Recent progress in colloid and surface chemistry with biological applications 9780841231207, 0841231206, 9780841231191

Table of contents :
Content: 1. Langmuir Monolayer Properties of Fluorinated Fatty Alcohols and Dipalmitoylphosphatidylcholine (DPPC) 2. A Review of Hydrophilization of Oxidized Nanocarbons3. Interactions between Graphene Oxide and Biomolecules from Surface Chemistry and Spectroscopy 4. The Role of Langmuir Monolayers To Understand Biological Events 5. a-Helix Facilitates Proteins To Form Langmuir Monolayer: Surface Properties and Orientation Studies of Aequorin and a-Synuclein at the Air-Water Interface 6. How To Decipher Protein and Peptide Selectivity for Lipids in Monolayers 7. Single-Molecule Fluorescence Microscopy Methods in Chromatin Biology 8. Review: Physicochemical Structure Effects on Metal Oxide Nanoparticulate Cytotoxicity 9. Dynamic Light Scattering Coupled with Gold Nanoparticle Probes as a Powerful Sensing Technique for Chemical and Biological Target Detection10. Conducting Polymeric Nano/Microstructures: From Fabrication to Sensing Applications 11. Organic-Inorganic Supramolecular Gels and Contrast Agents for Magnetic Resonance Imaging Based on the Surfactant-Covered Polyanionic Clusters 12. Diverse Near-Infrared Resonant Gold Nanostructures for Biomedical Applications 13. Bioanalysis within Microfluidics: A Review 14. Plasmonics in Analytical Spectroscopy 15. Quantitative Comparative Techniques of Infrared Spectra of a Thin Film16. Protein Response to Chromophore Isomerization in Microbial Rhodopsins Revealed by Picosecond Time-Resolved Ultraviolet Resonance Raman Spectroscopy: A Review 17. Biophysical Methods for the Studies of Protein-Lipid/Surfactant Interactions 18. Synchrotron-Based X-ray Methods as Powerful Tools for the Characterization of Monolayers at the Air/Liquid Interface

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Publication Date (Web): December 8, 2015 | doi: 10.1021/bk-2015-1215.fw001

Recent Progress in Colloid and Surface Chemistry with Biological Applications

In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Publication Date (Web): December 8, 2015 | doi: 10.1021/bk-2015-1215.fw001

In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

ACS SYMPOSIUM SERIES 1215

Publication Date (Web): December 8, 2015 | doi: 10.1021/bk-2015-1215.fw001

Recent Progress in Colloid and Surface Chemistry with Biological Applications Chengshan Wang, Editor Middle Tennessee State University Murfreesboro, Tennessee

Roger M. Leblanc, Editor University of Miami Coral Gables, Florida

American Chemical Society, Washington, DC Distributed in print by Oxford University Press

In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Publication Date (Web): December 8, 2015 | doi: 10.1021/bk-2015-1215.fw001

Library of Congress Cataloging-in-Publication Data Names: Wang, Chengshan (Professor of chemistry), editor. | Leblanc, Roger M., editor. Title: Recent progress in colloid and surface chemistry with biological applications / Chengshan Wang, editor, Middle Tennessee State University, Murfreesboro, Tennessee, Roger M. Leblanc, editor, University of Miami, Coral Gables, Florida. Description: Washington, DC : American Chemical Society, [2015] | Series: ACS symposium series ; 1215 | Includes bibliographical references and index. Identifiers: LCCN 2015045531 (print) | LCCN 2015048193 (ebook) | ISBN 9780841231207 (alk. paper) | ISBN 9780841231191 () Subjects: LCSH: Surface chemistry. | Biochemistry. | Chemistry--Research. | Alcohols. | Colloids. | Monomolecular films. Classification: LCC QD506 .R36 2015 (print) | LCC QD506 (ebook) | DDC 541/.345--dc23 LC record available at http://lccn.loc.gov/2015045531

The paper used in this publication meets the minimum requirements of American National Standard for Information Sciences—Permanence of Paper for Printed Library Materials, ANSI Z39.48n1984. Copyright © 2015 American Chemical Society Distributed in print by Oxford University Press All Rights Reserved. Reprographic copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Act is allowed for internal use only, provided that a per-chapter fee of $40.25 plus $0.75 per page is paid to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. Republication or reproduction for sale of pages in this book is permitted only under license from ACS. Direct these and other permission requests to ACS Copyright Office, Publications Division, 1155 16th Street, N.W., Washington, DC 20036. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law. PRINTED IN THE UNITED STATES OF AMERICA In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Publication Date (Web): December 8, 2015 | doi: 10.1021/bk-2015-1215.fw001

Foreword The ACS Symposium Series was first published in 1974 to provide a mechanism for publishing symposia quickly in book form. The purpose of the series is to publish timely, comprehensive books developed from the ACS sponsored symposia based on current scientific research. Occasionally, books are developed from symposia sponsored by other organizations when the topic is of keen interest to the chemistry audience. Before agreeing to publish a book, the proposed table of contents is reviewed for appropriate and comprehensive coverage and for interest to the audience. Some papers may be excluded to better focus the book; others may be added to provide comprehensiveness. When appropriate, overview or introductory chapters are added. Drafts of chapters are peer-reviewed prior to final acceptance or rejection, and manuscripts are prepared in camera-ready format. As a rule, only original research papers and original review papers are included in the volumes. Verbatim reproductions of previous published papers are not accepted.

ACS Books Department

In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Editors’ Biographies

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Dr. Chengshan Wang Dr. Chengshan Wang got his Ph.D. in Chemistry Department of University of Miami at 2008. After his postdoc experience in California State University, Los Angles, Dr. Wang has been an Assistant Professor now in Chemistry Department of Middle Tennessee State University since 2011.

Dr. Roger M. Leblanc Dr. Roger M. Leblanc got his Ph.D. at Université Laval in 1968 and accomplished his postdoc research in Davy Faraday Research Lab in 1970. From 1971 to 1993, Dr. Leblanc worked in the Department of Chemistry and Biology of Université du Québec à Trois Rivières as a Professor and the Chairman of the Department from 1971 to 1975. Since 1994, Dr. Leblanc has been a Professor at the Chemistry Department of University of Miami. He is also the Chair of the Chemistry Department from 1994 to 2002, and again from 2013 to present.

© 2015 American Chemical Society In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Publication Date (Web): December 8, 2015 | doi: 10.1021/bk-2015-1215.pr001

Preface This issue of the ACS Book Series has been designed to survey selected recent advances in colloids and surface chemistry, and to highlight some connections between fundamental research and some important biological relevance. Current research at both the air-water interface and at nanoparticle interfaces are featured. The topics are arranged in a natural progression from non-biologically to biologically relevant topics, although the line in discussing these topics is blurred. This blurred boundary illustrates the natural interdisciplinary character and strength of the field of surface science at air-liquid or liquid-solid interface. The chapters in the book are divided into three sections: the surface properties of surfactant molecules, the surface modification of nanoparticles and state-of-theart spectroscopic methods to enhance our understanding of membrane-mimetic chemistry, imitating certain aspects of biological membranes. The surface properties of surfactant molecules cover the first six chapters. Chapter 1 deals with the interaction of synthesized fluorinated amphiphiles to understand their interaction with dipalmitoyl phosphatidylcholine Langmuir monolayer, a model of the pulmonary surfactants in mammals. The drug carriers in drug delivery systems have been investigated in Chapter 2, by investigating the water-dispersibility of nanocarbon materials in the aqueous phase. In particular, graphene oxide dispersed in the aqueous phase has been examined in Chapter 3, in terms of the nature of the interaction of the carbon nanomaterial with lipids and proteins. Biological surface science is a broad, interdisciplinary subfield of surface science, where properties and processes at biological and synthetic surfaces and interfaces are investigated. The Langmuir monolayer as a bio-membrane model system was studied with the objective to understand biological phenomena in Chapters 4, 5 and 6. The binding properties’ of proteins and peptides to a lipid monolayer are provided in these three chapters. The surface modification of nanoparticles is grouped in six chapters. To map chromatin structure at a nucleosomal resolution, a single molecule-based fluorescence microscopic technique has been developed and is discussed in Chapter 7. The role of nanoparticle surface charge and the relative number of binding sites, as well as the cytotoxicity of nanoparticles are covered in Chapter 8, whereas, the gold nanoparticle probes with the size measurements by a Dynamic Light Scattering assay have been discussed in Chapter 9. The fabrication of conducting polymeric structure at the nanoscale was developed using different lithographic methods, which are discussed in Chapter 10. The progress of a recently developed supramolecular strategy for organizing nano-sized fine particles into soft material system is presented in Chapter 11.

ix In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Finally, near-infrared resonant gold nanostructures have been applied for cancer imaging and cancer therapy in Chapter 12. State-of-the-art spectroscopic methodologies have been recently developed and cover the last group of six chapters. A series of integrated microfluidic chips and systems that can execute airborne microbe capture, enrichment and continuous-flow high-throughput bioanalysis have been developed, and are covered in Chapter 13. The basic physical model of plasmon enhancement is discussed in Chapter 14, with the objective to present the plasmon enhanced work based on shell-isolated nanoparticles in Raman scattering and in fluorescence. The analysis of molecular adsorbates on flat surface is described in term of Surface Infrared Spectroscopy and discussed in Chapter 15. Picosecond time-resolved UV resonance Raman spectroscopy has been covered in Chapter 16 with the objective to probe the structural dynamics of specific sites in protein structure, in particular, the chromophore isomerization in microbial rhodopsin. The most popular biophysical methods employed to characterize protein-lipid/surfactant interactions are outlined in Chapter 17. For probing the lateral ordering in condensed Langmuir monolayer at the Angstrom level, grazing incidence X-ray diffraction has been applied to various single-chain and double-chain amphiphiles in Chapter 18. The main elements of this 18 chapter book highlight recent advances in exploring the behaviors and the biological applications of surfactant molecules, nanomaterials, and biomacromolecules at the surface and interfaces. Particularly, several chapters cover the studies of the dynamic physicochemical interactions, which govern the properties and behaviors between nanomaterials surfaces and the surfaces of biological components, such as peptides, proteins, phospholipids, DNA and biological fluids. The authors of these 18 chapters are trying to understand and explore such interfaces from the perspective of surface and colloidal chemistry with the state-of-the-art spectroscopic methods. One can expect that a better understanding at the surface and interfaces will contribute significantly to the development and applications of surfactants and nanomaterials in the biological systems. The Editors would like to express their gratefulness to Eric Waidely in his help in proof-reading the chapters included in this book.

Chengshan Wang Middle Tennessee State University - Chemistry 1301 E. Main Street P.O. Box 68 Murfreesboro, Tennessee 37132, United States

Roger M. Leblanc Department of Chemistry, University of Miami 1301 Memorial Drive Coral Gables, Florida 33146, United States

x In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Chapter 1

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Langmuir Monolayer Properties of Fluorinated Fatty Alcohols and Dipalmitoylphosphatidylcholine (DPPC) Hiromichi Nakahara, Takayoshi Yamada, Chihiro Usui, Shunichi Yokomizo, and Osamu Shibata* Department of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo, Nagasaki 859-3298, Japan *E-mail: [email protected]. Phone: +81-956-20-5686. Fax: +81-956-205686. URL: http://www.niu.ac.jp/~pharm1/lab/physchem/indexenglish.html.

The authors have newly synthesized fluorinated amphiphiles with relatively short perfluorocarbon chains to understand their interaction with biomembranes. This chapter describes the monolayer miscibility of perfluorobutylated (F4H11OH) or perfluorohexylated long-chain alcohols (F6H9OH and F6H11OH) with DPPC, which is a major component of native pulmonary surfactants in a mammal. The two-component monolayer has been elucidated from the thermodynamic and morphological aspects. The surface pressure (Π)−molecular area (A) and surface potential (ΔV)−A isotherms for the systems were measured on 0.15 M NaCl at 298.2 K. From the isotherm data, a plot of an excess Gibbs free energy change of mixing versus mole fraction and a two-dimensional phase diagram were constructed to elucidate the miscibility between the two components. The miscibility is also supported by the in situ fluorescence microscopy (FM) and ex situ atomic force microscopy (AFM) after transfer on a mica substrate. Herein, the fluidization of DPPC monolayers containing a small amount of F4H11OH and F6H9OH is induced by increasing surface pressures. On the other hand, the incorporation of F6H11OH undergoes the solidification of DPPC monolayers. The control

© 2015 American Chemical Society In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

of phase states of DPPC monolayers is very important for a pulmonary replacement therapy.

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Introduction A molecule substituted with fluorine is endowed with unique properties such as higher hydrophobicity as well as lipophobicity (1), higher gas-dissolving capacity (1, 2), less chemical and biological activities, lower surface tension, and higher fluidity (1, 3). These fascinating properties indicate a potential towards use and application in medicine, disease diagnosis, and therapy (1, 4–6). Indeed, it has been reported that fluorinated materials are potentially applicable as blood substitutes (6–10) and effective additives to lung surfactant preparations (6–14). Despite these favorable properties, there is danger (especially for highly fluorinated compounds) of accumulation in the environment and in the human body (15–18). Among them, the anion type of perfluorooctanoic acid (PFOA) is gaining attention in terms of its sorption by soils and sediments. However, perfluorooctyl bromide (PFOB), which has been developed as the blood substitutes, has been shown to have short organ retention times (1, 2). With the aim of the reduction in the retention time and of the acquirement of the unique properties, the partially fluorinated (or semifluorinated) materials have been investigated using Langmuir monolayers at the air-water interface (19–23). Among the various techniques to investigate the properties of fluorinated amphiphiles, Langmuir monolayers are a considerably simple and optimal model in mimicry of biomembranes. In particular, due to the reduction of one dimension, the monolayer technique shows the advantage of understanding the mutual interaction between two or more components. Furthermore, with the both in situ and ex situ methods such as Brewster angle microscopy (BAM), fluorescence microscopy (FM), atomic force microscopy (AFM), and grazing incidence X-ray diffraction (GIXD), the phase behavior and interaction can be visualized and elucidated in more details. As the simplest partially fluorinated compound in terms of chemical structures, semifluorinated alkanes (CnF2n+1CmH2m+1 or FnHm) have been studied for more than two decades (24). It is a quite surprising property for the alkanes to form Langmuir monolayers on the aqueous subphase because hydrogenated n-alkanes form a droplet instead of spreading as monolayers. Goldmann et al. have subsequently examined the monolayer made of pure FnHm to clarify the FnHm−FnHm and FnHm−substrate interactions (25). Nevertheless, the interactions between fluorocarbon chains and between fluorocarbon and hydrocarbon chains seem to still remain unknown factors. Recently, we have studied the monolayer behavior of partially fluorinated long-chain alcohols (CnF2n+1CmH2m+1OH or FnHmOH) and their interaction with dipalmitoylphosphatidylcholine (DPPC) (20, 26–28). The fluorinated alcohols are less affected by the subphase pH than the compound containing carboxylic or amino groups and show a relatively simple chemical structure among fluorinated amphiphiles. In addition, DPPC is a major component of native pulmonary surfactants (29, 30). The film of pulmonary surfactants at the air-alveolar fluid interface undergoes successive changes in fluidity and rigidity 2 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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during compression and expansion. DPPC contributes significantly to the rigidity of pulmonary surfactant films. However, such films can exhibit slow adsorption and poor diffusion at the surfaces. The fluidity of DPPC monolayers is perturbed by the addition of F8HmOH molecules, which depends on their fluorination degree (20, 26–28). Indeed, the F8H5OH incorporation causes a fluidizing effect on DPPC monolayers. On the other hand, the addition of longer F8HmOH (m = 7, 9, and 11) shows an opposite effect of solidification. Especially, in the binary DPPC/F8H7OH system, the two components interact less at low surface pressures whereas a strong interaction occurs at high surface pressures. Thus, fluorinated amphiphiles have potential of controlling the fluidity of DPPC monolayers, however, the interaction between these chains is still documented poorly. The degree of fluorination in partially fluorinated chains may produce an unexpected interaction with lipids. In these days, FnHmOH (n < 8) in the solid state has been reported to show the irregular and interesting behavior for its melting points (19): the melting point of FnHmOH (n < 8) is lower than that of the corresponding hydrogenated alcohols. This unexpected finding is considered to be attributed to the specific properties of fluorocarbons. Furthermore, the study on the monolayer of amphiphiles with fluorocarbon shorter than 8 has been performed rarely (31). Therefore, this article describes recent efforts devoted to understanding the interaction and miscibility of two-component monolayers of long-chain alcohols (F4H11OH, F6H9OH, and F6H11OH) and DPPC. The thermodynamical elucidation of the binary miscibility in the monolayer state was performed by using isotherm data such as surface pressure (π)−molecular area (A) and surface potential (ΔV)−A curves. The phase behavior with respect to monolayer composition and surface pressure was visualized with FM, and AFM.

Experimental Section Materials (Perfluorobutyl)undecanol (F4H11OH), (Perfluorohexyl)nonanol (F6H9OH), and (Perfluorohexyl)undecanol (F6H11OH) were synthesized as reported previously (see Scheme 1) (19). L-α-dipalmitoylphosphatidylcholine (DPPC; purity >99%) was obtained from Avanti Polar Lipids (Alabaster, AL). The fluorescent probe 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4yl)amino]hexanoyl]-sn-glycero-3-phosphocholine (NBD-PC) was from Avanti Polar Lipids. These lipids were used without further purification. Chloroform (99.7%) and n-hexane (>98.5%) were purchased from Kanto Chemical Co., Inc (Tokyo, Japan) and Merck KGaA (Uvasol, Darmstadt, Germany), respectively. Methanol (99.8%) and ethanol (>99.5%) were from nacalai tesque (Kyoto, Japan). These were used as spreading solvents. The chloroform/methanol (2/1, v/v) mixtures for F4H11OH and n-hexane/ethanol (9/1, v/v) for F6H9OH and F6H11OH were used as spreading solvents. Sodium chloride (nacalai tesque) was roasted at 1023 K for 24 h to remove all surface active organic impurities. The substrate solution was prepared using thrice distilled water (surface tension = 72.0 mN m–1 at 298.2 K; electrical resistivity = 18 MΩ cm). 3 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Scheme 1. Chemical Structures of (a) F4H11OH, (b) F6H9OH, and (c) F6H11OH

Methods Surface Pressure–Area Isotherms The surface pressure (π) of monolayers was measured using an automated homemade Wilhelmy balance. The surface pressure balance (Mettler Toledo, AG-245) had a resolution of 0.01 mN m–1. The pressure-measuring system was equipped with filter paper (Whatman 541, periphery = 4 cm). The trough was made from Teflon-coated brass (area = 720 cm2), and Teflon barriers (both hydrophobic and lipophobic) were used in this study. Surface pressure (π)–molecular area (A) isotherms were recorded at 298.2 ± 0.1 K. The spreading solvents were allowed to evaporate for 15 min prior to compression. The monolayer was compressed at a speed of ~0.10 nm2molecule–1min–1. The standard deviations (SD) for molecular surface area and surface pressure were ~0.01 nm2 and ~0.1 mN m–1, respectively (28, 32, 33).

Surface Potential–Area Isotherms The surface potential (ΔV) was recorded simultaneously with surface pressure, when the monolayer was compressed and expanded at the air-water interface. It was monitored with an ionizing 241Am electrode at 1–2 mm above the interface, while a reference electrode was dipped in the subphase. The electrometer (Keithley 614) was used to measure the surface potential. The SD for the surface potential was 5 mV (34, 35).

4 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Fluorescence Microscopy (FM) The film balance system (KSV Minitrough) was mounted onto the stage of an Olympus microscope BX51WI (Tokyo, Japan) equipped with a 100W mercury lamp (USH-1030L), an objective lens (SLMPlan50×, working distance = 15 mm), and a 3CCD camera with a camera control unit (IKTU51CU, Toshiba, Japan). A spreading solution of the co-solubilized samples was prepared, doped with 1 mol% of the fluorescence probe (NBD-PC). Image processing and analysis were carried out using the software, Adobe Photoshop Elements ver. 7.0 (Adobe Systems Incorporated, CA). The total amount of ordered domains (dark contrast regions) was evaluated and expressed as a percentage per frame by dividing the respective frame into dark and bright regions. For the percentage, resolution (or discrimination threshold) was 0.1% and the maximum SD was 8.9%. More details on FM measurements were provided in the previous paper (11, 33).

Atomic Force Microscopy (AFM) Langmuir-Blodgett (LB) film preparations were carried out with the KSV Minitrough. Freshly cleaved mica (Okenshoji Co., Tokyo, Japan) was used as a supporting solid substrate for film deposition (vertical dipping method). At selected surface pressures, a transfer velocity of 5 mm min–1 was used for singlelayer deposition. The film-forming materials were spread on 0.15 M NaCl at 298.2 K. The transfer occurs so that the hydrophilic part of the monolayer is in contact with mica while the hydrophobic part is exposed to air. LB films with a deposition rate of ~1 were used in the experiments. The AFM experiments were performed in the air at room temperature. The AFM images were obtained using an SPA 400 instrument (Seiko Instruments Co., Chiba, Japan) at room temperature in the tapping mode, which provided both topographical and phase contrast images. Other details about AFM measurements have been mentioned previously (33).

