluorescent dyes produce in the same region a strongly amplified effect in fluorescence without detectable change in SPR signal. In this configuration, which requires angle-resolved incident light (Neumann et al. 2002), the sensitivity of fluorescence-based response can be much higher than that of SPR. Both of them allow not only providing equilibrium measurements but also studying the association-dissociation kinetics with the use of flow cells. An immense variety of potentially useful nanocomposites can be constructed exhibiting enhanced emission (see Chap. 12). An interesting possibility, for example, can be realized when the dye is incorporated into silica core of the beads that are coated by silver layer to generate porous but continuous metal nanoshells (Zhang et al. 2006b). The other possibility is to construct nanocomposite composed of silver core and the outer shell of organic dyes with a spacer layer between them (Roy et al. 2012). Being brightly emissive, such particles are simultaneously protected from the

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quenching effect of the solvent. The synthesis of hybrid nanoprobes composed of gold nanorods, CdSe/ZnS quantum dots and iron oxide nanoparticles was reported (Basiruddin et al. 2011). They were shown to combine plasmonic, fluorescent and magnetic properties into a single molecular construct. In this regard, the approach based on metal particle enhancement has a strong potential for application in high throughput screening and other assays dealing with great number of samples and determining many parameters. Such possibility of multiplex detection was demonstrated for the DNA microarrays, in which the surfacebound silver nanoparticles were used as fluorescence enhancers (Sabanayagam and Lakowicz 2007). A special field of research and development is the near-field microscopy that, together with other microscopic methods will be discussed in Volume 2, Chap. 15. In this method, a plasmonic tip-enhanced near-field fluorescence probing can be used, and it allows achieving a single-molecular sensitivity (Höppener and Novotny 2012). Focusing on evanescent-wave and plasmonic effects, we have to recognize that there are many factors influencing this enhancement and, notably, the interplay between enhancement and quenching. These factors can depend on the emitter (quantum yield, lifetime), on the metal (plain surface or nanoparticles, particle size and shape, their resonance absorption energy) and on both of them (their orientation and distance). In the case of fluorescent molecules located at very short distances from a metal surface, the quenching takes place. Electromagnetic-field enhancement occurs at longer distances. As a result, there is an optimal dye to metal distance for fluorescence enhancement. With good understanding of this phenomenon, switching between enhancement and quenching can provide the broadest dynamic range hardly available to any other sensing technique. It is presently certain that the phenomenon of metal enhancement of light scattering and of enhancement of fluorescence is of general origin. It appears irrespective of the nature of the excited state (singlet or triplet) and of the way of excitation (optical or in a chemical reaction). This adds many new dimensions to research and practical use of this phenomenon. Such bright perspective stimulates detailed studies in all aspects, including the sizes, shapes and composition of metal nanostructures, and also the nature, spectroscopic properties and location of fluorophores. The role of augmentation of optical intensity incident on the dye molecule through near field enhancement, of modification of the radiative decay rate of the emitter and of increasing the coupling efficiency of the fluorescence emission to the far field through nanoparticles scattering deserve detailed studies. An immense variety of nanocomposites exhibiting plasmon-enhanced emission can be constructed. They can be made of similar and dissimilar emitters but with necessary participation of plasmonic nanoparticles in their special arrangement. This allows achieving important goal to increase the output signal and thus to achieve lower limits of target detection. Some novel aspects have to be listed. They are the increase of brightness in super-resolution imaging (nanoscopy) (Willets et al. 2017)

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and of the power of light-emitting diodes (Gu et al. 2011). The ability of plasmonic nanostructures to squeeze light into sub-wavelength volumes is in the design of nanolasers (Wang et al. 2017).

13.5 Microwave Acceleration of Metal-Enhanced Emission In continuous attempts to improve the sensing technologies, the scientists approached, and some achieved already, the single-molecular level of detection. With the implication of materials and techniques based on plasmonic effects, great steps have been made in this direction. For obtaining much benefit from these achievements, we have to resolve one more problem – to increase the speed of analysis. When the time-consuming steps of sample pre-treatment and after-treatment are eliminated or substantially reduced, then the assay duration becomes the critical step limited by the sensor-target incubation time. Diffusion of the partners in solutions, their binding requiring sometimes high activation energies, competition with target analogs for binding sites—all that increases the time of analysis. The process of recognition between the target and receptor involves many trial-and-error steps on molecular level, so that many minutes and sometimes hours are needed for establishing the equilibrium between free and bound target. The slow rate is particularly characteristic for the most popular assays involving macromolecules, in which the multi-point specific contacts need to be established. Those are the DNA hybridization assays and immunoassays. The problem is how to speed up the analysis. The low-power microwave heating is increasingly used in different techniques of chemical synthesis for accelerating the reaction rates (Tokuyama and Nakamura 2005). Microwaves, with the frequency of 0.3–300 GHz, lie between the infrared and radio-frequency electromagnetic radiation. They are strongly absorbed by mobile dipoles in the medium, so that polar systems of any size are selectively heated, and non-polar remain transparent to this radiation. Home kitchen microwaves, operating with frequency 2.45 GHz work based on this principle of dielectric heating (Kaur and Singh 2016). In this case also the noble metal nanostructures may provide their highly localized contribution. For metals, the attenuation of microwave radiation arises from the motions of extremely mobile conductance electrons. The charge carriers in the metal, which are displaced by the oscillating electric field, are subjected to resistance due to collisions with the lattice phonons. They provide energy to the lattice resulting in the local heating. This so-called Ohmic heating of the metal nanoparticles occurs in addition to the heating of any polar medium, such as water surrounding the nanoparticles (de Castro and Fernández-Peralbo 2012; Silva et al. 2017). Microwave heating can be useful for synthesis of plasmonic nanoparticles in mild conditions (Joseph and Mathew 2015). Essentially, the localized heating around the metal nanostructures can be achieved without heating of the whole volume. This accelerates the local diffusion and specific binding (recognition) between macromolecules close to them. Thus, the speed of