Results and Discussion Thermal Properties Thermal properties of saturated fatty acids or alcohols are expected to depend on their aliphatic chain lengths due to the simplicity of chemical structures (36). For instance, the melting point (Tm) of the acids (or alcohols) increases linearly with respect to the elongation of their chain lengths. Fluorination of the whole hydrogen atoms in a tail group of saturated fatty acids changes or improves their original properties such as melting point, vapor pressure, etc. The Tm values of the perfluorinated fatty acids also increase linearly against hydrophobic chain length although a slope of Tm versus carbon number for the perfluorinated acids is twice smaller than the corresponding hydrogenated acids (37). This fact means that the high fluorination provides original compounds with thermal stability. The variation in melting point allows us easily to estimate the difference in the intermolecular interactions such as dipole-dipole 5 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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interaction and van der Waals interaction between the hydrocarbon−hydrocarbon and fluorocarbon−fluorocarbon chains. In this regard, partially fluorinated amphiphiles are also expected to be endowed with such properties. In fact, the melting point of ω-(perfluorooctyl)alkanol (or F8HmOH) is larger than that of the corresponding hydrogenated alcohol (19); the melting point of perfluorooctylated fatty alcohols increases by ~1 K compared to the corresponding hydrogenated alcohols. However, the melting points of F4H11OH and F6HmOH are lower than those of the corresponding hydrogenated alcohols with the same hydrophobic chain length (19). This may be attributed to dipole-dipole interaction, molecular weight, and restricted motion at the CF2−CH2 linkage, which are open to be disputable. Nevertheless, the irregular phenomenon suggests that the dipole-dipole and van der Waals interactions between the hydrophobic chains of F4H11OH and F6HmOH are different from FnHmOH (n ≥ 8). Langmuir Monolayer of Pure Systems The surface pressure (π)–molecular area (A) and the surface potential (ΔV)–A isotherms of F4H11OH, F6H9OH, F6H11OH, and DPPC monolayers on 0.15 M NaCl at 298.2 K are shown in Fig. 1. The other alcohols with hydrophobic chains shorter than 15 are difficult to form an insoluble (or stable) monolayer at the air-water interface under the same condition due to their high solubility into water (19). At 298.2 K, F4H11OH (curve 1) forms a typical disordered monolayer corresponding to the liquid-expanded (LE) phase of hydrogenated lipid monolayers. A collapse pressure (πc) of F4H11OH monolayers is ~44 mN m−1 at 0.24 nm2. The extrapolated area of highly packed states on the π–A isotherm is ~0.40 nm2, which is based on the cross-sectional area of F-chains (~0.30 nm2) (1). The F4H11OH and F6H9OH (curve 2) monolayers initially have a π value of more than 1 mN m−1 at large molecular areas (A = ~2.0 nm2), where the monolayer is in a disordered phase. However, the isotherm for F6H9OH monolayers has a kink or a transition pressure (πeq) at ~8 mN m−1 (indicated by a dashed arrow), which means a phase transition of the disordered state to an ordered state corresponding to liquid-condensed (LC) phase of the lipid monolayers (19). On further compression, the monolayer collapses at ~47 mN m−1. Considering the same hydrophobic chain length between F4H11OH and F6H9OH, it is found that the replacement of −(CH2)2− by −(CF2)2− improves the rigidity of monolayers. Incidentally, the F4H11OH monolayer comes to indicate the phase transition at the temperature lower than 298.2 K. More detailed elucidation on the phase transition for F4H11OH and F6H9OH monolayers (e.g. entropy and enthalpy changes) has been described in the previous paper (19). Similarly to the thermal behavior of the solid state mentioned above, the slope (∂πeq/∂T) for F4H11OH and F6H7OH monolayers are more sensitive to temperature: the thermal sensitivity of F4H11OH monolayers is twice as large as that of the corresponding fatty alcohol monolayers. On the other hand, F6H11OH including a longer hydrophobic chain has a tendency to form more rigid monolayers. During the compression from ~1 mN m−1 to the collapse pressure of ~52 mN m−1, F6H11OH (curve 3) monolayers keep being in an ordered phase without the transition. The molecular areas at the constant surface pressure (e.g., 15 mN m−1, Fig. 1) indicate that the enhancement 6 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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of fluorination in hydrophobic chains and their elongation produce the condensing effect on monolayers; F4H11OH < F6H9OH < F6H11OH. This result is also supported by that for F8HmOH (m = 5, 7, 9, and 11) monolayers (20, 26–28). That is, it can be said that the F-moiety rather than the hydrocarbon (H) moiety in FnHmOH molecules dominates over the monolayer ordering and orientation at the close-packed state for the monolayers. DPPC monolayers (curve 4) have an LE/LC phase transition at ~11 mN m–1 (dashed arrow) and a collapse pressure at ~55 mN m–1, which has been discussed elsewhere (11, 33, 38).

Figure 1. The π–A and ΔV–A isotherms of F4H11OH (curve 1), F6H9OH (curve 2), F6H11OH (curve 3), and DPPC (curve 4) monolayers on 0.15 M NaCl at 298.2 K.

The surface potential (ΔV) of monolayers can be considered as a combination of dipole moments of molecules in the subphase (layer 1), polar head group (layer 2), and terminal group of hydrophobic chain (layer 3). Independent dipole moments and effective local dielectric constants are attributed to each of the three layers. Thus, the ΔV–A isotherms of the monolayers indicate changes in molecular orientation upon lateral compression. It is widely accepted that the ΔV–A isotherm of monolayers consisting mainly of H-chains or F-chains exhibits positive or negative variation against lateral compression, respectively (28, 39, 40). The negative ΔV value of F-chains is attributed to the electronegativity of a fluorine, which has been discussed thoroughly (21, 22, 39–42). The ΔV value decreases with decreasing molecular areas and finally approaches a minimum ΔV value (ΔVmin) at the close-packed state, where monolayers begin to collapse. 7 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

The ΔVmin value for F4H11OH monolayers is around −650 mV. On the other hand, both the values for F6H9OH and F6H11OH monolayers are concentrated to nearly −700 mV. In this connection, F8HmOH (m = 5, 7, 9, and 11) monolayers indicate almost ΔVmin = −750 mV independent of total chain lengths (20, 26–28). Considering few contributuns of dipole moments (layers 1 and 2) to the ΔV value, these result becomes important evidence that F-moiety (accurately, terminal CF3− group) in FnHmOH molecules is exposed to the air similarly to typical lipid monolayers and that the monolayer ordering and orientation depend on the F-moiety rather than the H-moiety at the close-packed state for the monolayers.

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Two-Component Monolayers with DPPC The π–A and ΔV–A Isotherms The two-component interaction and miscibility between DPPC and the fluorinated fatty alcohols in the monolayer state can be elucidated from the π–A and ΔV–A isotherms measured by varying the monolayer composition. The isotherms of binary monolayers often lie in compositional order between the isotherms of each pure monolayer due to the extensive variable of molecular area (or surface concentration). The π–A and ΔV–A isotherms for the binary DPPC/F4H11OH, DPPC/F6H9OH, and DPPC/F6H11OH monolayers are shown in Fig. 2. Herein, the binary monolayers including both H-chains and F-chains indicate the more distinct and larger variation in ΔV–A isotherms due to their opposite ΔV signs (20, 26–28). The criteria for assessing the interaction of binary monolayers are based on a variation in πeq as well as πc with regard to composition (43). In particular, the ΔV–A isotherm is useful in determination of the pressures (πeq and πc). The disordered/ordered phase transition on the π–A and ΔV–A isotherms is indicated by dashed arrows (19, 44). In Fig. 2A, the π–A isotherms shift to smaller areas with increasing the mole fraction of F4H11OH (XF4H11OH) and accordingly the πeq value increases. The increment in πeq means that there is a fluidizing effect of F4H11OH on DPPC monolayers (28). In addition, the πc value apparently changes as a function of XF4H11OH. These variation in πeq and πc suggests a miscibility between DPPC and F4H11OH. The ΔV–A isotherms shift more considerably from positive to negative values (~600 to −600 mV for ΔVmin) with regard to XF4H11OH. Shown in Fig. 2B are the isotherms for DPPC/F6H9OH monolayers. Both the pressures (πeq and πc) vary with the mole fraction of F6H9OH (XF6H9OH). However, the mode of πeq variation is somewhat complicated differently to the DPPC/F4H11OH system. This is attributed to the fact that both pure components in the DPPC/F6H9OH system exhibit the phase transition. Nevertheless, in the small XF6H9OH region, it is found that the addition of F6H9OH to DPPC monolayers induces the fluidizing effect. Whereas, the π–A isotherms for the DPPC/F6H11OH system in the larger XF6H11OH region indicate the reduced πeq value as the mole fraction of F6H11OH (XF6H11OH) increases (Fig. 2C). This means a solidifying effect of F6H11OH on DPPC monolayers. In the case of the binary DPPC/F8HmOH monolayers (20, 26–28), the incorporation of F8HmOH also generates fluidizing or solidifying effects depending on m: fluidization for m = 5 and solidification for m = 7, 9, 11. The boundary factor to 8 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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induce the fluidizing or solidifying effects must be related deeply to a mismatch between the hydrophobic chain lengths of DPPC and the fluorinated alcohols. It is noticed that the additional effect of the alcohols on DPPC monolayers changes in direct opposition only by two methylene groups. Consequently, the miscibility for the three systems here is suggested by the fact of the variation in surface pressures (πeq and πc) against mole fraction.

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Figure 2. The π–A isotherms of the two-component DPPC/F4H11OH (A), DPPC/F6H9OH (B), and DPPC/F6H11OH (C) monolayers on 0.15 M NaCl at 298.2 K. Adapted with permission from reference (44). Copyright 2015 Japan Oil Chemists’ Society.

Excess Gibbs Free Energy Change The mutual interactions between DPPC and the fluorinated alcohols in the monolayer state can be analyzed thermodynamically with the excess Gibbs free ), which is calculated with the following equation energy change of mixing ( (Eq. (1)) (43):

where Ai and Xi are the molecular area and mole fraction of component i, respectively, and A12 is the mean molecular area of the binary monolayer. For identical interactions between the two components, is zero. In this state, they are either ideally mixed in the monolayer or are not mixed completely, resulting in patch-like packing state (45, 46). A negative value indicates that an attractive interaction exists between the two components. The − XF4H11OH plot for DPPC/F4H11OH at representative surface pressures is shown in Fig. 3A. The values in 0 < XF4H11OH ≤ 0.7 are almost negative at all surface pressures. In addition, they decrease with an increase in surface pressure. The −XF4H11OH profile reaches −850 J mol−1 at XF4H11OH = 0.3 as a minimum at 10 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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45 mN m−1. This indicates that the attractive force between hydrophobic chains of DPPC and F4H11OH is enhanced upon compression and their affinity at high surface pressures becomes the largest value at XF4H11OH = 0.3. At XF4H11OH = 0.9, values exhibit positive values, which means existence of a repulsive the force or a steric hindrance between the two components. However, the values are relatively small in terms of the interaction between the two components. Thus, the monolayer at XF4H11OH = 0.9 is considered to take a looser packing. As for the DPPC/F6H9OH system (Fig. 3B), the values are negative at all surface pressures over the whole XF6H9OH and reach nearly −1600 J mol−1 at XF6H9OH = 0.6 as the surface pressure increases to 45 mN m−1. On the other hand, the DPPC/F6H11OH system indicates the minimum value of around −600 J mol−1 at XF6H11OH = 0.3 (Fig. 3C). It is noticed that the value for the DPPC/F6H9OH system is more than twice as small as that for DPPC/F6H11OH system, which indicates that DPPC molecules prefer F6H9OH in miscibility to F6H11OH. This is considered to be due to the similarity in monolayer phase states between DPPC (LE/LC) and analysis is based on the additivity rule F6H9OH (disordered/ordered). The so that it does not provide direct relationships with interaction modes such as fluidization and solidification.

Figure 3. Excess Gibbs free energy changes of mixing ( ) of the binary DPPC/F4H11OH (A), DPPC/F6H9OH (B), and DPPC/F6H11OH (C) monolayers as a function of XFnHmOH at typical surface pressures on 0.15 M NaCl at 298.2 K. Adapted with permission from reference (44). Copyright 2015 Japan Oil Chemists’ Society.

Two-Dimensional Phase Diagram Two-dimensional phase diagrams, which illustrate variations between the phases of monolayers with respect to surface pressure and composition under the thermodynamically equiliburium state, are constructed by plotting the πeq and πc values for the binary monolayers against composition at 298.2 K. In the 11 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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DPPC/F4H11OH system (Fig. 4A), the πeq values are kept almost constant below XF4H11OH = 0.3. However, they change positively as XF4H11OH increases from 0.3 to 0.7. As shown in Fig. 4B, the πeq value in the DPPC/F6H9OH system increases with increasing XF6H9OH smaller than 0.6. Then, it decreases to the πeq value of pure F6H9OH (~8 mN m−1). On the other hand, for the DPPC/F6H11OH system (Fig. 4C), the reduction in πeq is caused with an increase in XF6H11OH. These variations in πeq against monolayer composition are a confirmation of miscibility between the two compositions and suggest the interaction modes of fluidization (DPPC/F4H11OH and DPPC/F6H9OH) and solidification (DPPC/F6H11OH). In the high surface pressure region, the experimental πc values also vary against mole fraction. If the surface pressure remains constant with regard to mole fraction, two components can be said to be immiscible with each other. Accordingly, the two components are found to be miscible in the monolayer state.

Figure 4. Two-dimensional phase diagrams based on the variation of the transition pressure (πeq: open circle) and collapse pressure (πc: solid circle) on 0.15 M NaCl at 298.2 K as a function of XFnHmOH. The dashed lines were calculated according to Eq. (2) for ξ = 0. The solid line at high surface pressures was obtained by curve fitting of experimental collapse pressures to Eq. (2). “M” indicates a mixed monolayer formed by DPPC and FnHmOH species, whereas “Bulk” denotes a solid phase of DPPC and FnHmOH (“bulk phase” may be called “solid phase” not monolayer state). Adapted with permission from reference (44). Copyright 2015 Japan Oil Chemists’ Society.

The coexistence phase boundary of the monolayer/bulk states of the molecules spread on a surface can be theoretically simulated using the Joos equation (47, 48) under the assumption of a regular surface mixture:

12 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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where and denote the respective molar fractions of components 1 and 2 in the two-component monolayers; and are the respective collapse pressures of components 1 and 2; is the collapse pressure of the two-component monolayer at a given composition of (or ); ω1 and ω2 are the corresponding molecular areas at the collapse; ξ is the interaction parameter. As seen in Fig. 4, the solid curve at higher surface pressures was drawn by adjusting ξ in Eq. (2) to obtain the best fit for the experimental values of collapse pressures. All the systems here are of the positive azeotropic type, which indicates the stronger interaction between heterogeneous monolayers. The interaction energy can be calculated as the following equation,

where z is the number of nearest neighbors, equal to 6, in a close-packed monolayer, and the interaction energy is Δε = ε12 – (ε11 + ε22)/2 (48); εij denotes the potential energy of interaction between components i and j. In the small mole fraction region, the ξ value for the DPPC/F4H11OH system is largest among the three systems. This is found to depend on the fluidity of monolayers for pure components: F4H11OH > F6H9OH > F6H11OH. In the large mole fraction region, the two components nearly cause the ideal interaction or very weak interaction due to the small ξ value (near zero). On the other hand, the ξ value for the DPPC/F8HmOH systems except for m = 5 is quite larger in magnitude in the small XF8HmOH region. That is, the values for the F8HmOH systems are almost 2−3 times larger in magnitude than those for the F4H11OH and F6HmOH systems. It is suggested that the interaction mode of alcohols with F-chains shorter than 8 is quite different from that of F8HmOH. Considering that the ξ value is estimated at the close-packed state of monolayers, it is found that the F-chains are not restrained strongly by the hydrophobic chains of DPPC. That is, it can be said that the F4- and F6-moiety in the alcohols possess the degree of freedom for the molecular motion at the linkage between H- and F-chains compared to a F8HmOH molecule (44).

Fluorescence Microscopy (FM) FM observations at the air-water interface can provide morphological information on the phase behavior of monolayers in relatively high resolution and magnification. FM measurements require a fluorescent probe, which is incorporated into the monolayer. The bright and dark contrasts are respectively assigned to LE (or disordered) and LC (or ordered) phases of lipid monolayers (49), which is based on the fact that the probe generally forms disordered monolayers at the interface due to its spatially bulky moiety (or fluorescent part). However, there is a worry of the possibility that the probe (or dye) affects the original phase behavior of monolayers. Thus, the resulting FM image should be checked in validity that the incorporation of FM probes has no influence on the original isotherm and the original domain shape in the image captured with Brewster angle microscopy (BAM). The BAM image is observed in situ at the 13 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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air-water interface and the capture requires no exogenous materials. The BAM observation is simply based on the difference in refractive index of monolayer phases caused by a change in the molecular density or packing at the surface (50, 51). It has been reported that the addition of less than 1 mol% FM probes produces few effects on the monolayer behavior (38, 52). Thus, the FM images presented here have been checked by being compared to the corresponding BAM images to have no influence of the probe on the original phase behavior (data not shown).

Figure 5. Fluorescent micrographs of the binary DPPC/F4H11OH monolayers on 0.15 M NaCl (298.2 K) at 12, 15, and 25 mN m−1. The monolayers contain 1 mol% of fluorescent probe (NBD-PC). The scale bar in the lower right represents 100 μm. Reproduced with permission from reference (44). Copyright 2015 Japan Oil Chemists’ Society.

The FM micrographs of the binary DPPC/F4H11OH monolayers are shown in Fig. 5. As for the DPPC monolayer, the images exhibit LC domains coexistent with LE regions in a clear and sharp contrast. The LC domain with counterclockwise arms typically at 15 mN m−1 is characterized at DPPC monolayers (20, 28, 53–55). In common, a domain formation is controlled by balance of a line tension at the boundary between disordered and ordered domains and a long-range dipole-dipole interaction between ordered domains (49, 56–61). When a small amount of F4H11OH is added into DPPC monolayers (XF4H11OH = 0.1), the ordered domain become larger in size and the domain shape changes to a nearly circular or bean-like form. This means that the F4H11OH addition 14 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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enhances contribution of the line tension at the phase boundary. However, the further addition induces the size reduction of ordered domains and modification of the shape as shown at XF4H11OH = 0.3 and 0.4. At 25 mN m−1 (for XF4H11OH = 0.3) and 15 mN m−1 (for XF4H11OH = 0.4), the specific modification of ordered domain shape is observed: the edge of the ordered domains changes into disordered phase. These phenomena correspond to the fluidizing effect on DPPC monolayers (28). Moreover, it indicates that a fluidizing effect of F4H11OH on DPPC monolayers is induced by surface pressures. This effect is considered to be characterized at the incorporation of fluorinated amphiphiles with F-chains shorter than 8 because there are many papers on the fluidizing effect induced by surface compositions in the binary systems containing the fluorinated amphiphiles with longer F-chains (23, 28, 31, 41). Apparently, the present system exerts not only the surface pressure-induced effect but also the surface composition-induced effect, which is shown in Fig. 4. At XF4H11OH > 0.4, the FM image remains homogeneously bright regardless of surface pressure (data not shown).

Figure 6. Fluorescent micrographs of the binary DPPC/F6H9OH monolayers on 0.15 M NaCl (298.2 K) at 12, 15, and 25 mN m−1. The monolayers contain 1 mol% of fluorescent probe (NBD-PC). The scale bar in the lower right represents 100 μm. 15 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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In the binary DPPC/F6H9OH system at XF6H9OH ≤ 0.1 (Fig. 6), the phase behavior is almost the same as the DPPC/F4H11OH system in terms of the domain shape. With the further addition of F6H9OH (XF6H9OH = 0.2), the surface pressureinduced fluidization, which corresponds to unclearness of the edge of ordered domains, is observed at 25 mN m−1. The fluidizing effect is observed in smalleramount addition compared to the F4H11OH system, which implies that the F6 moiety tends more to disturb the hydrophobic chain in DPPC at middle surface pressures. On the other hand, the DPPC/F6H11OH system does not exert the surface pressure-induced fluidization as seen in Fig. 7. The solidifying effect is, rather, observed with increasing XF6H11OH to 0.2. Interestingly, the ordered domain fuses with each other at XF6H11OH = 0.2. The domain fusion indicates attenuation of repulsive force between the domains, which is based on the long-range dipoledipole interaction. This is considered to result from the reduced polarization or dispersed dipole moment inside each ordered domain. Thus, the domain fusion and growth in size strongly support the mutual miscibility between DPPC and F6H11OH in the monolayer state.

Figure 7. Fluorescent micrographs of the binary DPPC/F6H11OH monolayers on 0.15 M NaCl (298.2 K) at 12, 15, and 25 mN m−1. The monolayers contain 1 mol% of fluorescent probe (NBD-PC). The scale bar in the lower right represents 100 μm. 16 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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17 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 8. Surface-pressure dependence of the ratio of ordered domain area (dark contrast) in FM images of the binary DPPC/F4H11OH (A), DPPC/F6H9OH (B), and DPPC/F6H11OH (C) monolayers. Adapted with permission from reference (44). Copyright 2015 Japan Oil Chemists’ Society.