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receptor-target binding can be increased, which is highly needed for increasing efficiency of operation of sensors. The combination of metal enhancement and localized heating by microwaves makes the sensing both very sensitive and rapid (Aslan and Geddes 2005; Santaus and Geddes 2018). It was demonstrated that for both popular and time-consuming DNA hybridization assays and immunoassays with the application of microwaves the incubation times can be reduced manifold, from hours to seconds. Thus, it was shown (Aslan et al. 2006; Aslan et al. 2007a) that by combining the metal enhancement effects of silver nanoparticles on dye emission with low-power, localized microwave heating, a model DNA hybridization assay to detect 23-mer targets can be kinetically complete within 20 s. This gives the acceleration up to 600-fold, as compared to identical hybridization assay run without microwaves. Such treatment does not lead to any destruction or denaturation of interacting partners. Simultaneously, by metal enhancement, a greater than ten-fold increase in assay sensitivity was achieved. The speed of assay together with high sensitivity is especially important in the identification of such dangerous bioagents, as Anthrax (DNA) or Ebola (RNA) viruses. The limiting factors in current immunoassays are also their low sensitivity and slow speed. For increasing the sensitivity, additional operations (such as catalytic amplification) are frequently introduced, and they still further increase the time of test duration. Therefore, the microwave-heated metal-enhanced technique is a technology offering substantial improvement. This approach allows reducing the incubation times in immunoassays from many minutes to few seconds with dramatic increase of sensitivity (Aslan et al. 2007c). In view of these findings, it is highly probable that this new technology will fundamentally change the ways by which the immunoassays and other diagnostic tools are employed in clinical medicine, adding both speed and sensitivity.

13.6 Sensing and Thinking. Plasmonics, Is that Sole Search for Brightness? In present days, we are on the most romantic step of exploration of light-matter interaction. It has led us to observation of phenomena that are seemingly unusual but so important in practical applications, such as evanescent waves, plasmonic scattering, plasmonic rise of absorbance and the metal enhancement of fluorescence. A lot has been done, and superiority over traditional detection techniques is clearly seen. Meantime it is still difficult to predict whether a particular sized metal structure will strongly quench or strongly enhance the fluorescence or other type of emission of a definite molecular dye or nanocomposite, and if this happens, to what extent. Still, we expect that these new achievements will develop into new simple and cheap in performance technologies available for everyday user. With these devices possessing very fast response, we have to forget about “incubation steps” or “catalytic

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amplification” in detecting antibodies, DNA analysis or checking for harmful agents. We have the tools; let us get use of them! The reader can respond to the following questions: 1.

Analyze the properties of evanescent wave. What is the distance dependence for the evanescent wave excitation of dyes? Compare it with the sizes of macromolecules, nanoparticles, cells. 2. How the evanescent-wave excitation can be realized in sensing? Suggest several examples on molecular recognition of different targets. 3. What is the connection between surface plasmon resonance and fluorescence enhancement in plasmon resonance conditions? Can these two types of observation be combined? 4. What is special in metal nanoparticles, so that they provide the effect of enhancement of fluorescence? What is the dependence of this effect on the particle size? 5. Explain a complicated dependence of dye fluorescence on the distance to plasmonic plain surface. Why this distance can be considered as a key controlling parameter in sensing? Will the same regularity be valid on interaction with plasmonic nanoparticles? 6. Which properties of plasmonic nanoparticles are revealed in their light absorption and scattering in solutions? 7. What is the difference between light absorption and extinction? What is actually measured with spectrophotometer? 8. How the size and shape of plasmonic nanoparticle influences the efficiency in fluorescence enhancement? 9. Explain the difference between propagating and localized plasmons. Which of them can be employed in homogeneous fluorescence assays and how? 10. Explain the mechanism of paradoxical result that the enhancement of fluorescence intensity can be coupled with shortening of lifetime. 11. What spectroscopic changes occur on association of plasmonic nanoparticles and how these changes are used for sensing? 12. Why the microwave heating can be local and focused on only metal particles in solutions? How can it decrease the duration of the assays?

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