Figure 9. Typical AFM topographic images of the binary DPPC/F4H11OH monolayers for XF4H11OH = 0.5 at 20 and 35 mN m−1. The scale bar in the lower right represents 500 nm. The cross-sectional profiles along the scanning line (white line) are given just below the respective AFM images. The height difference between the arrows is indicated in the cross-sectional profile. Reproduced with permission from reference (44). Copyright 2015 Japan Oil Chemists’ Society. 18 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 10. Typical AFM topographic images of the binary DPPC/F6H9OH monolayers for XF6H9OH = 0.3 at 20 and 35 mN m−1. The scale bar in the lower right represents 500 nm. The cross-sectional profiles along the scanning line (white line) are given just below the respective AFM images. The height difference between the arrows is indicated in the cross-sectional profile. A percentage of the ordered domain per frame of FM images is plotted as a function of surface pressure in Fig. 8. As for the DPPC/F4H11OH system (Fig. 8A), the ordered domain ratio in the range of 0 ≤ XF4H11OH ≤ 0.2 increases monotonously and finally approaches ~95% upon compression, which suggests that the small-amount incorporation of F4H11OH does not induce the fluidizing 19 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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effect. However, the percentage at XF4H11OH = 0.3 and 0.4 does not reach the value near ~95% due to the fluidizing effect on the ordered domains. In the case of the DPPC/F6H9OH system (Fig. 8B), the percentage at XF6H9OH = 0, 0.05, and 0.1 reaches nearly 100% with an increase in surface pressure. However, at XF6H9OH = 0.2, it is found that the ordered domain transfers rapidly into disordered phases within 5 mN m−1 beyond π = 20 mN m−1. The monotonous increase in the percentage occurs in the binary DPPC/F6H11OH system from XF6H11OH = 0 to 0.2 (Fig. 8C). The profile shows no reduction with respect to surface pressure in percentage as opposed to the DPPC/F4H11OH and DPPC/F6H9OH systems. Considering the fact that the fluidizing effect induced by surface pressures is not observed for the binary DPPC/F8HmOH system (20, 26–28), it is suggested that the compact corn-swing motion of F4- and F6-moieties in the alcohols as the fulcrum in the CH2−CF2 linkage disturbs or disperses the ordered domain at the phase boundary to transfer it to the disordered state at middle surface pressures.

Atomic Force Microscopy (AFM) The in situ microscopic observation at the air-water interface has limitations of resolution and magnification to catch the clear two contrasts at least on the image. As mentioned above, the phase morphology of monolayers at high mole fractions of the alcohols could not be visualized with BAM and FM in the present study. Therefore, the phase behavior and distribution have been observed at the nano-meter scale with AFM, which is herein the ex situ microscopy employing the Langmuir-Blodgett (LB) technique. However, the LB technique includes a possibility of changes of the native monolayer structures at the interface by the electric charge between the samples and substrates, and by the physical factors during the deposition procedure. AFM images may therefore not provide completely correct information on the phase behavior at the air-water interface but nevertheless allow us to understand phase morphologies that can’t be caught with in situ microscopic techniques (BAM and FM). The representative AFM images at XF4H11OH = 0.5 in the DPPC/F4H11OH system are shown in Fig. 9. There are many stripes (bright contrast) composed mainly of DPPC (44). The height difference between the stripe and the surrounding network (dark contrast) composed mainly of F4H11OH is ~0.5 nm irrespective of an increase in surface pressure from 20 to 35 mN m−1. These stripes are quite small in size compared to the ordered domains shown in Fig. 5. This is attributed to the transformation of ordered domains into disordered domains, which is induced by F4H11OH. Shown in Fig. 10 are the AFM micrographs at XF6H9OH = 0.3 in the DPPC/F6H9OH system. The image at 20 mN m−1 indicates the coexistence state of bright and dark domains. The bright domain is made mainly of DPPC because of the molecular length and the fact that the domain reduces in size and its occupied ratio in a frame also decreases at XF6H9OH = 0.5 (data not shown). Moreover, similarly to the DPPC/F4H11OH system, the height difference of ~0.5 nm remains regardless of surface pressure. However, the bright stripe is fused upon further compression to 35 mN m−1. The fusion of the stripes do not support the surface pressure-induced fluidization at the nano-meter scale. This may be because that F6H9OH dissolves 20 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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into the DPPC-rich domain by the surface pressure increase on the basis of the binary miscibility. This dissolution is also supported by the fact of reduction in occupied area of the domain with dark contrast with increasing surface pressures (Fig. 10). The AFM topographic images for the DPPC/F6H11OH monolayers shown in Fig. 11 are apparently different from the former two systems. As seen in the images at 10 mN m−1, the dark region increases in occupied area with increasing XF6H11OH. Therefore, it is found that the bright contrast is expressed by the DPPCrich domains. At XF6H11OH = 0.3, the DPPC-rich domain disturbs the formation of the network of F6H11OH at 10 mN m−1. The network is more difficult to be formed by the rise in surface pressure. Conparison to the AFM images in Fig. 10 allows us easily to understand the solidifying effect of F6H11OH on DPPC monolayers (XF6H11OH = 0.3). When F6H11OH is added further (XF6H11OH = 0.5 and 0.7), the F6H11OH network comes to be formed to disperse the DPPC-rich domain finely. Nevertheless, the ratio of bright domains per frame is kept high irrespective of the fine dispersion of DPPC-rich domains. The similar solidification have been reported for the systems containing partially fluorinated amphiphiles (20, 26).

Figure 11. Typical AFM topographic images of the binary DPPC/F6H11OH monolayers for XF6H11OH = 0.3, 0.5, and 0.7 at 10 and 35 mN m−1. The scale bar in the lower right represents 500 nm. The cross-sectional profiles along the scanning line (white line) are given just below the respective AFM images. The height difference between the arrows is indicated in the cross-sectional profile. 21 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Conclusion The lateral interaction between DPPC and partially fluorinated alcohols having perfluorobutyl or perfluorohexyl moieties has been elucidated employing the monolayer technique at the air-water interface. The two-component DPPC/F4H11OH, DPPC/F6H9OH, and DPPC/F6H11OH systems are found thermodynamically and morphologically to be miscible in the monolayer state. The mode of interaction for all the systems presented here is very similar in interaction parameter and energy, which are estimated from the isotherm at high surface pressures. However, the miscibility behavior at low and middle surface pressures is significantly different among the three systems. The DPPC/F4H11OH and DPPC/F6H9OH systems indicate a fluidizing effect on DPPC monolayers. In particular, at the specific compositions, the fluidization is induced by the increment in surface pressure. On the other hand, a solidifying effect on DPPC monolayers occures in the DPPC/F6H11OH system. Considering the difference in total carbon number between F6H9OH and F6H11OH, it is found that only the two methylene groups are the key to the two opposite effects. Generally, the amphiphiles with the Fn moiety (n ≤ 8) are accumulated less in the human body and the environment compared to perfluorinated and highly fluorinated amphiphiles. Therefore, perfluorobutylated or perfluorohexylated compounds are hoped for a potential use and application in medical and pharmaceutical fields as well as in industrial and materials science. With the aim of application particular in artificial pulmonary surfactant preparations, it is quite important to control the softness and hardness of pulmonary surfactant monolayers, especially DPPC. Herein it is demonstrated that the rigidity of DPPC monolayers is possible to be adjusted by the addition of the partially fluorinated amphiphiles. Moreover, the rigidity can be controlled by composition of monolayers as well as lateral pressure. These findings will necessitate a further understanding of interaction of fluorinated compounds with biomembranes and their constituents.

Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research 26350534 from the Japan Society for the Promotion of Science (JSPS).

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10. Krafft, M. P.; Rolland, J. P.; Vierling, P.; Riess, J. G. New J. Chem. 1990, 14, 869–875. 11. Nakahara, H.; Lee, S.; Krafft, M. P.; Shibata, O. Langmuir 2010, 26, 18256–18265. 12. Gerber, F.; Krafft, M. P.; Vandamme, T. F. Biochim. Biophys. Acta 2007, 1768, 490–494. 13. Gerber, F.; Krafft Marie, P.; Vandamme Thierry, F.; Goldmann, M.; Fontaine, P. Biophys. J. 2006, 90, 3184–3192. 14. Gerber, F.; Krafft, M. P.; Vandamme, T. F.; Goldmann, M.; Fontaine, P. Angew. Chem., Int. Ed. 2005, 44, 2749–2752. 15. Riess, J. G. Curr. Opin. Colloid Interface Sci. 2009, 14, 294–304. 16. Goss, K. U. Environ. Sci. Technol. 2008, 42, 456–458. 17. Burns, D. C.; Ellis, D. A.; Li, H.; McMurdo, C. J.; Webster, E. Environ. Sci. Technol. 2008, 42, 9283–9288. 18. Higgins, C. P.; Luthy, R. G. Environ. Sci. Technol. 2006, 40, 7251–7256. 19. Nakahara, H.; Nakamura, S.; Okahashi, Y.; Kitaguchi, D.; Kawabata, N.; Sakamoto, S.; Shibata, O. Colloids Surf., B 2013, 102, 472–478. 20. Nakahara, H.; Krafft, M. P.; Shibata, A.; Shibata, O. Soft Matter 2011, 7, 7325–7333. 21. Broniatowski, M.; Dynarowicz-Łatka, P. Langmuir 2006, 22, 2691–2696. 22. Broniatowski, M.; Dynarowicz-Łatka, P. Langmuir 2006, 22, 6622–6628. 23. Lehmler, H.-J.; Bummer, P. M. Colloids Surf., B 2005, 44, 74–81. 24. Gaines, G. L., Jr. Langmuir 1991, 7, 3054–3056. 25. Fontaine, P.; Fauré, M.-C.; Bardin, L.; Filipe, E. J. M.; Goldmann, M. Langmuir 2014, 30, 15193–15199. 26. Nakahara, H.; Hirano, C.; Shibata, O. J. Oleo Sci. 2013, 62, 1029–1039. 27. Nakahara, H.; Hirano, C.; Fujita, I.; Shibata, O. J. Oleo Sci. 2013, 62, 1017–1027. 28. Nakamura, S.; Nakahara, H.; Krafft, M. P.; Shibata, O. Langmuir 2007, 23, 12634–12644. 29. Yu, S.-H.; Possmayer, F. J. Lipid Res. 2003, 44, 621–629. 30. Veldhuizen, R.; Nag, K.; Orgeig, S.; Possmayer, F. Biochim. Biophys. Acta 1998, 1408, 90–108. 31. Lehmler, H.-J.; Jay, M.; Bummer, P. M. Langmuir 2000, 16, 10161–10166. 32. Nakahara, H.; Tsuji, M.; Sato, Y.; Krafft, M. P.; Shibata, O. J. Colloid Interface Sci. 2009, 337, 201–210. 33. Nakahara, H.; Nakamura, S.; Hiranita, T.; Kawasaki, H.; Lee, S.; Sugihara, G.; Shibata, O. Langmuir 2006, 22, 1182–1192. 34. Nakahara, H.; Shibata, O.; Rusdi, M.; Moroi, Y. J. Phys. Chem. C 2008, 112, 6398–6403. 35. Nakahara, H.; Shibata, O.; Moroi, Y. Langmuir 2005, 21, 9020–9022. 36. CRC Handbook of Chemistry and Physics, 91st ed.; CRC Press: Boca Raton, FL, 2010; pp 2610. 37. Nakahara, H.; Shibata, O. J. Oleo Sci. 2012, 61, 197–210. 38. Nakahara, H.; Lee, S.; Shibata, O. Biophys. J. 2009, 96, 1415–1429. 39. Broniatowski, M.; Miñnes, J.; Dynarowicz-Łatka, P. J. Colloid Interface Sci. 2004, 279, 552–558. 23 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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40. Broniatowski, M.; Sandez Macho, I.; Miñnes, J., Jr.; Dynarowicz-Łatka, P. J. Phys. Chem. B 2004, 108, 13403–13411. 41. Hiranita, T.; Nakamura, S.; Kawachi, M.; Courrier, H. M.; Vandamme, T. F.; Krafft, M. P.; Shibata, O. J. Colloid Interface Sci. 2003, 265, 83–92. 42. Shibata, O.; Krafft, M. P. Langmuir 2000, 16, 10281–10286. 43. Gaines Jr., G. L. In Insoluble Monolayers at Liquid-Gas Interfaces; Interscience Publishers: New York, 1966; pp 281−288. 44. Nakahara, H.; Ohmine, A.; Kai, S.; Shibata, O. J. Oleo Sci. 2013, 62, 271–281. 45. Shah, D. O.; Schulman, J. H. J. Lipid Res. 1967, 8, 215–226. 46. Marsden, J.; Schulman, J. H. Trans. Faraday Soc. 1938, 34, 748–758. 47. Savva, M.; Acheampong, S. J. Phys. Chem. B 2009, 113, 9811–9820. 48. Joos, P.; Demel, R. A. Biochim. Biophys. Acta 1969, 183, 447–457. 49. McConnell, H. M. Annu. Rev. Phys. Chem. 1991, 42, 171–195. 50. Hénon, S.; Meunier, J. Rev. Sci. Instrum. 1991, 62, 936–939. 51. Hönig, D.; Möbius, D. J. Phys. Chem. 1991, 95, 4590–4592. 52. Wang, C.; Li, C.; Ji, X.; Orbulescu, J.; Xu, J.; Leblanc, R. M. Langmuir 2006, 22, 2200–2204. 53. Weis, R. M.; McConnell, H. M. Nature 1984, 310, 47–49. 54. Scholtysek, P.; Li, Z.; Kressler, J.; Blume, A. Langmuir 2012, 28, 15651–15662. 55. Leiske, D. L.; Meckes, B.; Miller, C. E.; Wu, C.; Walker, T. W.; Lin, B.; Meron, M.; Ketelson, H. A.; Toney, M. F.; Fuller, G. G. Langmuir 2011, 27, 11444–11450. 56. Benvegnu, D. J.; McConnell, H. M. J. Phys. Chem. 1993, 97, 6686–6691. 57. Benvegnu, D. J.; McConnell, H. M. J. Phys. Chem. 1992, 96, 6820–6824. 58. McConnell, H. M. J. Phys. Chem. 1990, 94, 4728–4731. 59. Moy, V. T.; Keller, D. J.; McConnell, H. M. J. Phys. Chem. 1988, 92, 5233–5238. 60. Keller, D. J.; Korb, J. P.; McConnell, H. M. J. Phys. Chem. 1987, 91, 6417–6422. 61. Keller, D. J.; McConnell, H. M.; Moy, V. T. J. Phys. Chem. 1986, 90, 2311–2315.

24 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Chapter 2

A Review of Hydrophilization of Oxidized Nanocarbons Publication Date (Web): December 8, 2015 | doi: 10.1021/bk-2015-1215.ch002

Masaki Ujihara*,1 and Toyoko Imae1,2 1Graduate

Institute of Applied Science and Technology, National Taiwan University of Science and Technology, 43 Keelung Road, Section 4, Taipei 10607, Taiwan 2Department of Chemical Engineering, National Taiwan University of Science and Technology, 43 Keelung Road, Section 4, Taipei 10607, Taiwan *E-mail: [email protected].

Nanocarbons, e.g. fullerene, carbon nanotubes, and graphene, can be oxidized to add solubility and dispersibility to them in aqueous media. The functional groups introduced by the oxidation are further modified by esterifications, amidation, and the other reactions to improve the dispersibility. Thus obtained water-dispersible nanocarbons can be used for the drug carriers in drug delivery systems. While these water-dispersible nanocarbons are obtained by top-down methods, bottom-up approaches to synthesize nanocarbons have been recently developed. The precisely designed compounds as the model of nanocarbons are revealing the behaviors of nanocarbons, and functionally-oriented nano-sized carbon materials, named “carbon quantum dots”, have been examined as imaging agents. In either approach, effects of detailed structures on the functions of nanocarbons are still under investigation.

© 2015 American Chemical Society In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Introduction From the latter part of the twentieth century, carbon materials with new molecular structures have been discovered one after another. Fullerene (buckminsterfullerene, C60) was discovered in 1985, and the higher fullerene (C70, C76, and higher) have been reported today (1, 2). A carbon nanotube (CNT) was formally discovered in 1991, although tubular structures in carbon materials were reported earlier (3). Then, graphene was prepared and characterized in 2004 (4). These discoveries of the novel carbon materials were sensational and stimulated researcher’s motivation. Today, many nanocarbons have been reported, such as nanoribbon and nanohorn (5, 6). With these nanocarbons, the conventional carbon materials, such as graphite and carbon blacks, have been reviewed as the starting materials to prepare the nanocarbons (7, 8). While these starting materials are used in “top-down” approaches, which decompose the materials to nano-size, “bottom-up” approaches, which build up the nano-materials from low molecular compounds, have been recently gathering attention. The nanocarbons obtained by the bottom-up approaches are examined as model compounds for fundamental research and used as functional “carbon dots”. After the discovery of new carbon materials, their superior properties in electrical and mechanical fields were reported, and they were anticipated to extend the frontiers in many fields (9, 10). Nanocarbons have dimensions of several nm to hundreds nm, which are suitable for biological applications: Their large surface areas allow high activity for sensing, and their small sizes can lead to penetration into tissues (11, 12). However, their applications were not rapidly developed, despite the high expectations. One of the reasons for this lagging is the low dispersibility of carbon materials. Because these carbon materials easily agglomerate, their applications are limited. Due to the carbon material’s poor dispersibility in aqueous media, serious issues arise for biomedical applications. To improve the dispersibility of nanocarbons in aqueous media, the addition of proper solubilizing groups or other chemical modifications are required. In this review article, several methods to disperse nanocarbons for easier handling will be introduced. The oxidation of nanocarbons to introduce hydrophilic groups, the chemical modifications of the oxidized nanocarbons, and synthesis of nanocarbons from dispersible materials will be discussed.

Oxidation of Nanocarbons and Their Dispersibility in Water The nanocarbons can be classified into two groups. The materials consisting of only carbon (e.g. fullerene, CNT, and graphene) are basically hydrophobic, and then introduction of hydrophilic moieties is required to disperse them. On the other hand, some nanocarbons synthesized from hydrophilic compounds (e.g. carbon dots) can retain their hydrophilic moieties and then be dispersible in aqueous media. In this section, the chemical modifications of the former group are discussed. The chemical modification is a strong approach to make the nanocarbons soluble/dispersible into the media. Fullerene derivatives have been studied, and 26 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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their synthesis methods are well established (13). Fullerene is hydrophobic, but the introduction of hydrophilic moieties can make fullerene derivatives watersoluble (14). Today, many chemical modifications for water-soluble fullerene derivatives have been reported, and they can be referred elsewhere (15). One of the modifications is carried out by the oxidation reactions onto the fullerene. Typically, the oxidation of fullerene introduces hydroxyl groups into the molecular structure and makes it water-soluble. The oxidation of fullerene is carried out by the reaction with fuming sulfuric acid (up to 12 hydroxyl groups) and successive H2O2 (up to 40 hydroxyl groups) (16, 17). When the oxidation reaction is performed with H2O2 in the presence of phase-transfer catalyst, the reaction is facilitated and the C60(OH)44 can be obtained (18). These water-soluble fullerenes are commercially available as the radical sponge®, which is used in cosmetics as an antioxidizing agent (19). The CNTs can be chemically modified to enhance their dispersibility, too. The CNTs are considered to be used in biological fields as the absorber of near-infrared light: The CNTs introduced in the tissue absorb the near infrared light and generate reactive oxygen species (ROS) and heat for photodynamic and photothermal effects, respectively (20). Some researchers demonstrated that the CNTs could be used as drug carriers after chemical modifications to improve the dispersibility (21). Many methods to modify the CNTs have been proposed, and the oxidation is a popular one. When the oxidation reaction targets the defects (e.g. 5-membered rings) in the single walled CNT (SWCNT), similarly to that for fullerenes, the ends of SWCNT are selectively modified, because they have similar structures to the hemispheres of fullerenes (22). The hydroxyl groups also can be introduced on the ends of CNTs selectively; however, this reaction can open the SWCNT when the reaction condition is strong enough (23). That is, the framework of the ends can be destroyed by oxidation and the carboxyl groups are introduced, while the oxidation of fullerene should retain the “buckyball” framework and introduce only hydroxyl groups (16–19). Although the selectivity is useful to modify the ends of SWCNT without degradation of the characteristics of SWCNT, the open-end SWCNT can’t be dispersed in water even after shortening into 100 to 300 nm in length (24). The hydrophobic interaction and the π-π interaction between the sidewall of SWCNT are still strong, and then surfactants are needed to avoid flocculation of the open-end SWCNT in water (24). In biological applications, the length of SWCNT should be shortened enough to decrease the interactions of sidewalls to inhibit the undesired agglomeration and adsorption. The shortened SWCNT derivatives can be dispersed in aqueous media well, and then indicates less toxicity (25). Compared to the bundles of SWCNT, the spherical aggregates of SWCNT, named the single walled carbon nanohorn (SWCNH) have better dispersibility. The SWCNH has dimension in the range of 50-100 nm in diameter and can be dispersible in common organic solvents without chemical modification, and mild oxidation on the tips leads good dispersibility in water (26, 27). These characteristics of SWCNH are due to a geometric effect: The spherical aggregates (SWCNH) interact each other by their tips, and then the π-π interaction between their sidewalls is suppressed. The oxidation on the edges allows a good water-dispersibility of the SWCNH in aqueous media, because the contacting area in SWCNH toward water is limited to the tips. Therefore, the oxidized SWCNH can be used for biological applications. 27 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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The tips of SWCNT are opened by the mild oxidation, and the oxidized SWCNT encapsulates drug molecules inside as a microcapsule for drug delivery systems (DDS) (28). On the other hand, the reaction on multi-walled CNT (MWCNT) have options for chemical reactions. Because the MWCNTs consist of concentric multiple layers, the inner layers can retain the length and functions of pristine CNT, while the outer layers are used as the sacrifice layer (29–32). The oxidized MWCNTs can be served for further modifications with biomaterials, such as enzymes and antibodies, and then used for the electrical sensors (29–32). The oxidation of edges and sidewall of MWCNT can be controlled by the reaction conditions. Namely, the edges can be selectively removed by the reaction using warmed HNO3 (33), and the mixture of HNO3/H2SO4 leads to the oxidation of whole CNT structures (34–36). These oxidation reactions introduce oxygen functional groups to the MWCNT, and then the oxidation can be confirmed by the FT-IR absorption spectroscopy: Typically, absorption band of carboxyl groups appears around 1750 cm-1 (34). The differences are present in the appearances of the products: The product after HNO3 treatment is a powder that can ready be filtrated, whereas the product after treatment with HNO3/H2SO4 becomes a paste that causes clogging during filtration. The excess oxidation on sidewall can result in longitudinal cleavage of the tubular structure (Figure 1). When the oxidation reaction on the sidewall is accomplished by usage of strong oxidizer (e.g. KMnO4), the tubular structure is “unzipped” to be the nano-ribbon structures of graphene oxides (37).

Figure 1. TEM image of multi-walled carbon nanotubes after oxidation using concentrated HNO3 at 100 °C for 24 h. Arrows indicate opened parts in nanotubes. Reproduced with permission from reference (29). Because the plane structure of condensed aromatics is chemically stable, the oxidation of graphene requires strong reaction conditions, as the reaction 28 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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on sidewalls of MWCNT does. The synthesis method of graphene oxide (GO) using the mixture of KMnO4, HNO3, and H2SO4 was established by Hummers in 1958 (38), and this method was superior to the previously-existing methods using KClO3/HNO3 (Brodie, 1859) and KClO3/HNO3/H2SO4 (Staudenmaier, 1898) in the safety and reproducibility (39, 40). As the result of severe oxidation, Hummers’ method provides the water-dispersible GO, while the graphite oxide obtained by Brodie method is swelled by alcohols (41). Therefore, Hummers’ method has been used as a standard method to obtain the GO. However, this method still requires a long time to accomplish the oxidation, and then some modifications and improvements have been reported (42–44). As significant developments, the typically modified Hummers’ method (Kovtyukhova et al, 1999) using K2S2O8 and P2O5 in H2SO4 was reported (42), and the improved Hummers’ method (Marcano et al, 2010) was performed in a mixture of KMnO4/H2SO4/H3PO4 (9:1) (43). In these methods, NaNO3 was alternated by phosphoric acid, and then, the gas generation of NOx and undesired nitration can be avoided. Today, the synthesis methods of GO are still developing to shorten the reaction time and decrease the waste (44). The GO are usually exfoliated from the graphite oxides, and then, the size of GO sheets represents that in the graphite oxides. Typically, the size of commercially available GO is in the range from several hundred nm to several μm, and it can be decreased to smaller than 100 nm (hereafter, named nano-GO) by ultrasonication (45). When large GO was ultrasonicated, the nano-GO of 100 nm in diameter was obtained with a good reproducibility (46) (Figure 2). The size of nano-GO can be varied by the precursors and the reaction conditions, too. For example, the GO prepared from a carbon black (amorphous carbon) after a weak oxidation condition (refluxed in concentrated HNO3) takes a few nm in diameter, and it can be soluble in water as an adsorbent for aromatic compounds (47–49). The adsorption ability of this nano-GO depended on the size of particles, namely, the larger nano-GO could adsorb more adsorbate, 2-naphthol and the other aromatic compounds (48, 49). This suggests that the nano-GO with very small dimensions can be used as the drug carrier, although the space to load the drug molecules should be ensured (50, 51). As another approach, the nano-GO of several nm can be prepared by the successive oxidation after the conventional GO preparation, too (52). Thus, the size of GO can be designed for the intended usage in the range from a few nm to several hundred nm. After oxidation, the nanocarbons become water dispersible; however, their dispersibility is differed by environmental effects, such as pH and coexisting ions. The carboxyl groups introduced by the oxidation reaction are ionized to R-COOand then increase the water dispersibility at neutral to alkaline conditions; however, they can decrease hydrophilicity and bind together in the acidic condition (Figure 3): The threshold is around pH 4 as expected from pKa of carboxylic acids (46, 48). The polyvalent ions, e.g. Ca2+, Mg2+, and polyamines can crosslink the oxidized nanocarbons via the ionic interactions between carboxylate groups (45, 53) (Figure 4). Therefore, the oxidized nanocarbons should be used in neutral and alkaline conditions without polyvalent ions. Otherwise, their carboxyl groups should be capped by further modifications, as explained in the next section. 29 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 2. TEM images (A and C) and size distributions (B and D) of graphene oxide before (A and B) and after (C and D) ultrasonication by an ultrasonic horn for 4 hs. Five samples were performed, and their size distribution became less than original GO after the ultrasonication (original GO: 416.4 ± 27.9 nm, nano-GO: 96.2 ± 2.3 nm). Reproduced with permission from reference (46).

Figure 3. Zeta potential of graphene oxide dispersed in aqueous solutions. Reproduced with permission from reference (46). 30 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 4. Dispersions of graphene oxide without (A) and with (B) poly(amido amine) (PAMAM) dendrimer (generation 4, amine terminated). Reproduced with permission from reference (53).

Chemical Modification of Oxidized Nanocarbons The oxidation reactions introduce many functional groups into nanocarbons. Typically, epoxide, carbonyl, carboxyl, and hydroxyl groups can be observed in the nanocarbons as known as Lerf-Klinowski model for GO (Figure 5) (42, 43, 54). These functional groups can be used for the further chemical modifications to improve the water solubility and to functionalize the oxidized nanocarbons. The reactions in this section are basically applicable commonly to the other oxidized nanocarbons, such as oxidized CNTs. The epoxy groups are highly reactive to the nucleophilic species, such as amines. The GO reacts with primary amines at room temperature in an aqueous medium (55, 56). This reaction can be used to immobilize the other molecules directly on the GO, and then the immobilization of aminated cyclodextrin (CD) was examined to add the molecular recognition properties to GO (56). The CD/GO nanocomposites could catch the guest molecules in aqueous dispersions, and then they could be used for the drug delivery system and the sensing. In the other directions, the reaction of epoxides with amines in GO can result in the reduction of GO (57, 58) and the N-doping to graphene (57–59). The covalent bonding formation between amines and GO via epoxy reaction is applicable to immobilize the other biomaterials, e.g. proteins; however, the reaction of GO with polyamines can cause the cross-linking of GO and then lead to undesired aggregations and precipitations. As mentioned in section 2, the polyamine causes aggregation by its ionic interaction with carboxylates. Then, the epoxy groups covalently bind the polyamines, and the aggregate becomes irreversible (see Figure 4). Therefore, the reaction of GO with amines should be carried out in alkaline conditions to avoid aggregation. 31 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 5. Structural model of graphene oxide (Lerf-Klinowski model). Hydroxyl groups in graphene sheet are perpendicular to the sheet. Reproduced with permission from reference (54). Copyright 1998 American Chemical Society. The immobilization of amines can be performed in the milder reaction using carboxyl groups, too (26, 27, 29–36, 50). The carboxyl groups are activated for amidation reaction using the condensation reagents, and typically water-soluble carbodiimide is used in aqueous media. In this case, the reaction with amines should be processed in the alkaline conditions to avoid the aggregation, as mentioned above: The aggregates can be covalently bonded by the amide linkages via the initial aggregation formed by the ionic interactions. The aggregate of nano-GO cross-linked by the PAMAM dendrimer with amine terminals was shown in Figure 6A. On the other hand, the activation of carboxyl groups is applicable to the esterification reaction. The reaction of GO with PAMAM dendrimer with hydroxyl terminals did not result in the aggregation (Figure 6B). The ionic interactions between the GO and alcohols were weak, and then the initial aggregation was not formed. Therefore, the reaction proceeded homogeneously in the dispersion. While the amidation of amines can be performed in aqueous media, non-aqueous reaction is suitable for the reactions with alcohols. In the non-aqueous media, the other activation reactions such as the reaction with SOCl2 in organic solvents are applicable to activate the carboxyl groups (34, 35). Then, the amines and alcohols are applied to the GO dispersion to obtain the corresponding amides and esters, respectively (46, 50, 51, 53). Using these methods, biomaterials, e.g. proteins, can be directly immobilized on the nanocarbons via their own amino groups (29), or amine groups are introduced as the linkers to the desired materials which do not have amine group, e.g. CD (36, 50). The esterification is popular to introduce the polyethylene glycols (PEGs) on GO to design the carrier of DDS (45). The PEGylation can improve the 32 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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water dispersibility of GO by the hydrophilicity of PEG, and the dispersibility is stabilized by capping the carboxyl groups which are pH-sensitive and cross-linked by the polyvalent cations (see section 2). In the biological systems, the PEGylation on GO can extend the circulating life as the PEG covers the GO from the immune system (60). Thus, the esterification of PEG and the other hydrophilic polymers, e.g. the hydroxyl-terminated PAMAM dendrimer, can extend the biological applications of GO. Moreover, the other functional groups can be introduced to the GO by the reactions of carboxyl groups. Typically, the GO is functionalized by folic acid (FA) for cancer targeting after the modification with the hydroxyl-terminated PAMAM dendrimer, and then the obtained FA/dendrimer/GO nano-composites are examined for the DDS (51, 61).

Figure 6. TEM images of nano-sized GO after reaction with amine-terminated PAMAM dendrimer (generation 4) (A) and hydroxyl-terminated PAMAM dendrimer (generation 4) (B), respectively. Reproduced with permission from reference (46).

However, the chemical modifications on nanocarbons, such as the esterification of oxidized CNT with heparin, can significantly decrease the loading amount of drugs which bind on the CNT by the π-π interaction, because of the steric hindrance and competitive adsorption of the introduced groups on CNT (62). Therefore, the modification of oxidized nanocarbons should be designed to achieve the high loading. The hybridization with CD can be a solution to enhance the capacity for drug loading as the CD provides new binding sites for hydrophobic drugs (36, 50). For another example, the heparin was further hybridized with polyglycolic acid (62). This co-polymer provided a microenvironment as hydrophobic moiety and hydrogel, which can retain drug molecules in the vicinity of CNT. The other polymers, such as the dendrimers and hyperbranched polymers, can also be used to encapsulate the drugs and nanoparticles (27, 30–32, 34, 35, 50, 51, 59). In the viewpoints of solubilization processes, the different interactions between drug molecules and solubilizers can result in the selective solubilization: The π-π interaction of aromatic compounds 33 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

can stabilize their adsorption on GO (50, 51), while the macrocyclic moieties, typically CD, encapsulates small and hydrophobic ones (36, 50); typically aliphatic compounds can be solubilized. The hydrogel can allow the retention and slow releasing of hydrophilic drugs (27, 50, 51). Thus, the solubilization and releasing behaviors of drugs on GO derivatives should be verified; however, the research on these behaviors are yet limited.

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Nanocarbons Synthesized by Bottom-Up Methods The GO and the other oxidized nanocarbons reviewed in the previous sections are prepared by top-down methods; decomposed to be nano-sized from the bulk materials such as graphite. Recently, another approach, bottom-up methods, is focused to design the nanocarbons. The bottom-up methods to design the nanocarbons today can be classified into two types: (1) precise syntheses, and (2) functionally-oriented designs. The precise syntheses allow well-controlled introduction of hydrophilic moieties, and therefore, the water-dispersibility will be designed by these bottom-up methods. The precise syntheses of nanocarbons have been examined for the effective synthesis of fullerene (63), SWCNTs (64), and graphene derivatives (65–68). Apart from that, the precise synthesis can allow the control of size and functional groups of nanocarbons, in viewpoints of solubility and dispersibility, the synthesis methods are used to introduce concavity and convexity into the surfaces of nanocarbons to decrease their π-π interaction. Several model compounds of graphene are illustrated in Figure 7.

Figure 7. Precisely synthesized model compounds of graphene. (A) warped nanographene (C80H30), (B) tetrabenzo[8]circulene, and (C) ovalene (C32H14). Rings with 5, 7, and 8 members were marked by their numbers (66–68). The “warped nanographene” (C80H30) was synthesized from the corannulene (C20H10), which possesses a 5-membered ring (66). The obtained compound has a 5-membered ring in the center and five 7-membered rings, which result in the negative Gaussian curvature. These warped graphenes can be dissolved in common organic solvents as expected; however, another nanographene synthesized with negative curvature, tetrabenzo[8]circulene (containing an 8-membered ring), has poor solubility in the common solvents and indicates limited solubility in 1,2-dichlorobenzene (solubility: 10 mg / mL) (67). This 34 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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different solubility suggests the geometric effects on solubility of nanocarbons. Importantly, the interaction of nanocarbons with surrounding media is affected not only by the geometric effects, but also by the electron state of nanocarbons: Typically, the dispersibility of SWCNTs is differed by their chirality (69–71). Amines can adsorb on the metallic SWCNT (69). The pre-solubilized SWCNTs by surfactant, such as sodium dodecylsulfate, are mixed in the agarose gel, and then the semiconductive SWCNT is selectively trapped in the gel (70). The similar effect is observed for the selective solubilization of semiconductive SWCNT by polyfluorene derivatives, too (71). Now, the warped nanographenes are semiconductive, and therefore, the effects of electron states should be considered (66, 67). While the warped nanographenes can be models of the defects in graphene, the graphene-like compound with low-molecular weight and plane structure, ovalene (C32H14), can be considered as the model of the part of graphene surrounded by the defects (isolated domains in graphene) (68). The ovalene is sparingly soluble in toluene but soluble in 1-methylnaphthalene, and it is fluorescent. The GO contains many isolated domains in its sheet, and these domains behave as semiconductors (72). Therefore, the study on interactions of the synthesized nanographene can elucidate that of GO with small molecules adsorbed on it. Beyond that, the designed nanocarbons can take unique structures, such as helicene (73), and their solubility and dispersibility should be important to understand functionality of carbon materials. On the other hand, new nanocarbons functionally-oriented have recently been reported as “carbon dots” or “carbon quantum dots (CQDs)” to emphasize their functionality (74, 75). The concept of CQDs is wide and focusing on the functions as nano-sized semiconductors: Therefore, the nano-sized GO and the other nanocarbons mentioned above can be included in the group of CQDs. However, the CQDs newly reported in these few years are synthesized by the bottom-up approaches using various materials which do not include the condensed aromatic rings. The starting materials can be synthetic compounds; however, the natural compounds such as sugar (76), citric acid (77), milk (78), fruit juice (79), and fruit peel (80) can be used in the viewpoints of biocompatibility and green chemistry. These precursors are processed in harsh conditions, typically heated around 200 °C in aqueous medium in the autoclave, and then suffer hydrothermal reactions, which include messy chemical processes, e.g. dehydration, condensation, rearrangement, cyclization, aromatization, and oxidation reactions; as conventionally summarized as the caramelization (81, 82), Maillard reaction (83), and carbonization (84). In these reactions, condensed aromatic rings, which characterize the CQDs, are formed and developed. The products generally appear brown solutions and contain nanoparticles (CQDs) of several nm. The coexisting low-molecular compounds (such as phenols) and large particles in the products can be removed by dialysis and centrifugation, and then the yield of CQDs in a process depends on the definition of the desired CQDs (80). Their chemical structures are ill-defined and contain condensed aromatic rings. The aromatic moieties are segregated by carbonyl, carboxyl and hydroxyl groups; these functional groups stabilize t he CQDs dispersed in aqueous media. These functional groups can be chemically modified to improve their dispersibility in living tissues as the other nanocarbons can be (see the 35 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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session 3) (85). The characteristics of chemical structures formed in the process are strongly depending on the precursors and the other coexisting materials: For example, the CQDs obtained from proteins contain many amine groups, and then such CQDs can be used as the N-doped CQDs (79). On the other hand, there is no nitrogen source in citric acid itself, and the hydrothermal reaction can be processed with amines, such as ethylenediamine, to synthesize the N-doped CQDs (78). The chemical structures of CQDs are not yet cleared; however, the functionalization of CQDs seems to be achieved by embedding functional groups originally contained in the precursors, e.g. porphyrin rings from botanical extracts. The material design of CQDs should be developed in terms of organic chemistry, although the functionality and applications of obtained CQDs have been mainly discussed. The CQDs can be used for biological research as the imaging agents, taking their advantages of low acute toxicity and fluorescent properties (86). The fluorescent properties of CQDs depend on the chemical structures, and the ill-defined structures of CQDs provide the variety of electron states in the CQDs, as appeared as multicolor fluorescence (87) (Figure 8). The CQDs have multiple excitation bands, and their emission bands are varied by many factors, such as coexisting materials (88). Therefore, the CQDs can be used as the multifunctional probes and sensors, although their characteristics are not yet clearly understood.

Figure 8. Fluorescence spectra of CQDs prepared from citrate, ethylenediamine, and iron compounds. (a) excitation spectrum at emission at 443 nm. (b) emission spectrum at excitation at 324 nm. (c) emission spectrum at excitation at 373 nm. Reproduced with permission from reference (87).

Conclusions In this review, derivatives of nanocarbons (fullerene, carbon nanotubes, and graphene) were introduced in viewpoints of solubility and dispersibility. The nanocarbons are attracting each other via π-π interactions, and then the interactions should be weakened by the fragmentation of aromatic regions to be dispersed in solvents. Moreover, the nanocarbons are basically hydrophobic, and then the hydrophilic moieties should be introduced in the molecular structures. Oxidation reactions are preferable to satisfy the both of fragmentation and 36 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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hydrophilization. The oxidation on nanocarbons provides several functional groups into their molecules: Typically, epoxy, carboxyl, and hydroxyl groups are introduced, and they can be further modified to improve the functionality and stability in the dispersion. The epoxy group can directly react with amines, and the carboxyl groups are used for amidation and esterification with the assistance of condensation agents. Thus, the oxidized nanocarbons can be dispersed in aqueous media, and the remaining aromatic regions can load hydrophobic moiety in molecules. This solubilization behavior of nanocarbon derivatives is used for the DDS. Recent studies on new nanocarbons designed by bottom-up approaches are also reviewed. The precise syntheses of warped nanographenes suggest that the proper curvature in molecules can decrease their π-π interactions and control their solubility. These studies using the precisely designed model compounds will verify the solubilization mechanisms of nanocarbons and the drug-loading process in the DDS. Another bottom-up approach is the preparation of ill-defined CQDs via messy reactions of low molecular compounds. This approach is convenient and suitable to obtain multifunctional fluorescent probes; however, their behaviors are not systematically understood yet. The nanocarbons, whichever prepared by the top-down or the bottom-up approaches, are now gathering attentions in the biological fields, because they are considered to be less toxic than the conventional QDs such as CdSe nanoparticles: The low toxicity of nanocarbons has been examined for their acute effects, but their chronic effects is not yet confirmed. Meanwhile, the nanocarbons have been revealed as generation source of the ROS in body, and this behavior can be hazardous but applicable as anticancer drugs (20, 25, 89). The nanotoxicology has only just begun (90), and the biological effects of nanocarbons need further research, similar to the health effects of coffee, which includes the oxidized nanocarbons.

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Chapter 3

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Interactions between Graphene Oxide and Biomolecules from Surface Chemistry and Spectroscopy Shanghao Li,1 Zhili Peng,1 Xu Han,1 and Roger M. Leblanc*,1 1Department

of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, Florida 33146, United States *E-mail: [email protected]. Tel.: +1-305-284-2194. Fax: +1-305-284-6367.

Graphene oxide (GO), an oxidized form of graphene, holds a similar single-atom-thick structure to graphene, but possesses plenty of oxygen−containing functional groups, such as carboxyls on the edges and hydroxyls and epoxies on the basal plane. GO possesses extraordinary properties, including low cost manufacturing, rich colloidal properties, high adsorption, and strong fluorescence quenching. GO has also recently been demonstrated to have advantageous applications in the biomedical field and in biosensing. Nevertheless, one critical question needs to be addressed before any actual applications can be discussed: how does GO interact with biomolecules? In this chapter, we will approach this question and briefly summarize the recent progress from the views of surface chemistry and spectroscopy methodology.

Introduction Graphene oxide (GO) is a two−dimensional, atomically thin carbon nanomaterial with functional oxygen−containing groups, such as carboxyl groups at the edges, hydroxyl and epoxide groups mainly at the basal plane, and some C=C sp2 domains (1, 2). Compared with other nanomaterials, the extremely large surface area on both sides, one−atom thickness (1.1 ± 0.2 nm), abundant functional groups, and good dispersion in water render GO as an ideal solid substrate to bind biomolecules through both covalent and non−covalent interactions (3, 4). The history of GO related research can be extended back to © 2015 American Chemical Society In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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about 150 years ago on some of the earliest studies involving the chemistry of graphite oxide (5). The recent decades have witnessed great research progress of GO in biological applications, such as biosensing (6–8), controlled drug delivery (including peptides, proteins, nucleic acids and anticancer drugs) (9–11), cellular uptake (12, 13), microscopic imaging (14–16), and photothermal treatment for cancers and Alzheimer’s disease (17–19). Although great progress has been achieved in research for the potential applications of GO in the biological systems, one significant question needs to be considered before any actual applications: How does GO interact with biomolecules, i.e. amino acids, peptides, proteins, nucleic acids, and lipids? Studies of the interactions between these biomolecules and GO can provide further supports and insights for the potential applications of GO. Unfortunately, there is very limited information available to answer such a fundamental and important question. Hence, the purpose of this chapter is intended to summarize the recent progress of the interacting studies between biomolecules and GO from a viewpoint of surface chemistry and spectroscopy.

Structure and Characterization of GO Although tremendous efforts have been made in the research of GO, the exact atomic structure of GO currently remains largely unknown and under debate (5, 20, 21). Usually, the nature of the functional groups in GO strongly depends on the reaction conditions, such as starting materials, preparation time, and reaction temperatures. In fact, GO may be considered as a family of carbon containing nanomaterials, rather than a single compound, due to its complex structure. We will briefly discuss the structure and characterization of GO from the most commonly used methods, such as spectroscopy and microscopy. The GO sheet consists of a carbon network with both sp2−hybridized carbon atoms in hexagonal rings (i.e. graphene−like C=C) and carbon atoms bearing oxygen functional groups (i.e. C–OH, C-O-C, C=O, and -COOH) (5, 22). The sp2−hybridized graphene−like domains are often found in the sheets of GO, while the oxygen−containing functional groups are mainly dispersed at the basal plane or the defects of the GO sheet (Figure 1A). Various spectroscopic and microscopic techniques have been applied to characterize the structure and morphology of GO. Figure 1B shows a typical UV-vis absorption spectrum of GO aqueous dispersion. It displays a maximum absorption at 229 nm due to the π−π* transition of aromatic C=C bonds and a shoulder around 300 nm due to the n−π* transition of C=O bonds (23–25). As displayed in Figure 1C, the Fourier transform infrared spectroscopy (FTIR) of GO synthesized by the strong acid oxidation method shows in the structures of GO the groups of hydroxyl (C–OH, 3000–3600 cm-1), ketone (C=O, ~1750–1850 cm-1), carboxyl (COOH) (~1600–1750 cm-1), sp2-hybridized C=C (in-plane stretching, ~1500–1600 cm-1), and epoxide (C–O–C, ~1280–1330 and 800–900 cm-1) (26, 27). GO has a couple of bands in the Raman spectrum (Figure 1D), i.e. the in-phase vibration of sp2–hybridized carbon networks (G band) at ~1590 cm-1 and a disorder band caused by the oxygenated groups at the edges (D band) at ~1350 cm-1 (28). The 44 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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integrated intensity ratio of the D- and G-bands (ID/IG) is often used to indicate the oxidation degree in the structure of GO. The morphology of GO has been widely studied using atomic force microscopy (AFM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM) (Figure 2). Among these techniques, AFM is likely the most widely used tool in the determination of the surface topography of GO with lateral dimension and precise height profile. While the size distribution of GO sheet obtained from microscopic studies is quite diverse, the height of single layer GO is usually around ~1 nm (18, 24, 29–31).

Figure 1. (A) Proposed chemical structure; (B) UV-vis absorption; (C) FTIR spectroscopy; and (D) Raman spectroscopy of graphene oxide. Figure 1B is reproduced with permission from reference (23). Copyright 2012 American Chemical Society; Figure 1C is reproduced with permission from reference (26). Copyright 2013 Institute of Physics.

Interaction between GO and Amino Acids Interaction between amino acids with GO has attracted significant attention in research because of the ubiquitousness of the amino acids and the potential biomedical applications of GO in biological systems. α-Amino acids are biologically essential organic compounds composed of amine (–NH2) and carboxylic acid (–COOH) functional groups at the α position, along with a side-chain. They are the basic building-blocks of peptides and proteins. Depending on the properties of the side-chain group (i.e. polar, non-polar, acidic, 45 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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basic, and aromatic), the interaction between amino acids and GO is driven by various forces, such as electrostatic, hydrophobic, hydrogen bond and/or van der Waals interactions (32).

Figure 2. (a) A tapping mode AFM image of graphene oxide (GO) sheets on mica surface, (b) the height profile of the AFM image, (c) TEM image of the GO, and (d) SEM image of the GO. Reproduced with permission from reference (31). Copyright 2011 Ivyspring International Publisher.

Adsorption Study Zhang et al. investigated experimentally the possible adsorption of 20 amino acids to the surface of GO (Table 1) (32). The adsorption was determined by the concentration changes before and after incubation with GO. It was found that the interaction strength between GO and the amino acids in phosphate buffered saline (PBS) at pH 7.4 followed the order Arg > His > Lys > Trp > Tyr > Phe (Table 1) (32). Their experiments showed that the other 14 amino acids have little adsorption on the surface of GO. As expected, the amino acids Arg, His and Lys with positive charge have strong electrostatic interactions as the main driving forces with negatively charged GO surface. Compared with Arg and Lys, His has an imidazole (C3H4N2) ring substituent, which has been demonstrated from molecular modeling studies that the imidazole ring has strong π–π interaction (33). 46 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Therefore, besides electrostatic interactions, π–π interaction also contributes to the adsorption of His to the surface of GO. Theoretical studies also display that the aromatic rings of the amino acids (e.g. Trp, Tyr and Phe) prefer to orient in parallel with respect to the plane of graphene via the π–π interactions (33). The π–π binding strength with graphene surface follows the trend: Trp > Tyr > Phe > His depending on the polarizability of the amino acid. As mentioned above on its structure, GO has some intact graphene–like islands which facilitate the adsorption between the GO nanosheet and the aromatic amino acids (Trp, Tyr, Phe and His) via the π–π interaction.

Table 1. The Concentrations of the Tested Amino Acids (nmol/mL) before and after Incubation of GO in PBS at pH 7.4 and the Corresponding Concentration Ratios (Revised from Reference (32)) Amino Acid

Before incubation (nmol/mL)

After incubation (nmol/mL)

Ratio

Arg

87.80

55.52

0.6323

His

77.30

55.32

0.7157

Lys

93.89

69.91

0.7446

Trp

125.02

95.17

0.7612

Tyr

92.81

81.05

0.8733

Phe

94.89

89.34

0.9415

Other 14 amino acids

--

--

0.9722~1.0262

Fluorescence Quenching of Trp or Tyr by GO Fluorescence spectroscopy and quenching are widely used to investigate the interaction of substances with molecules due to its sensitivity, low cost and ease for operation. These techniques can sensitively detect the changes of the local environment of the fluorophore by simply measuring the fluorescence signal (34). It has been known that GO can quench the emission of fluorescent molecules or nanoparticles, such as organic dye molecules (35, 36), fluorescent labels (29, 37), and quantum dots (QD) (24, 38), through Förster resonance energy transfer (FRET) from the fluorescent part to GO. The fluorescent dyes or labels usually contain aromatic rings and the quenching is via non−covalent interactions, such as electrostatic interactions, hydrogen bonding, hydrophobic and π−π interactions between GO and the dye molecules or fluorescent labels (29, 39, 40). Based on the structure and component of fluorescent amino acids (Trp or Tyr), there should be a non−covalent interaction between them and GO, quenching the fluorescent assay. Indeed, the experiments showed the strong quenching effect of GO. 47 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Experimentally, the amino acid Phe is also fluorescent, but it is not suitable for the quenching study due to its low quantum yield (41). Therefore, Phe will not be mentioned in this part. When mixed with GO, the fluorescence intensity of Trp was strongly quenched without shifting the emission peak (Figure 3A). Similar fluorescence quenching phenomenon was also observed in Tyr assay.

Figure 3. Fluorescence quenching of Trp by GO. (A) The fluorescence quenching of Trp (10-6 M) by mixing with different concentrations of GO. (B) The quenching of Trp (F0/F, black) and fluorescence lifetime ratio (τ0/τ, red) as a function of GO concentrations. Reproduced with permission from reference (23). Copyright 2012 American Chemical Society. After the correction of the “inner filter effect” (23, 42, 43), Stern−Volmer plot of F0/F against the concentration of GO is showed in Figure 3B (black color), where F0 and F are the fluorescence intensity at the maxima in the absence and in the presence of GO, respectively. If the quenching mechanism is static only, the fluorescence lifetime should remain the same, as this process does not affect the excitation state of the fluorophore (43). Hence, the static quenching can be described by the classical Stern−Volmer plot with linear fit. However, the fluorescence lifetime of Trp increases slightly with linearity when GO is added (red line, Figure 3B), and the plot of F0/F against GO has an upward curvature (black line, Figure 3B). Similar observation was also found for Tyr. These observations indicate that the quenching of Trp and Tyr by GO is mainly static quenching, slightly combined with dynamic quenching.

Hydrophobic Interaction Study between Trp or Tyr and GO Since both Trp and Tyr have a hydrophobic moiety, they may have hydrophobic interaction with the hydrophobic part of GO in the process of quenching. Triblock copolymer Pluronic F127 (PF127) was utilized in the experiment with a hope to block this interaction due to the fact that PF127 was previously shown to have strong hydrophobic interaction with GO (44, 45). PF127 consists of a central hydrophobic block of polypropylene glycol flanked by two hydrophilic blocks of polyethylene glycol. The hydrophobic segments 48 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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have previously been shown to interact with the hydrophobic part of GO while the hydrophilic chains extend to the aqueous solution (44, 45). When mixed GO with PF127, the hydrophobic moiety of GO is proposed to be covered by the hydrophobic part of PF127, screening the hydrophobic interaction between GO and Trp. As shown in Figure 4A, the mixture of GO:PF127 (1:1, w/w) has a lower quenching efficiency than the corresponding GO concentration in the absence of PF127. Since PF127 itself does not affect the fluorescence of Trp, the observation of reduced quenching supports the assumption that the added PF127 blocks the hydrophobic interaction between GO and Trp. Lower quenching effect of the mixture of GO and PF127 is also observed for Tyr.

Figure 4. Sterm–Volmer plot of Trp under different conditions. (A) 10-6 M Trp against the concentration of GO alone, and the mixture of GO:PF127 (1:1, w/w). (B) 10-6 M Trp against the concentration of GO at pH 5.6 and 9. Reproduced with permission from reference (23). Copyright 2012 American Chemical Society.

Electrostatic Interactions between Trp or Tyr and GO Electrostatic interactions can also exist between GO and Trp during quenching. The carboxylic groups of GO are readily deprotonated with negative charge when GO is dispersed in pure water at pH 5.6. If electrostatic interactions are important for the quenching, decreased quenching efficiency between GO and Trp at basic pH is expected, as Trp is also negatively charged (the isoelectric point for Trp is 5.9). As expected, the value of F0/F is lower at pH 9 compared with that at pH 5.6 (Figure 4B), indicating the existence of electrostatic interactions in the quenching process. Similar to Trp, the quenching efficiency between GO and Tyr at pH 9 is also lower than that at pH 5.6.

Interaction between Peptides or Proteins and GO As GO has recently been exploited for drug delivery, biomolecule detection, and near−infrared photothermal treatment for cancers and Alzheimer’s disease (17–19), the understanding of the interaction between GO and peptides or proteins 49 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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is fundamentally essential, especially for those drug− or disease−related peptides or proteins. Similar to the studies mentioned above, fluorescence spectroscopy is a simple method to investigate the interaction between peptide/proteins and GO. The quenching property of GO can also be used for protein detection by measuring the fluorescence signal with or without target proteins. Furthermore, the unique nanostructure of GO with large specific surface area makes this material an ideal platform for protein immobilization through non-covalent binding. In this part, we will focus on the interaction between GO and peptides or proteins through the measurement of their intrinsic fluorescence, fluorescence quenching and immobilization.

Intrinsic Fluorescence of Peptides and Proteins The intrinsic fluorescence of a peptide or protein is a mixture of the fluorescence from individual fluorescent amino acid residues (i.e. Trp, Tyr and Phe). Among them, most of the intrinsic fluorescence emissions of a folded protein are due to excitation of Trp residues, with some weak emissions due to Tyr and even weaker emission from Phe (46). Typically, the emission peak of Trp depends on the polarity of the local environment around it and can be used as a probe of the conformational state of a protein (46, 47). Furthermore, Trp is a relatively rare residue found in proteins; many proteins contain only one or a few Trp residues. Therefore, Trp fluorescence can be used as an intrinsic measurement for the conformational and environmental change of the residues. Aβ40 is the most abundant form of Aβ peptides, and its fibril formation is associated with the development of Alzheimer’s disease. Human islet amyloid polypeptide (hIAPP) is a 37 amino acid residue peptide and its amyloid deposits are the major source of fibrils found in the islets of Langerhans of type 2 diabetic patients (48, 49). Both have only one fluorophore, the Tyr residue at position 10 for Aβ40 and position 37 for hIAPP. When mixed with GO, the intrinsic fluorescence of Tyr in the amyloid beta 1-40 (Aβ40) and human islet amyloid polypeptide (hIAPP) is quenched (Figure 5A) (23). While the emission peaks of both peptides are not shifted, the quenching indicates that there is binding interaction between GO and Aβ40 or hIAPP. The higher quenching of Aβ40 should not be due to the charge differences between Aβ40 and hIAPP, because the slightly negatively charged Aβ40 (isoelectric point 5.4) in principle should have weaker electrostatic interactions with GO than the positively charged hIAPP (isoelectric point 8.8). Instead, the structural differences between Aβ40 and hIAPP are likely the reason for the observed difference between their quenching efficiency. The single Tyr in Aβ40 has six aromatic amino acid residues in its vicinity (three phenylalanines and three histidines). Strong π−π and/or hydrophobic interactions may thus exist between these residues and GO. However, the Tyr residue in hIAPP is located at the negatively charged C−terminus, and there are no aromatic residues in this domain for the interaction with GO. Therefore, the quenching efficiency of hIAPP is lower than that of Aβ40. 50 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 5. The Stern-Volmer plot of different proteins aqueous solution quenched by GO at pH 5.6. (A) 10-5 M hIAPP and 10-5 M Aβ40 against the concentration of GO; (B) 10-6 M BSA and 10-6 M HSA against GO. Reproduced with permission from reference (23). Copyright 2012 American Chemical Society. GO also strongly quenches the fluorescence of bovine serum albumin (BSA) and human serum albumin (HSA), with a slightly higher quenching efficiency for HSA, as shown in Figure 5B. The quenching difference between these two proteins is likely related to their respective number of Trp residues. BSA has two Trp (Trp135 and Trp214) and HSA only one (Trp214) (50). It could also be due to protein conformational changes as a result of their interaction with GO, leading to a decrease of their fluorescence intensity. We also studied the fluorescence of other proteins, such as lysozyme and pepsin, and quenching was always observed in our experiments. Therefore, it is possible that GO is a universal quencher for intrinsic fluorescence of proteins.

Protein Detection Using the Fluorescence Quenching of GO If the peptide or protein detection system using GO is based on the fluorescence signal, the process in most cases follows the following pattern: (1) fluorescence quenching of the probe due to the strong non-covalent binding between the fluorescent moiety and GO; (2) target recognition by the probe detaching the fluorescent moiety from GO surfaces; (3) fluorescence recovery of the probe. In principle, by substituting the corresponding probe for the target, the same strategy can be easily extended to detect different peptides or proteins. In fact, this process in literature has been widely used in the field of analytical chemistry to detect proteins. Dong et al. designed a platform for effective sensing of thrombin by FRET from quantum dots (QDs) as donors to GO as an acceptor (24). The QDs were first conjugated to a molecular beacon which can recognize thrombin. The strong interaction between the molecular beacon and GO resulted in the fluorescence quenching of QDs. Once the molecular beacon was bound to thrombin, the distance between the QDs and GO became larger, weakening the interaction between GO and QDs. Therefore, the fluorescence of QDs was recovered. 51 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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To increase the sensitivity of protein detection using peptide-protein binding, Wang et al. utilized the fluorescence quenching property of GO to selectively detect cyclin A2, a prognostic protein indicator in many early-stage cancers (51). The fluorescence of the dye-labeled peptide probe, FITC-HAKRRLIF, is efficiently quenched when mixed with GO. Similar to what we have mentioned above about the interaction between aromatic amino acids and GO, the authors also proposed that non-covalent binding (i.e., electrostatic interactions, hydrophobic interaction, and π-π interaction) contributes to the efficient quenching of the dye by GO. Once the target protein, cyclin A2, is added, the fluorescence of dye was recovered.

Protein Adsorption on the Surface of GO GO used as a matrix for enzyme immobilization was first reported by Zhang et al. (3) Horseradish peroxidase and lysozyme molecules were immobilized onto the surface of GO via non-covalent bonding without any cross-linking reagents by simply mixing these components. The immobilized enzyme can be observed clearly by AFM imaging. Apparently, protein immobilization onto the surface of GO results from the synergic effect of different types of interactions. However, this process is dominated by the electrostatic interactions between negatively charged GO sheets and enzyme molecules. Lysozyme (LYZ) content is relatively high in biological fluid samples, such as tears, milk, saliva, urine and blood serum (52–54). Lysozyme is a small globular enzymatic protein with 129 residues linked by four disulfide bridges. This enzyme is part of the innate immune system, damaging the bacterial cell walls by hydrolyzing the peptidoglycan (55). GO has been previously used for the purpose of analyte detection and quantification from the biological fluid. To detect tetracyclines from milk samples, Liu et al. applied GO to enrich the analyte and then detect the target by MALDI−TOF mass spectroscopy (56). In another study, Song et al. constructed a system of GO-fluorescein isothiocyanate-labeled peptide for the detection of matrix metalloproteinase 2 in complex serum samples (57). Yan et al. studied both in vitro and in vivo biocompatibility and cytotoxicity of GO when it was intravitreally injected into rabbit eyes (58). Their preliminary results showed that GO has good intraocular biocompatibility with little cytotoxicity on cell viability, cell morphology and membrane integrity. Due to the high abundance of lysozyme in these biological fluid samples, one has to consider the possible interaction and adsorption between lysozyme and GO in the experiments. Our group recently investigated the interaction between GO and lysozyme and the possible applications of this interaction in separation and selective adsorption of lysozyme (59). Surprisingly, compared to BSA and HSA (Figure 5B), a more dramatic quenching of lysozyme is observed under the same concentration with the increase in the content of GO (Figure 6A). In the presence of 10 µg/mL GO, the value F0/F of lysozyme increases to proximately 26.5. The fast reduction of fluorescence intensity indicates a much stronger interaction between GO and 52 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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lysozyme. To determine the nature of interaction between GO and lysozyme, the quenching has been investigated at pH 5.6, 10 and 12. Considering the isoelectric point of lysozyme is about 11, its charge strongly depends on the pH of the solution (60). Thus lysozyme is more positively charged at pH 5.6 than pH 10. From Figure 6A, lysozyme at lower pH has higher quenching effect by the negatively charged GO, indicating that the strong quenching of lysozyme by GO is predominantly due to attractions between lysozyme and GO. Other methods were used to further characterize the interaction between GO and lysozyme, i.e. zeta potential, dynamic light scattering and AFM imaging. At pH 5.6, the zeta potential of 5 µg/mL GO is -38.85 mV, however, the value increases to -9.05 mV in the presence of 14.3 µg/mL lysozyme. In order to find out the trend of zeta potential change in the GO/lysozyme mixture, a titration experiment of zeta potential was performed with a constant GO concentration at 5 µg/mL (Figure 6B). As more lysozyme is present, the zeta potential value of GO/lysozyme mixture shifts toward the more positive. When lysozyme is added to 20 µg/mL, the zeta potential reaches about 0 mV, probably corresponding to the maximum loading of lysozyme on the surface of GO. As shown in Figure 6C, in the GO/lysozyme mixture, the hydrodynamic diameter distribution depends on the pH and the concentration of GO. The size of GO/lysozyme decreases as the pH increases from 5.6 to 12 at each corresponding concentration of GO, indicating a weaker interaction at higher pH. In fact, the size of the mixture at pH 12 is almost the same as that of pure GO (data not shown). This may be due to the electrostatic repulsion between lysozyme and GO at pH 12 which are then both negatively charged. AFM was used to directly observe the morphology of GO/lysozyme at pH 5.6 on a freshly cleaved mica surface. It seems that lysozyme “glues” GO sheets together with uneven height from 3.5 nm to more than 20 nm (Figure 6D). We have so far demonstrated that strong interaction between GO and lysozyme results from the electrostatic interactions as described above, but it is worth noticing that some weak interactions may also exist, such as π-π interaction, hydrophobic interaction, and hydrogen bonding.

Adsorption and Desorption of Lysozyme on GO We demonstrated above that the strong interaction between GO and lysozyme is predominately resulting from electrostatic interactions. In our experiment, this interaction was so strong that we could use GO to remove and separate lysozyme from aqueous solution (59). Briefly, GO is added into a lysozyme aqueous solution to form a GO/lysozyme assembly, and then NaCl is added to precipitate GO/lysozyme. After centrifugation, the fluorescence (Figure 7A) and UV-vis absorbance (Figure 7B) of lysozyme in the supernatant almost completely disappears. Therefore, GO could be an excellent adsorbent material to remove lysozyme from aqueous solutions. In the pellet, the adsorbed lysozyme can be released by adding NaOH and CaCl2, which separate lysozyme from the surface of GO (59). 53 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 6. (A) F0/F of 10-6 M lysozyme against the concentration of GO at pH 5.6, 10 and 12. (B) Zeta potential of GO/LYZ aqueous solution against LYZ concentration at pH 5.6. The concentration of GO was fixed at 5 µg/mL. (C) Mean hydrodynamic diameter of GO/LYZ mixture. (D) AFM image of GO/LYZ mixture. The scale bar at the bottom right is 1 µm. Reproduced with permission from reference (59). Copyright 2014 American Chemical Society.

Figure 7. (A) Fluorescence spectra and (B) UV−vis absorption spectra of lysozyme before and after adsorption by GO. Reproduced with permission from reference (59). Copyright 2014 American Chemical Society. 54 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Selective Adsorption of Lysozyme One may use the strong interaction between lysozyme and GO to selectively adsorb lysozyme from a mixture of proteins on the basis of their electrostatic interactions. If two proteins are positively charged and have similar isoelectric points (pI), both will be adsorbed by GO without selectivity. Fortunately, very few natural proteins or enzymes bear as many positive charge as lysozyme. To study the selective adsorption of lysozyme by GO, we mixed lysozyme (LYZ, pI 11, 14.3 kDa) in 0.1 M phosphate buffer at pH 7 with ovalbumin (OVA, pI 4.9, 43 kDa), bovine serum albumin (BSA, pI 5.3, 68 kDa), or human serum albumin (HSA, pI 4.7, 66.5 kDa) (59). After GO and sodium chloride are added subsequently, the mixture was centrifuged to obtain the supernatant. In a binary protein mixture (i.e. LYZ/BSA, LYZ/HSA, or LYZ/OVA), the supernatant after centrifugation was characterized by spectroscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS−PAGE). The obtained fluorescence and UV−vis absorption spectra of the mixture after adsorption by GO were very similar to those of the control experiments using BSA, HSA or OVA alone (59). Experiments from the SDS-PAGE showed that the band of lysozyme at ~15 kDa disappeared upon addition of GO. Similarly, in a ternary of protein mixture, as shown in Figure 8, the SDS-PAGE displays that the band of lysozyme disappeared upon addition of GO (see Lane 2 and 4). These observations confirm that lysozyme was selectively adsorbed by GO, leaving other proteins in the solution (i.e. BSA, HSA, and OVA).

Interactions between Graphene Oxide and Oligonucleotides DNA and RNA based oligonucleotide nanotechnology plays an important role in biological research, and have attracted attention for decades (61, 62). The information encoded within the well-known Watson-Crick structure of DNA and RNA is the key to a variety of biological phenomena. However, there still remain huge challenges regarding their biological applications, such as detection limit, cost efficiency, and biocompatibility. Due to the remarkable chemical and physical properties of GO, the hybridized nanomaterials consisting of DNA or RNA with GO display a high potential for new generations of biosensors, drug delivery systems, molecular machines, and devices in many other fields (63–65). In an effort to investigate the role of interactions between oligonucleotides and GO, covalent and non-covalent binding strategies are generally employed to attach DNA or RNA to the GO, as illustrated in Figure 9. The fluorescence quenching of the probe by GO is due to the strong binding between the fluorophore and GO. When the target recognition happens, the fluorescent probe will be detached from GO surfaces, resulting in the recovery of the fluorescence of the probe. For example, Berry’s group reported biological applications of graphene based device in 2008 (66). In this study, the terminal amine group of single stranded DNA (ssDNA) covalently bonded to the carboxylic group from GO, which was confirmed by hybridizing it with its conjugated fluorescent labeled DNA probe. The preference of DNA tethering on thicker layers and on wrinkles 55 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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of GO were observed. In addition to covalent binding approach, non-covalent binding methods using van der Waals, π-π stacking and electrostatic interactions are more widely utilized for DNA detection (67–69).

Figure 8. Electrophoresis of a ternary mixture of LYZ/OVA/HSA and LYZ/OVA/BSA separated by 12 % SDS−PAGE. Protein marker (Lane M), LYZ/OVA/HSA control (Lane 1), LYZ/OVA/HSA adsorbed by GO (Lane 2), LYZ/OVA/BSA control (Lane 3), LYZ/OVA/BSA adsorbed by GO (Lane 4). Reproduced with permission from reference (59). Copyright 2014 American Chemical Society.

Figure 9. i) Fluorophore attached DNA was absorbed onto GO, and its fluorescence was quenched because of FRET. ii) Specific sequence ssDNA was added to the surface, which leads to the fluorescence restoration. 56 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Studies have shown that nucleobases, nucleosides, DNA and RNA can be absorbed to graphene based materials. Rao’s group studied the interaction of DNA base pairs with graphene by isothermal titration calotrimetry (ITC) (70). Their data have shown experimental binding energies of Guanine (G) > adenine (A) > cytosine (C) ≈ thymine (T). Moreover, Lu et al. immobilized dye labeled ssDNA onto the GO via the ionic interaction (11). The fluorescence of the conjugated organic dye (fluorescein-based dye) was quenched after adsorption onto the surface of GO (Figure 9A). When the target DNA was added (Figure 9B), the fluorescent dye would be detached from GO surface due to the formation of the complementary DNA between ssDNA and the target DNA, resulting in the reappearance of the fluorescence (Figure 9C). Recently, thiolated DNA was employed to assemble gold nanoparticles into two dimensional materials on top of the GO (71). DNA was coated onto the basal plane of GO by electrostatic interactions. This hybrid nanostructure showed good water solubility and exhibited a high potential for applications in catalysis, nanoelectronics, and biosensing platforms. Similarly, Huang’s group modified metal nanoparticles with DNA, followed by depositing it onto the GO via π-π stacking (16).

Interaction between GO and Lipids GO has been extensively studied for various biomedical applications (4, 72), and one novel direction of these studies is the development of new nanocomposite materials of GO with lipid membranes. However, detailed studies regarding the interactions between GO and cell membranes or model membrane systems is rather limited. In this part, we will focus on the latest studies on the interaction between GO and lipids.

Orientation of GO in the Presence of Lipid Monolayers at the Air−Water Interface Due to the unique structure of GO, which has an extremely thin layer (~1 nm) with large surface area and irregular shape, it is important to know how GO orientates itself when interacting with cell membranes. Our group recently investigated the interaction between GO and model membranes using the Langmuir monolayer technique that was applied at the air-water/aqueous interface and revealed the possible nature and orientations of GO when interacting with different lipids (45). Langmuir monolayer at the air−water interface is a typical two−dimensional (2–D) surface chemistry approach, widely applied for the structure and property studies of proteins and lipids at the air−water interface (73–75). Because of the deprotonation of carboxyl groups of GO sheets (76, 77), electrostatic interactions are expected to occur between negatively charged GO and charged headgroups of lipids. The air−water interface is an ideal platform for the study of interaction between lipids and GO, as the lipid molecules have readily oriented themselves at the interface with polar/charged groups merged 57 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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in the aqueous phase while hydrophobic moiety facing toward the air phase. Furthermore, Langmuir monolayer of lipid is a well-accepted in vitro model to mimic biological membranes (78), which can be considered as two weakly assembled Langmuir monolayers. Five different lipids were used in this study: positively charged dioctadecyldimethylammonium bromide (DODAB), 1,2-distearoyl-snglycero-3-ethylphosphocholine chloride salt (DSEPC); zwitterionic 1,2distearoyl-sn-glycero-3-phosphocholine (DSPC), and negatively charged 1,2-distearoyl-sn-glycero-3-phosphate sodium salt (DSPA) and stearic acid (SA). The same 18-carbon chain alkyl groups in the nonpolar tail with different charged head groups are purposely chosen to rationalize the possible interactions. The study revealed that the interactions are governed by electrostatic forces between the polar head groups of the lipids and GO, evidenced by the fact that GO successfully incorporated into the monolayer of DODAB and DSEPC (positive charged), but not DSPC, DSPA and SA (neutral/negative charged). It was further elucidated that the shielding effect from the substructure of the head group is also an important fact when considering the interactions between GO and lipids. Collectively considering the possible orientations of GO and experimental data, we proposed that an “edge-in” fashion is more likely to occur where GO penetrated into the DODAB lipid monolayer (Figure 10a and 10b). However, “face-in” fashion to penetrate the monolayer is not likely to happen for DSEPC monolayer (Figure 10C and 10D), probably due to the possible electrostatic shielding from sn-glycero-3-ethylphospho groups.

Interactions Between GO and Lipid Bilayers Frost et al. investigated the interactions between GO and liposomes (79). They prepared supported lipid bilayers with different charges on SiO2 surfaces by using extruded liposomes with a mean diameter of 80-90 nm. The model negatively charged liposomes, POPC/POPS (3:1), were prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS); and positively charged liposomes, POPC/POEPC (3:1), were prepared from POPC and 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC). The model membranes were then exposed to an aqueous suspension of GO, and then monitored by the quartz crystal microbalance with dissipation monitoring technique (QCM-D). It was found that when the negatively charged POPC/POPS (3:1) membrane surface is exposed to GO, there is no sign of adsorption of GO based on the QCM-D signal, which could be attributed to the electrostatic repulsion between the lipid membrane head groups and GO. In contrast, when the positively charged POPC/POEPC (3:1) membrane was used, there is a clear signal from the QCM-D indicating the flat adsorption of the GO flakes on the membrane and covers most of the surface. The finding once again demonstrates the importance of electrostatic forces between GO and lipid head groups when studying their interactions. Further 58 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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experiments found that after the adsorption of GO onto the positively charged POPC/POEPC (3:1) lipid membrane, an interesting multilayered structure could be obtained with further sequential additions of POPC/POEPC (3:1) liposomes and GO to the surface (Figure 11).

Figure 10. Schematic diagrams of the possible orientations of GO when interacting with different types of lipids monolayer at the air-water interface. Reproduced with permission from reference (45). Copyright 2013 American Chemical Society.

Antibacterial Activity of GO Toxicity of synthetic carbon nano-materials, such as fullerenes and carbon nanotubes have been extensively studied while only a few toxicity investigations on GO related materials are available. It is believed that the physical properties of GO such as solubility, dispersion and size would strongly influence their antibacterial activities. Recent investigations have showed that GO could exhibit strong antibacterial activities (80–83) The physical damages on the bacterial cell membranes by the sharp edges of GO nanosheets are believed to be responsible for the antibacterial activity observed in these studies. The interaction between GO and the bacterial membrane could result in destruction of membrane structure and the leakage of RNA from the cell (membrane stress) (83). Another mechanism was proposed that graphene-based materials might induce oxidative stress on 59 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Publication Date (Web): December 8, 2015 | doi: 10.1021/bk-2015-1215.ch003

bacterial cells (80). A three-step antibacterial activity mechanism was also proposed (81). The process begins with the bacterial deposition onto GO resulting in direct contact between bacteria and GO, which is followed by a disruptive interaction with bacterial, inducing membrane stress. The last step would involve the disruption of a specific bacterial process by oxidizing a vital cellular structure or component, inducing oxidation stress. These studies demonstrated that GO could be used as potential antibacterial materials in daily life to protect the public health.

Figure 11. Schematic representation of the formation of a POPC/ POEPC (3:1) lipid membrane and subsequent adsorption of GO. Further addition of POPC/POEPC (3:1) liposomes and GO results in a multilayered structure. The figure is not drawn to scale. Reproduced with permission from reference (79). Copyright 2012 American Chemical Society.

Conclusion Graphene oxide (GO) holds promising applications in biological and biomedicine fields due to its unique physical and chemical properties as a novel 2-dimensional nanomaterial. It is fundamentally important to investigate the interactions between GO and biomolecules for any practical applications. From the views of surface chemistry and spectroscopy, we briefly summarized the recent progress of the interaction studies between GO and biomolecules, including amino acids, peptides, proteins, oligonucleotides and lipids. Because of its unique one-atom-thick structure with plenty oxygen-containing groups and graphene-like domains, GO surface has high adsorbility of biomolecules via non-covalent binding, particularly electrostatic and π−π interactions. When the biomolecules are adsorbed onto the surface of GO by these interactions, the fluorescence of the intrinsic fluorophores or the dye attached will be strongly quenched. When these biomolecules are detached from the GO surface by recognition or competition, the fluorescence will be restored. Many new biosensors are reported and fabricated on the base of this mechanism for the detections of peptides, proteins, DNA and RNA. The interaction between lipid model membranes and GO is governed primarily by electrostatic interactions, as the experiments showed that positively charged lipids have a strong binding effect on GO. Recent 60 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

investigations demonstrated that GO can exhibit strong antibacterial activities, which may be due to the physical damage or oxidation stress of the bacterial cell membranes caused by GO.

Acknowledgments

Publication Date (Web): December 8, 2015 | doi: 10.1021/bk-2015-1215.ch003

R.M.L. gratefully acknowledges the support of the National Science Foundation under Grant 1355317. All authors gratefully acknowledge the reviewers for the valuable and constructive suggestions and comments on this chapter.

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Chapter 4

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The Role of Langmuir Monolayers To Understand Biological Events Luciano Caseli,*,1 Thatyane Morimoto Nobre,2 Ana Paula Ramos,3 Douglas Santos Monteiro,4 and Maria Elisabete Darbello Zaniquelli3 1Institute

of Environmental, Chemical and Pharmaceutical Sciences, Federal University of Sao Paulo, Rua Sao Nicolau, 210, 2° andar, Centro, Diadema, SP, Brazil, 09913-030 2Physics Institute of Sao Carlos, University of Sao Paulo, Avenida Trabalhador Saocarlense, 400, Parque Arnold Schimidt, Sao Carlos, SP, Brazil, 13566-590 3Chemistry Department, Faculty of Philosophy, Sciences and Letters of Ribeirao Preto, Department of Chemistry, University of Sao Paulo, Avenida Bandeirantes, 3900, Monte Alegre, Ribeirao Preto, SP, Brazil, 14040-901 4Institute of Sciences, Engineering and Technology, Federal University of Jequitinhonha and Mucuri Valleys, Rua do Cruzeiro, 01, Jardim São Paulo, Teófilo Otoni, MG, Brazil, 39803-371 *E-mail: [email protected].

In this chapter, we present an overview about different roles of Langmuir monolayers as biomembrane models, contributing to understand biological phenomena or help develop biotechnological process. Beginning with experiments that allow the development of the concept of biomembrane organization, we then discuss the interaction of lipids with bioactive compounds, peptides (i.e., fragments of proteins) and proteins. The presence of nanoparticles at the lipidic interface was also discussed. Finally, this chapter also shows some aspects of Langmuir monolayers on the nucleation and growing of nanocrystals mimicking biomineralization processes.

© 2015 American Chemical Society In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Colloid and surface sciences have been developed for the preparation of new materials and surface characterization techniques. However, the colloidal systems self-assembled by biologically relevant lipids can be also used as biomimetic models, due to the resemblance with the organization found in biomembranes. In this context, colloids and surface sciences can provide information about specific processes or for the elucidation of diseases (1). These biomimetic systems can also improve or accelerate the development of new drugs or diagnostic protocols as discussed in the following (2, 3).

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Langmuir Monolayer as Biomembrane Model System Micelles, liposomes (LP), planar bilayer membranes (BLM) and Langmuir monolayers (LM) are examples of colloidal self-assembled systems employed as biomimetic models. In common, these systems have an organized amphiphilic interface separating the hydrophilic (aqueous phase) from the hydrophobic (air or oil phases). Micelles are systems in equilibrium with no lateral pressure. In these systems, the area occupied by a lipid molecule at the interface can be easily accessed. Phospholipids with two alkyl chains can produce self-assembled planar or spherical bilayer systems. However, they cannot form spherical micelles due to geometrical constraints. When small amounts of lipids are deposited on the surface of water or of any aqueous solution, they can be spread along the air-water interface and a layer is produced, forming LMs, which consists of an organized monomolecular film (4, 5). In this case, the polar head of the lipid molecules is oriented toward the aqueous phase and the hydrophobic tail is oriented toward the air phase. The repulsive force between lipid molecules is responsible for their spreading and the origin of a positive surface pressure, π = γo - γ , defined as the difference between the surface tension of the aqueous subphase before (γo) and after the spreading of the monolayer (γ). In order to develop a model for the liquid state, Irving Langmuir upgraded an apparatus (developed by Agnes Pockels) that is able to measure changes in π while a moving barrier compressed the air-liquid interface, changing the available area per molecule, A(6). With these two sets of data, π and A, a surface pressure-area isotherm obtained at a constant temperature is plotted. The attractiveness in this simple and elegant experiment is the possibility to correlate macroscopic data with intermolecular forces acting between molecules forming the LM. The monolayer can be submitted to an external lateral pressure by means of mobile barriers forcing the monolayer to reach more condensed states so as to counterbalance the lateral pressure exerted by the lipid molecules (7). During the compression, we can study the properties that depend on the molecular area, such as lipid surface density, electrical charge density, surface compressibility (surface Young modulus), and dynamic surface elasticity. As shown in Table 1, the above-mentioned properties are difficult to measure by another technique. Also, temperature, pH, and electrolytic composition of the aqueous phase can be changed and controlled during the experiments. Additionally, these monolayers can be transferred to solid supports by the Langmuir-Blodgett (LB) approach, enlarging the number of techniques to 66 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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characterize the monolayer. A scheme for an LM as model for cell membranes is shown in Figure 1. Gorter and Grendel, who are pioneers in models describing membranes, proposed in 1925 that membranes of erythrocytes were composed of a bimolecular leaflet of lipid molecules (8). This model was proposed based on data obtained from a Langmuir trough experiment involving the spreading of lipids from erythrocyte membranes extracted with acetone. Ever since, lipid LMs as model for biological membranes have been used to study mechanisms of biological events. A recent review emphasises the growing interest in LMs to study interactions in biointerfaces, correlating topics with the structure of water, biophysics of peptides and enhanced inorganic-organic composites (9). LMs were also object of a review on interfacial chemical reactions catalyzed by enzymes, particularly those involving lipid molecules, and coupling of polyelectrolytes to oppositely charged monolayers (10). Another importance is the interaction of lipids organized in LM with peptides and enzymes as well as nucleating and growing of nanoparticles. Such subjects will be presented in this chapter.

Table 1. Comparison of Colloidal Biomembrane Model Systems Feature / Model System

Liposome

Micelle

BLM

LM

Typical Average Thickness (nm)

2

5

5

2.5

Lipid surface density change

No

No

No

Yes

Electrical charge density change

No

No

No

Yes

Surface elasticity access

No

Yes

Yes

Yes

Figure 1. Scheme for a monolayer of lipids and membrane proteins representing a model for cell membranes. Some of the properties that can be assessed through characterization techniques are represented in this sketch. 67 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Biomembrane Organization The lipid bilayer model of Gorter and Grendel obtained from LM experiments represented a major key point in the development of the biomembrane models (8, 11). Despite most proteins are not able to accommodate in half a bilayer, or many can denature at the lipid air-liquid interface, several experiments could be conducted to show the interaction of enzymes with lipids and the consequences of the catalytic activity of the enzyme, and these results will be discussed in details in the following sections of this chapter. Also, it is worth mentioning the importance of the values of surface density and surface elasticity of the biomembrane model for the enzyme activity (3, 12), as well as the increase in elasticity promoted by cardiolipin of a LM biomembrane model (13). Cardiolipin levels in mitochondria are associated with Parkinson´s disease. Also, the constitution and mechanism of ion channels in biomembranes is extremely relevant. After the establishment of the correspondence between the lipid-water interface in a lipid monolayer of a bilayer and those of a lipid monolayer formed on mercury electrodes (14), electrochemical experiments involving lipid monolayers containing gramicidin forming channels have also shown the importance of the lipidic environments and the composition of these environments for the channel activity (15). Another aspect of the membrane organization is the so-called raft domains (16). This corresponds to lipid microdomains induced by preferential packing of sphingolipids and cholesterol, where membrane-anchored proteins (through glycosylphosphatidylinositol anchor – GPI proteins) are preferentially located (17). The clustering of GPI proteins as well as lipid distribution in membrane models, LM or BLM, are mostly studied by means of AFM and fluorescence microscopy (18). These domains play an important role in signal transduction, enzyme activity and membrane transport.

Peptides and Proteins in Langmuir Monolayers Peptides are molecular sequences of amino acid units with molecular weight lower than 10,000. These structures have been intensively studied in the last decades since they are involved in several biological mechanisms. For instance, they can initiate an infectious process or be involved in the diagnostic of some diseases (19–21). Understanding the interaction of peptides with cell membranes can explain numerous biological events, including pathologies. Also, peptide-cell membrane model interactions have explored in order to elucidate the mechanism of action of peptides that act as drugs. Furthermore, some peptides can permeate cell membranes to deliver drugs (22). In some circumstances, peptides can also be the drug themselves (23–25). In all cases, LMs have been shown to be an appropriate tool to mimic a cell membrane, with easy control of the composition and the molecular architecture. Mimicking biomembranes using LM allows the study of specific events involving peptides separately from the numerous ones occurring at the same time on the cell surface. 68 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Peptides can be obtained from desired regions of proteins of interest to better understand the role of this specific region on protein-membrane interactions. In some cases, peptides corresponding to the C- or N-terminus (26) or containing specific binding domains of proteins can be synthesized (27), and their interaction with LMs can elucidate the mechanism about how proteins anchor to the membrane. Sometimes, it is necessary to screen the complete sequence of the protein to identify regions of interest, and then one can synthesize those different “pieces”, as peptides, to determine which region is related to a specific biological event (19, 27). This is how the Langmuir technique has been employed to study Alzheimer´s disease (AD). Despite the different hypothesis for the origin of AD, there is a consensus about the involvement of amyloid-β (Aβ) protein in this pathology since patients with AD present Aβ insoluble toxic fibrils in the brain (28, 29). In the literature, Aβ protein was deconstructed in peptides and the role of different regions of the protein on the fibrillation/aggregation process was studied (29–37). Since the conversion of the protein into its toxic form involves changes in secondary structure (from α-helix to β-sheet), polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS) has been used to evaluate these systems (29). Also, microscopic techniques such as epifluorescence and Brewster angle microscopy (BAM) are useful to identify aggregates at the interface. In some cases, Aβ peptides were synthesized containing an aliphatic chain attached either to its C- or to its N- terminal region, as a strategy to keep the peptide at the air-water interface. The role of the membrane in the aggregation of Aβ-peptides was also investigated, focusing particularly on the membrane composition (36). Viral infection is also an important process studied by LMs (38–41). In general, after attaching to the host cells, a virus infects the host cells by entering into the cells and then introducing its genetic material. This attachment involves the interaction of proteins from the viral envelope with proteins and lipids on the cell membrane of the target cell. After that, viral entry occurs usually by fusion of the membranes. For the Human Immunodeficiency Virus (HIV), the literature shows that the N-terminus region of the gp41 protein in the viral envelope is a putative fusion peptide between HIV and target cells. The peptide containing 23 amino acid residues of the gp41 N-terminus was synthesized and its interfacial properties as well as its interaction with different lipids were characterized (39). The results indicated the conditions for peptide aggregation at the interface and showed that an oblique orientation of fusogenic peptides inserted into the membrane is necessary to cause membrane perturbations, which favours the initial steps of the fusion process. Antimicrobial peptides (AMPs) are a class of peptides widely studied by the Langmuir technique. The great interest in these compounds is due to their potential as antibiotics. AMPs can be isolated from plants, bacteria, amphibians and mammals, including humans. AMPs can be also synthesized or bioinspired by some peptides found in nature (42). It has been reported that some parameters play an important role in determining AMP activity: peptide primary sequence and secondary structure, charge, amphiphilicity, hydrophobicity (polar/nonpolar amino acid ratio), and the angle related to hydrophilic/hydrophobic helix surfaces (43–51). In order to address the mechanism about how the peptide works and to improve its antimicrobial activity, peptides with different sequences were 69 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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synthesized and their interaction with membranes was evaluated. In some cases, replacing a single residue can drastically affect the interaction of the peptide with the membrane and consequently this may affect its activity (45, 46). For example, increasing the positively charged residues can enhance the interaction between the peptide and the lipid monolayers, especially those built with anionic lipids, which are the major constituents of bacterial membranes. However, the presence of additional non-polar residues (such as tryptophan) also boosted the interaction of the peptide with the lipid monolayer. This indicates that hydrophobic interactions are also important to damage the membrane. Alteration in the primary sequence of the peptide can also change its secondary structure, resulting in a more (or less) rigid structure and the change of the conformational flexibility of the peptide, which drastically affects the depth of peptide incorporation into the lipid monolayer (44–46). A very important property of an AMP is its membrane selectivity: as more selective to bacterial membranes, as more potential this peptide will be able to act as an antibiotic. This selectivity means a major interaction with bacterial membranes and no interaction with mammalian cells. The membrane selectivity is widely investigated by using LMs. Considering that mammalian cells have a more neutral character, the phosphatidylcholine lipids (DPPC - dipalmytoylphosphatidylcholine, DOPC - dioleoylphosphatidylcholine, egg-PC, etc.) are usually preferred. As bacterial cells are negatively charged, it is possible to predict, by using different lipid compositions in the LM, whether an AMP is a good candidate as a bactericidal drug. In addition, the detailed mechanisms of the AMP interaction with biomembranes and the potential to improve its activity by modification of the peptide structure can be investigated by LM technique. As a result, studies on the interaction between proteins and lipids by using LMs are very useful to understand phenomena occurring at the cell membrane level. Because of the great advance in techniques that are able to characterize interfacial events, it is now possible to obtain more details to address the interaction between lipid membranes and peptides/proteins. LM technique is powerful because lots of parameters are variable and can be monitored. The following example concerns a LM formed for pathology study. With LM technique, it is probable not only to address which peptide (region of protein) is able to interact with the membrane, but also to elucidate how the membrane can modulate this interaction when the parameters (like pH and temperature) related to the subphase are changed. As for peptides, how the peptide inserts into membrane depends on the composition and the architecture of the membrane and this is relevant to understand the pathology of a certain disease. Thus, a considerable variety of proteins were studied by the LM technique. Depending on the properties of various proteins, different strategies for the LM film confection have been employed (19, 52–63). The first method is co-spreading, which consists in spreading protein and lipid from the same stock solution. It is useful for some hydrophobic proteins (and peptides). However, since the protein is solubilized in organic solvents (e.g., chloroform), protein denaturation is possible (63, 64) , although proteins can readapt their conformation when submitted to other conditions and have their active conformation restored. 70 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Regarding the proteins soluble in aqueous solution, the injection method (i.e., the second method) is popular. This method consists in injecting the protein in the aqueous subphase, underneath a pre-formed lipid monolayer. This method is similar to the biological environment and has the advantage of being able to obtain kinetic parameters. The ability of the protein to diffuse and adsorb at the lipid interface can be monitored in terms of surface pressure versus time, and parameters like diffusion coefficient (23) can be obtained. If the diffusion is very slow, the protein can be homogenized in the aqueous subphase prior to the lipid film formation, and the method (i.e., the third method) can be called “protein as subphase”. In these cases, diffusion from the bulk to the interface can be ignored. Besides the similarities between these two last methodologies, surface pressure-area isotherms involving the same protein can be significantly different depending on the strategy of incorporation. In fact, there is a debate in the literature about proteins incorporated at lipid monolayers by using the subphase method: it was reported that proteins at the bare interface can denature, at least partially, due to the exposure of hydrophobic regions towards the air. However, considering that proteins are generally stored in the solid state, the exposure of hydrophobic regions towards the air should not be the only factor responsible for the protein denaturation, but the overall changes of the polypeptide at the interface including from those protein groups in contact with the aqueous phase. Although some authors observed that after lipid spreading at the interface the original conformation of the protein can be restored (63), there is still no agreement about this fact. The fourth strategy is spreading the protein on the top of the interface after lipid spreading. This strategy can be called “top methodology” (i.e., the fourth method); the idea is to avoid the contact of the protein with the organic solvent. In this strategy, the problem is that the protein solution can carry some lipid molecules to the subphase by aggregation. The last strategy (i.e., the fifth method) to incorporate a protein into a lipid film is to increase the ionic strength of the subphase. Then the protein is forced to adsorb at the lipid interface due to the salting out effect (65). Again, this methodology can result in protein unfolding due to the high salt concentration. Considering all the limitations and peculiarities mentioned above, LMs is a suitable strategy to mimic the cell membrane environment. In this way, interactions of several proteins with lipids were investigated successfully, such as the previously mentioned interaction between lipids and proteins related to fibril formation in neurodegenerative diseases, mainly Alzheimer´s disease (AD) (19).

Enzymes Adsorbed in Langmuir and Langmuir−Blodgett films Enzymes are biological compounds able to catalyze chemical reactions. Except for catalytic RNA molecules, enzymes are polypeptides containing special structures to interact specifically with the substrates and can promote a new mechanism for a given reaction. This new mechanism will result in a relative shorter time of reaction because the activation energies of the reaction are lowered in the new mechanism and the conversion rate of the substrates is increased to 71 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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millions of times faster. Many metabolic processes in cells require enzymes to occur in rates able to sustain life. Enzymes are usually specific for certain substrates due to their threedimensional structures. This specificity may be useful for biotechnology since enzymes can be used as a component of a sensor for a specific substance. In the literature, enzymes have been immobilized in solid matrices to serve as biosensors (66, 67). For Langmuir-Blodgett (LB) films, the first reports are related to the spreading of pure enzymes at the air-water interface and their subsequent transfer to solid supports (68–70). The advantage of the LB methodology relies on the facts that such films can be produced in several kinds of solid supports, producing molecularly ordered arrays and with high control of the molecular architecture. These facts open the possibility to construct devices with fast response with a low quantity of enzyme. Also, the films can be constructed in controlled environments, such as temperature, pH, and pressure. Furthermore, the sensitivity and detection limit may be pre-determined by changing some aspects of the molecular architecture, such as number of layers, and substances co-adsorbed. These aspects are interesting for the fabrication of biosensors based on the direct transduction of biological signals, which make these films as materials called “bio-inspired”. Enzymes are usually sensitive to vicinity conditions, and it is a challenge to find environments able to conserve the structure of the biomacromolecules and maintain at least part of their biological activity. Enzymes adsorbed at a clean airwater interface are reported. (67, 68, 70). If a higher amount of enzyme needs to be adsorbed, a high concentration of ions in the aqueous subphase favours enzymes to migrate from the bulk to the surface because of the salting out effect (71) as discussed as the fifth method in the previous section. However, an excess of salt may cause tough effects on the structure of the enzyme (72). A current approach to avoid the loss of enzyme activity is to form mixed lipid-enzyme monolayers at the air-water interface to be subsequently transferred to solid supports by using the LB methodology (73–76). The main idea is that lipids, being amphiphilic, contain hydrophobic and hydrophilic groups that help the accommodation of the enzyme into the lipid layer in such way that the major structures of the macromolecule are kept. In this case, the method of insertion of proteins can be done by using the second method in the previous section. For that, the ability of the enzyme to penetrate into the lipid monolayer can be investigated by varying the initial surface pressure prior to the enzyme insertion. Usually, high surface pressures decrease the ability of enzyme adsorption because the enzyme penetration may be inhibited when the surface pressure increases. Plots of increase of surface pressure owing to enzyme penetration versus initial surface pressures usually give the so-called exclusion surface pressure, πe (77), obtained by the extrapolation of the straight line to the x-axis (Figure 2). For initial surface pressures equal to or higher than πe, the enzyme is reported to be unable to penetrate into the lipid monolayer. However, this fact may not be considered as definite evidence that the enzyme cannot interact with the monolayer since adsorption of the enzyme on the lipid polar heads may still occur, and this would not lead necessarily to increase the surface pressure of the monolayer (78). Also, some hydrophobic enzymes may provide a sudden increase of the surface pressure 72 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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due to a fast enzyme penetration, in an effect called “piston” by some authors (79, 80). Further accommodation of the enzyme may provide a slow decrease of the surface pressure until equilibrium is reached.

Figure 2. Example for the determination of exclusion surface pressure of a protein inserted below a lipid monolayer.

Another point to be discussed is how an enzyme incorporated in a lipid monolayer in a high initial surface pressure can be compared to an enzyme incorporated in low initial surface pressures and then subjected to compression until a desired surface pressure is reached. Although thermodynamic equilibrium is expected for both, the process may be considered to be in a low dynamic state. Also, as the enzyme must be injected in specific points below the lipid monolayer, it is experimentally difficult to distribute the enzyme homogeneously, and equilibrium must be achieved after complete lateral diffusion of the enzyme, which is commonly slower than enzyme diffusion from the aqueous subphase to the air-water interface. A strategy to avoid the concentration of enzymes in specific points on the surface has been to spread the enzyme and the lipid together from the same solution (81–84). However, the organic solvents usually employed 73 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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to dissolve the lipid may denature the enzyme, and dispersion of lipids in water may make the spreading on the interface difficult. Another strategy is to immobilize the enzyme on a pre-formed lipid LB film. The solid substrate is inserted into the enzyme solution allowing for enzyme adsorption (70, 85–87), which promotes specific interactions of the enzyme with hydrophilic or hydrophobic surfaces. However, the weak association of the enzyme with the lipid surface may be a major drawback since the enzyme can be desorbed when re-inserted in aqueous solutions (88). A recent study compares the enzyme activity of uricase immobilized in several architectures of stearic acid (SA) as LB films (78). Uricase was adsorbed in pre-formed SA LB films, in which the LB film exposed the hydrophobic or the hydrophilic part. In other architectures, uricase was transferred with SA from a mixed enzyme-lipid LM. For that 1 or 4 monolayers were transferred. Enzyme activity for 1-layer LB films was higher than for 4-layer LB films, demonstrating that the catalytic activity is more relevant for enzymes incorporated in the outmost layer. Also, enzyme activity is better when the enzyme is adsorbed on hydrophilic layers, rather than on a hydrophobic layer. Since enzymes are immobilized in solid matrices together lipids, this functionalized biomimetic membrane has been shown to be structurally stable and able to preserve the enzyme activity for long periods of time (89). Enzyme activities in solid matrices are usually partially retained in comparison to that in solution (17), owing to restrictions of the biomacromolecule in terms of mobility and possibility to conformational adaptations. Higher enzyme activities in comparison with enzyme solutions are usually reported with heme-enzymes, for which the hydrophobic environments provided by the lipids favor the accessibility of the catalytic substrate to the catalytic site of the enzyme (64, 90). Typical behaviors of the enzyme retained at the surface of the biomimetic membrane have demonstrated potential usefulness of such assemblies for investigations in biomimetic environments, with favourable orientation of recognition sites. Also, self-molecular assembly of biomolecules allows for the insertion of the enzyme at a specific geometry, improving the recognition properties (17, 64, 90, 91). Figure 3 shows a scheme for enzymes being incorporated in LMs of lipids and the subsequent transfer to solid supports as an LB film with the purpose of using it as a matrix for molecular recognition. Regarding this scheme, it must be considered that proteins may adsorb on the solid support immersed in the aqueous subphase prior to the step of removal of the support. For this reason, some tests must be performed in order to check if the immobilized enzyme comes only from the film at the air-water interface. A possible test is inserting the solid support in the aqueous subphase with the enzyme dissolved, but with no lipid monolayer present. The solid support is then removed and the possible adsorption of the enzyme analyzed.Another possible experiment regarding this fact is to transfer the monolayer with the enzyme adsorbed to another compartment with a pure water subphase by means of a shallow lateral slot communicating the two compartments. After the mixed monolayer is transferred, the solid support is removed from the pure water subphase. In this sense, Table 2 shows some recent reports of enzymes adsorbed in Langmuir and LB films. 74 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 3. Scheme for obtaining mixed enzyme-lipid LB film.

Table 2. Enzymes Incorporated in LMs and/or LB Films Enzyme Organophosphorus Acid Anhydrolase

Main Finding/Reaction Product

Architecture

Ref.

Pure Enzyme On The Air-Water Interface

Enzyme Activity Could Be Measured

Ref. (92)

Lysozyme

Pure Enzyme On The Air-Water Interface

Salt Ions Minimize The Water-Accessible Surface Area Of The Protein, Enhancing Protein Dehydration And Assisting In Protein Refolding And Association.

Ref. (65)

Glutamate Dehydrogenase

Behenic Acid

Glutamate

Ref. (88)

Laccase

HeptylBis(Thiophene) Carbazole And Tricosenoic Acid

2,2′-Azino-Bis(3Ethylbenzthiazoline6-Sulphonate) Abts

Ref. (93)

Phosphatase Alkaline

Dimyristoyl Phosphatidic Acid (Dmpa)

P-Nitrophenolphosphate

Ref. (87)

Urease

Dipalmitoyl Phosphatidyl Glycerol (Dppg)

Urea

Ref. (94)

Tyrosinase

Arachidic Acid

Pyrogallol

Ref. (95)

Alcohol Dehydrogenase (Adh)

Dimyristoyl Phosphatidic Acid (Dmpa)

Ethanol

Ref. (96) Continued on next page.

75 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Table 2. (Continued). Enzymes Incorporated in LMs and/or LB Films

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Enzyme

Main Finding/Reaction Product

Architecture

Ref.

Horseradish Peroxidase

Dppg

Hydrogen Peroxide

Ref. (64)

Catalase

Dimyristoyl Phosphatidic Acid (Dmpa)

Hydrogen Peroxide

Ref. (90)

Uricase

Stearic Acid

Uric Acid

Ref. (78)

Sucrose Phosphorylase

Dmpa

Sucrose

Ref. (54)

Penicillinase

Dmpa

Penicillin

Ref. (97)

Cellulase And Adh

Dppc

Detecting Ethanol

Ref. (98)

Hyaluronidase

Dppc

Hyaluronic Acid

Ref. (61)

God

Phospholipid Analogous Vinyl Polymer

Signal Intensity Increasing With The Number Of Deposited Layers.

Ref. (99)

Also, it is important to mention that several studies use lipases inserted in the aqueous subphase and employ the lipid monolayer as catalytic substrate (99–103). Usually, the action of the enzyme changes the properties of the monolayer, which can be accompanied by analyzing how surface pressure varies with time or how infrared spectra are changed (104).

Bioactive Compounds in Langmuir Films as Biomembrane Models There are several reports in the literature studying the interaction between bioactive drugs and LMs. These studies have the purpose not only to understand how membrane interacts with active drugs, but also to increase its selectivity. For some drugs, the membrane is not the primary target of bioactive compounds, as they usually bind to a receptor (e.g., a DNA or an enzyme) inside the cell. However, the interaction with the membrane is crucial for the drug incorporation and to determine its selectivity. Also, the great interest in investigating the interaction between drugs and lipids is due to the possibility of incorporating such molecules in liposomes for drug delivery, especially aiming at the reduction of the toxicity. Many reports use LMs to investigate the interaction of membrane with antimicrobials (105–116), antiparasitic (117, 118), antitumor (119, 120), anti-inflammatory (121, 122), antipsychotic (123–126), and coronary vasodilator drugs (127). A suitable strategy to investigate drug selectivity is to build LMs of a specific compound found in a certain organism or organelle. Dynarowicz-Latka and co-workers verified that the antimycotic amphotericin B (AmB) is more toxic for 76 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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fungi than for host cells by comparing the results for the Langmuir films built with different sterols: ergosterols from fungi, and cholesterols from animals (128). For antibiotics, researchers have proven the specificity of the molecules and attested its low toxicity by using the fact that bacteria membranes are negatively charged, while mammalian cell membranes are, in general, composed of a great percentage of zwitterionic lipids (128). Moreover, for some bacteria, the specific phospholipid composition can be studied to evaluate the interaction with some antibiotics. Also, the interaction of drugs with Gram-negative bacteria has been evaluated by using LMs of lipopolysaccharides (129), the major constituent of the outer leaflet of Gram-negative bacteria. Total lipid extracts can also produce Langmuir films, and this strategy has been used mainly for bacteria (130). Cerebrosides and gangliosides are also interesting molecules to be taken into account since they are found mainly in nerves and brain tissue membranes. In addition, it has been found that gangliosides play an important role in tumor progression (131), so it could be applied as a molecular target for antitumor molecules that can easily be assessed by LMs. A difficulty in establishing a common mechanism of action for the drugs arises from the fact that they present very different chemical structures, and there are few connections between structure and activity involving drugs and lipids. In this case, an alternative is to synthesize molecules inspired by pre-existent drugs and verify their effects in the membrane. Alkyllysophospholipids are synthetic analogs of lysophosphatidylcholines (LPC) with anti-tumor properties (132) that are synthesized replacing the acyl group with an alkyl group. In order to increase the metabolic stability of LPC, the analogs edelfosine, milefosine and erucylphosphocholine were synthesized, and their effects were evaluated in terms of their interaction with LMs (119, 120, 133–137). Some examples of compounds and their derivatives that were also studied by the Langmuir technique are the antipsychotic phenothiazine (derivatives studied: chlorpromazine and trifluoperazine) (124), the antibiotics tetracycline and oxytetracycline (138, 139) and the fluoroquinolones antibiotics ciprofloxacin and moxifloxacin (140, 141).

Incorporation of Nanoparticles in LM and LB Films Acting as Cellular Membrane Understanding the mechanism of interaction between nanoparticles (NPs) and cell membranes is a major topic for the development of new therapeutic agents. In this way, systems organized in nanometric scale are a potent strategy in many technological fields since it is possible to evaluate the influence of NPs in living organisms using artificial models. The importance of such studies lies in the fact that many NPs are potentially toxic and can cause hazardous effects (142, 143). In this sense, a possible way to elucidate how nanoparticles act in living organisms is to work with theoretical and experimental membrane models able to mimic living cells (9, 144). Such models include LMs as shown in Figure 4. The studies involving NPs and LM can be divided into two approaches. The first involves systems in which nanoparticles interact with biomimetic systems, i.e., monolayers of lipids at the air-water interface above aqueous dispersions of NPs 77 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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(145). The second includes NPs that form stable structures at liquid-air interfaces forming interfacial aggregates that resemble LMs. Both systems can be employed in several areas such as materials, medical and biological sciences. NPs can promote significant changes in the lipid monolayer structure by means of ionic (145) or hydrophobic (146) interactions, changing lipid surface packing and being inserted in the interfacial layer (147), or altering phase behavior (148–150). However, the exact mechanism of interaction of NPs and LMs remains poorly understood. In the literature NPs are reported to be inserted under the lipid monolayer as pre-formed dispersions (145) or injected into the aqueous subphase beneath a lipid monolayer that has been already spread (151, 152) , which sometimes complicates the comparison owing to different effects resulted from different ways of incorporation. Nevertheless, there have been efforts to describe in general some aspects related to NPs. Some properties intrinsic to NPs must be considered such as flotation forces (driven by gravity) and immersion forces (driven by wetting). Important about these capillary forces, which are not often considered, is that the former acts on large particles (92.2%; molecular mass 2865.6 g/mol) (further referred to as Cter-R9AP) was purchased from Peptide 2.0 (Chantilly, VA). 111 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Cloning and Expression of RP2 and tLRAT The human RP2 (30.8 kDa) construct cloned in the pGEX-4T3 plasmid to express a GST fusion protein (GST-RP2) was a kind gift from Dr. Alfred Wittinghofer (Max-Planck-Institut für Molekulare Physiologie, Germany). Human tLRAT (amino acids 31-196, 20.8 kDa, without its N- and C-terminal segments postulated to serve for its membrane anchoring) has been cloned in the plasmid pET11a as previously described (28). Briefly, RNA from freshly dissected retinal pigment epithelium was isolated with Tri-reagent (Sigma) and used for reverse transcription reaction with the RevertAid H minus first strand cDNA synthesis kit (Fermentas). A thrombin cleavage site and a His-tag of 10 histidines were also added to the C-terminus of tLRAT (tLRAT-His-tag) to facilitate its purification. The pET11a vector was linearized and subsequently ligated with the purified PCR product corresponding to tLRAT. Plasmid DNA of RP2 and tLRAT were transformed into E. coli BL21(DE3) RIPL (Novagen) and grown overnight in the LB medium until saturation. Then, fresh LB containing 50 μg/ml ampicillin and chloramphenicol was inoculated with the transformed cell culture and incubated at 37 °C under agitation (250 rpm) until A600nm of 0.3 (RP2) or 0.6 (tLRAT) is reached. Their expression was then induced with 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) followed by an incubation during 5 h at 37 °C (RP2) or for 5 h at 30 °C (tLRAT). Bacteria were then sedimented by centrifugation at 3275 x g for 25 min. RP2 was not acylated in our experiments. Purification of RP2 and tLRAT RP2 and tLRAT were purified as previously described (19, 29). Pellets of bacteria containing GST-RP2 were resuspended in buffer (50 mM Tris, 100 mM NaCl, 5 mM MgCl2, 3 mM β-mercaptoethanol, pH 7.5), sonicated and centrifuged at 20,000 x g for 1 hour. The supernatant was then loaded on a GSTrap FF column (GE Healthcare) of 1 ml that had been preequilibrated with the same buffer. After an extensive washing of the column (at least 10 column volumes of buffer), RP2 was cleaved from GST with thrombin directly on the column for 16 hours at room temperature. Pure RP2 was then eluted using the same buffer containing instead 500 mM NaCl. Thrombin was removed from the eluent by connecting a Hitrap Benzamidine FF sepharose column (GE Healthcare) to the GSTrap column. RP2 was concentrated and the buffer was changed to 5 mM phosphate buffer, 100 mM NaCl (pH 7.4) using Amicon Ultra15. Pellets of bacteria containing the tLRATHis-tag were first disrupted by 3 cycles of freeze–thawing in the lysis buffer (100 mM Tris, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, pH 7.8). After sonication, bacteria were centrifuged at 13,000 x g during 20 min at 4 °C. Supernatant was discarded and membranes were resuspended in the loading buffer (500 mM Tris, 5 mM imidazole, 0.1% sodium dodecyl sulfate (SDS), pH 7.8). These resuspended pellets were shaken for 1 h at room temperature to homogenize the suspension which was then centrifuged at 100,000 g for 30 min at 21 °C. The supernatant was then loaded on a 5 ml His-Trap column preequilibrated with 5 column volumes of loading buffer. Column was washed with 10 to 20 column volumes of washing buffer (500 mM Tris, 40 mM imidazole, 0.1% SDS, pH 7.8). Elution was achieved 112 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

with a buffer containing 500 mM Tris, 150 mM imidazole, 0.1% SDS, pH 7.8. In order to remove the SDS to perform monolayer measurements, the elution buffer was exchanged for a phosphate buffer (50 mM, pH 7.0) using an Econo-Pac 10DG column (Bio-Rad) previously equilibrated with this buffer. The purity of RP2 and tLRAT was larger than 98% as judged from polyacrylamide gel electrophoresis which was carried out using a Bio-Rad Mini-protean II electrophoresis cell with 15% acrylamide.

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Determination of the Binding Parameters of tLRAT, RP2, and Cter-R9AP The determination of the MIP and synergy is very useful to decipher protein and peptide selectivity for particular lipids (20, 25, 27, 29–57). The surface pressure (Π) was measured by the Wilhelmy method using a tensiometer from Nima Technology (Coventry, U.K.) for RP2 and a DeltaPi4 microtensiometer from Kibron Inc for tLRAT and Cter-R9AP. The experimental setup was placed in a Plexiglas box with humidity control at room temperature. A 1200 μL home-built round Teflon trough (RP2) and a 500 μL glass trough from Kibron Inc (tLRAT and Cter-R9AP) were used for the monolayer binding measurements (30, 32, 33). The subphase buffer was 50 mM phosphate buffer (pH 7) for tLRAT, 5 mM phosphate buffer, 100 mM NaCl (pH 7.4) in the case of RP2 and 50 mM Tris, 150 mM NaCl, 5 mM β-mercaptoethanol (pH 7.4) for Cter-R9AP. The monolayer was prepared by spreading a few microliters of a solution of phospholipids at the surface of the buffer until the desired initial surface pressure (Πi) was reached. The waiting period for the film to reach equilibrium varies between 20 and, at most, 60 min, depending on the Πi, the type of lipid, the speading volume and the lipid concentration. Then, RP2, tLRAT or Cter-R9AP was injected underneath the lipid monolayer until an optimal, saturating final concentration of 0.5 µM, 79 nM or 5 µM was achieved, respectively (30, 32, 33). The kinetics of protein binding onto the phospholipid monolayer was monitored until the equilibrium surface pressure (Πe) was reached. The MIP and synergy are determined by injecting the protein or peptide at different Πi values of the lipid monolayer as described previously (20, 29). No difference was observed when measurements were performed in the presence or the absence of N2 or Ar when using polyunsaturated phospholipids for at least 2h. In addition, the same protein or peptide adsorption isotherms were obtained in the presence or the absence of BHT in the phospholipid solution (29).

Results and Discussion Measurement of the Binding Parameters of Proteins or Peptides to Lipid Monolayers As can be seen in the inset of Figure 1, the surface pressure of the phospholipid monolayer increases after the injection of the peptide into the subphase until equilibrium is reached (Πe). The larger Πi is, the smaller the surface pressure increase (ΔΠ ; ΔΠ = Πe - Πi). For example, ΔΠ of 20.7, 15.2 and 5.9 mN/m have been obtained after the injection of Cter-R9AP into the subphase of a DSPE monolayer at Πi of 7.8, 15.4 and 26.4 mN/m, respectively (inset of Figure 1). 113 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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As a consequence, a negative slope is obtained when plotting ΔΠ as a function of Πi (Figure 1). This plot allows to determine the MIP by extrapolating the regression curve to the x-axis (20, 29). A MIP value of 34.3 ± 1.5 mN/m has thus been obtained for Cter-R9AP in the presence of a DSPE monolayer (Figure 1). The MIP corresponds to the maximum surface pressure up to which proteins or peptides can insert into the monolayer and beyond which no insertion takes place. The synergy between the lipid monolayer and the protein or peptide is calculated by adding 1 to the slope of the plot of ΔΠ as a function of Πi (Figure 1) (29). The synergy is >0 when a positive interaction between the protein or peptide and the lipid monolayer is observed, whereas the synergy is 560 nm for propidium iodide detection. Cells stained with annexin-V-fluous alone are considered apoptotic while double-stained cells (annexin-V-fluous + 149 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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propidium iodide) are considered as late apoptotic or necrotic cells. The data (Figure 6) is shown as dot plots (A), or percentage of HUVEC in each quadrant (B). The letters within each quadrant denote the following: N = necrotic cells, A = apoptotic cells, L = living cells. Results correspond to a representative experiment of 3 independent assays. The asterisks denote a P value < 0.01 as compared to control cells (TNF-α). After a 24-hr exposure the TiO2 NPs induces apoptic cell death in ~20% of the HUVECs and necrotic death in 60% of cells at all used concentrations, in comparison with the control cells. The increase in necrosis is more pronounced than that of TNF-α-induced necrosis. We attribute the negative surface charge of the TiO2 NPs to be the major governing factor inducing the observed cell death. Case II: ZnO NPs Can Be Reduced by Lowering the PZC and Relative Number of Surface Binding Sites ZnO NPs are used extensively in many nanotechnology applications, yet is among the most toxic of the fourth period transition metal oxides. Because of the raised environmental health concern, there is an impetus to produce safer ZnO NPs while preserving their unique optical, electronic and structural properties. One approach to achieve this end is to tune the NP surface charge and alter its structural binding sites that interact with the cellular matrix. A recent report by Punnoose et al. (46) has done precisely this, in accord with predictive trends observed in our study (vide supra). Two types of ZnO NPs were synthesized with different surface properties in terms of charge and relative number of surface binding sites in aqueous solution media. In comparing the two differently synthesized ZnO NPs, the one with the higher zeta potential (i.e., more positive surface charge, and relatively larger number of binding sites) results in a 1.5-fold increase in cytotoxicicity, in agreement with our predictive model for non-phagocytic cells. Two equally-sized (9.26 ± 0.11 nm) ZnO NP samples were synthesized from zinc acetate using a forced hydrolysis process with their surface chemical structures modified using different reaction solvents. The resulting structures, analyzed using FTIR spectroscopy, reveal the surface compositional structures of ZnO-I and ZnO-II NPs (Figure 7). The spectra show differing degrees of surface hydroxylation between the two NPs. Characteristic Zn-O vibrations at 478 cm-1 for ZnO-I and 444 cm-1 for ZnO-II (47). The Zn:O ratio of the NPs are influenced by surface bound chemical groups resulting in a 34 cm-1 difference in vibrational frequency. Broad adsorption at 3410—3420 cm-1 is due to attachment of hydroxyl groups to the NP surface, which are more pronounced in ZnO-I than for ZnO-II. Coordination of atoms on the surface of ZnO NPs differ from that of their bulk structure. Absorption at 2351—2353 cm-1 is due to absorbed CO2. ZnO-I exhibits two strong bands, associated with the carboxylate functional group at 1412 and 1595 cm-1, denoting symmetric and assymetric stretches, respectively (48–50). ZnO-II NPs show these same bands from surface adsorbed groups from the acetate precursors, but with somewhat lower intensity and slightly different frequency. In comparing the degree of absorption on the NP surfaces as a measure of the relative number of binding sites [in the same manner as in our study using XPS (Figure 4)], the ZnO-I NP 150 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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surface has a relatively higher relative number of surface binding sites than that of ZnO-II with which to interact with cells. Hence, a relatively higher cytotoxicity would be predicted for cells coming into contact with ZnO-I than for ZnO-II NPs. This prediction is confirmed by the cytotoxicity assays comparing the two ZnO NPs (vide infra). The higher isoelectric point of ZnO-I as compared to ZnO-II also contributes to its higher cytotoxicity. Figure 8 shows zeta potential plots of the two NPs as a function of pH. The zeta potential of the ZnO-I NPs is significantly higher than those values for ZnO-II NPs, at +42.6 mV and +12.5 mV, respectively, under pH = 7.5, which is near the physiological pH. Due to the higher isoelectric point, ZnO-I NPs would adopt a stronger net positive charge at physiological pH as compared to ZnO-II NPs. Therefore, ZnO-I would have a greater Coulombic attraction to cellular material, and hence exhibit greater cytotoxicity among non-phagocytic cells. This trend is also consistent with our PZC results indicating that NPs with higher PZC (i.e., isoelectric point) have a corresponding increase in cytotoxicity (Figure 3).

Figure 8. Zeta potentials of ZnO-I and ZnO-II NP measured as a function of pH. Reproduced with permission from reference (46). Copyright 2014 American Chemical Society. 151 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 9. (A) and (B) display the cell viability of Hut-78 cancer cells after treating with ZnO-I and ZnO-II nanoparticles for 24 hrs at the concentrations indicated, determined using PI-flow cytometry and Alamar Blue methods, respectively. Error bars represent the mean ± standard error of three replicate experiments. Viability between the 2 types are significantly different (*P < 0.05). Reproduced with permission from reference (46). Copyright 2014 American Chemical Society. (see color insert) To examine differences between the two ZnO NP cytotoxicities, Hut-78 lymphoma T cells were used for the assays. The non-phagocytic Hut-78 cancer cells were treated with each of the ZnO NPs using two different assays: a propidium iodide (PI) assay and an Alamar blue assay. In the PI-flow cytometry, ZnO NP concentrations of 0, 0.3, 0.6 and 0.9 mM were used with cell viability determined after a 24-hr exposure (Figure 9A). A rapid decrease in cell viability 152 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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is observed for increasing concentrations of both ZnO-I and ZnO-II. The ZnO-I NPs exhibits a more rapid reduction in cell viability than ZnO-II NPs. The IC50 of ZnO-I and ZnO-II are 0.37 mM and 0.56 mM, respectively. In addition, independent experiments using an Alamar Blue assay applying 0, 0.1, 0.3, 0.6 and 0.9 mM ZnO concentrations yield the same trend, but with slightly lowered IC50 values with 0.31 mM for ZnO-I and 0.45 mM for ZnO-II (Figure 9B). In both assays, ZnO-II shows a 1.5 times greater IC50 relative to ZnO-I, indicating that ZnO-I NPs are markedly more toxic than ZnO-II NPs. Hence, these cytotoxicity assays confirm the prediction that (i) a greater number of surface binding sites and (ii) higher isoelectric points contribute to greater toxicity of ZnO-I NPs as compared to ZnO-II NPs for non-phagocytic cells.

Conclusions In summary, the observed toxicity trend in this series of fourth period transition metal oxide NPs (TiO2, Cr2O3, Mn2O3, Fe2O3, NiO, CuO and ZnO) examined is not cell-type specific within non-phagocytic cells. Instead, cytotoxicity appears to predominantly be a function of NP isoelectric point (ρ = 0.78), relative number of available particle surface sites (ρ = 0.71) on the NP surface (with Spearman’s correlation rank in parentheses). As the A649 and BEAS-2B cell lines show identical trends in toxicity, we predict that the trend will be evident in other non-phagocytic cell lines. Particle surface charge is pH dependent, and may thus influence the rate and routes of their cellular uptake as well as subsequent partitioning between organelles. The relative number of available surface binding sites with cytotoxicity increases the likelihood of NP interaction with biomolecules such as DNA, RNA, protein and lipids. The observed correlation of (i) surface charge and (ii) relative number of binding sites is a useful predictor for relative toxicity as shown by its relevance in the above two discussed case studies. These two variables are important parameters to consider for risk assessment in biological systems.

References 1. 2. 3.

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34. Slinkard, W. E.; DeGroot, P. B. J. Catal. 1981, 68, 423–432. 35. Gonbeau, D.; Guuimon, C.; Pfister-Guillouzo, G.; Levasseur, A.; Meunier, G.; Dormoy, R. Surface Sci. 1991, 254, 81–89. 36. Wagner, C. D.; Riggs, W. M.; Davis, L. E.; Moulder, J. F.; Muilenbuerg, G. E. Handbook of X-ray Photoelectron Spectro-scopy; Perkin-Elmer Corporation: Eden Prairie, MN, 1979. 37. Khawaja, E. E.; Salim, M. A.; Khan, M. A.; Al-del, F. F.; Khatak, G. D.; Hussain, Z. XPS, Auger, electrical and optical studies of vanadium phosphate glasses doped with nickel oxide. J. Non-Cryst. Solids 1989, 110, 33–43. 38. Howng, W.-Y.; Thorn, R. J. J. Phys. Chem. Solids 1980, 41, 75–81. 39. Ertl, G.; Hierl, R.; Knozinger, H.; Thiele, N.; Urbach, H. P. Appl. Surf. Sci. 1980, 5, 49–64. 40. Otamiri, J. C.; Andersson, S. L. T.; Andersson, A. Appl. Catal. 1990, 65, 266–279. 41. Strohmeier, B. R.; Hercules, D. M. J. Catal. 1984, 86, 266–279. 42. Henrich, V. E.; Cox, P. A. The Surface Science of Metal Oxides; Cambridge University Press: New York, 1994. 43. Perry, D. L.; Tsao, L.; Taylor, J. A. Inorg. Chim. Acta 1984, 85, L57–L60. 44. Fröhlich, E. Int. J. Nanomed. 2012, 7, 5577–5591. 45. Montiel-Dávalos, A.; Ventura-Gallegos, J. L.; Alfaro-Moreno, E.; Soria-Castro, E.; Garcia-Latorre, E.; Cabañas-Moreno, J. G.; del Pilar Ramos-Godinez, M.; López-Marure, R. Chem. Res. Toxicol. 2012, 25, 920–930. 46. Punnoose, A.; Dodge, K.; Rasmussen, J. W.; Chess, J.; Wingett, D.; Anders, C. ACS Sustain. Chem. Eng. 2014, 2, 166–1673. 47. Farbun, I. A.; Romanova, I. V.; Terikovskaya, T. E.; Dzanashvili, D. I.; Kirillov, S. A. Russ. J. Appl. Chem. 2007, 80, 1798–1803. 48. Bian, S.-W.; Mudunkotuwa, I. A.; Rupasinghe, T.; Grassian, V. H. Langmuir 2011, 27, 6059–6068. 49. Max, J. J.; Chapados, C. J. Phys. Chem. A 2002, 106, 6452–6461. 50. Max, J. J.; Chapados, C. J. Phys. Chem. A 2004, 108, 3324–3337.

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Chapter 9

Dynamic Light Scattering Coupled with Gold Nanoparticle Probes as a Powerful Sensing Technique for Chemical and Biological Target Detection Nickisha Pierre-Pierre and Qun Huo* NanoScience Technology Center, Department of Chemistry in College of Science, and Burnett School of Biomedical Science in College of Medicine, University of Central Florida, 12424 Research Parkway Suite 400, Orlando, Florida 32826 *E-mail: [email protected]. Tel: 407-882-2845.

Dynamic light scattering (DLS) is an analytical technique used routinely for nanoparticle size measurement. Gold nanoparticles are known for their exceptionally strong light scattering properties. By combining the strong light scattering properties of gold nanoparticle probes with the size measurement capability of DLS, a new chemical and biological sensing technique termed nanoparticle-enabled dynamic light scattering assay (NanoDLSay) was developed. Gold nanoparticles can be surface-modified with various chemical ligands, antibodies or other binding molecules to form gold nanoparticle probes. Binding of specific chemical or biological target analytes with the gold nanoparticle probes leads to nanoparticle cluster formation, and subsequently, an average particle size increase of the assay solution. Such particle size increases can be measured by DLS, and correlated to the quantitative information of target analytes. NanoDLSay is a single-step homogeneous solution assay, easy to perform, of low cost, and has excellent sensitivity and reproducibility. So far, this technique has been applied for quantitative detection and analysis of a wide range of chemical and biological targets, including proteins, DNAs, viruses, carbohydrates, small © 2015 American Chemical Society In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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chemicals, toxic metal ions, food and environmental toxins. In this review, we present both a tutorial explanation on the principle and key factors involved in NanoDLSay technique and a literature update of the field. The analytical performance of NanoDLSay is compared with other sensing techniques. In the Future Outlook section, we discuss further work that needs to be conducted to broaden the applications of NanoDLSay in chemical and biological sensing and quantitative analysis, and to move these applications from laboratories to real world settings.

Introduction Since its initial commercialization in the 1970s, dynamic light scattering (DLS) has become a laboratory technique used routinely for particle size measurement and analysis (1). DLS is suitable for measuring the size of particles in the range from a few nanometers to 3-5 microns. Despite its extensive use as a particle characterization tool, DLS was not traditionally considered as suitable for quantitative detection and analysis. As early as 1975, Cohen et al. proposed a particle agglutination assay for chemical and biological detection using DLS (2–4). The principle of the assay is quite simple: specific chemical ligands or biological sensing molecules such as antibodies can be attached to the surface of microparticles such as polymer beads. Upon binding with target analytes, the polymer beads are agglutinated together into clusters and aggregates. The agglutination of polymer beads leads to an increase of the average particle size of the assay solution. There should be a quantitative correlation between the average particle size of the assay solution and the concentration of the target analytes. Therefore, by measuring the average particle size of the assay solution using DLS, the target analyte can be detected and its concentration can be determined. However, despite some early attempts to apply this technique for the immunoassay, this technique never found practical applications and was quickly abandoned. The fundamental barrier that prevented the successful application of this DLS-based sensing and molecular assay technique lies within the insufficient light scattering property of the particle probes. DLS is a technique that measures the “average” size of a particle solution based on the collective light scattering intensity fluctuation caused by all particles in solution. Samples, particularly biological fluid samples such as blood, blood serum/plasma or urine, often contain a large number and amount of colloidal particles, and these particles also scatter light intensely. When mixing a sample solution with the particle probe solution, DLS detects the light scattering from both the particle probes and particles in the sample solution. When the light scattering intensity from the particles originating from the sample solution is comparable to the light scattering intensity of the microparticle probe, the measured average particle size of the assay solution reflects the effect of the sample matrix. In other words, the average particle size 158 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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of the assay solution does not solely reflect the agglutination of microparticle probes. In order to avoid such interference from sample matrix, the light scattering intensity of the particle probe must substantially exceed the background scattering from the sample matrix. Blood, blood serum/plasma and urine often contain biomolecular complexes, vesicles, and other colloidal particles that have a light scattering intensity comparable to the polymer beads. The light scattering intensity of polymer beads-based probes is not strong enough to suppress the sample background scattering. As a result, the originally proposed DLS-based particle agglutination assay is heavily interfered by sample background and the quantitative analysis is not reliable.

Figure 1. Schematic illustration of NanoDLSay. A: A chemical ligand or biomolecule that can specifically recognize the target analyte is conjugated to AuNPs to form a AuNP probe. Upon mixing with a sample that contains target analytes, the AuNP probes are clustered together, leading to an average particle size increase of the assay solution. B: Average particle size increase of the assay solution using dynamic light scattering (DLS). C: The establishment of a calibration curve for quantitative analysis using a set of standard solutions. The concentration of an unknown sample may be obtained by referring to the calibration curve. 159 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Table 1. A List of Toxic Metal Ions Detected by NanoDLSay Target analyte

Detection limit

AuNP probe

Sample source

Authors

AsIII

3 ppt

Glutathione (GSH), dithiothreitol (DTT), and cysteine (CYS) modified AuNPs

Groundwater

Kalluri et al. (9)

Pb (II)

100 ppt

Glutathione (GSH) modified AuNPs

Paints, plastics, water samples

Beqa et al. (10)

Pb (II)

0.20 (East lake water) and 0.22 (Yangtze water) pM; Drinking water 0.25 pM

Aza-crown-ether-modified silver nanoparticles

Ground water, Drinking water

Zhang et al. (11)

Pb 2+

6.2 pM

Unmodified AuNPs coupled with Pb2+-dependent DNAzyme

Groundwater

Miao et al. (14)

Pb 2+ ions

0.025 mM

Glutathione mediated self-assembled chains of gold nanorods

Pure water spiked with metal ions

Durgadas et al. (12)

Hg (II)

0.43 nM

Oligonucleotide conjugated AuNP probe

Groundwater

Xiong et al. (15)

0.1 nM

Hg2+ aptamer probe conjugated AuNPs

Lake water

Maet al. (16)

Hg

2+

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Target analyte

Detection limit

AuNP probe

Sample source

Authors

2, 4, 6 Trinitrotoluene

100 pM

Para-aminothiophenol modified AuNPs

TNA dissolved in mixed ethanol/acetonenitrile

Dasary et al. (17)

2, 4, 6 Trinitrotoluene

0.4 pM

1,2-Ethylenediamine (EDA) capped AuNPs

Surface water

Lin et al. (18)

Adenosine

7 x 10-9

DNA-conjugated AuNPs

Pure solution

Yang et al. (19)

Glucose

38 pmol/L

Oligonucleotide functionalized AuNPs

Human serum

Miao et al. (20)

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Table 2. A List of Small Chemicals and Biomolecules Detected by NanoDLSay

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Table 3. List of Food and Environmental Toxins Detected by NanoDLSay Detection Limit mL-1

AuNP Probe

Sample Source

Authors

Gold nanorod-AFB1-BSA conjugate

Peanut samples

Xu et al. (28)

Aflatoxin B1

0.16 ng

Aflatoxin M1

37.7 ng/L in Buffer, 27.5 ng/L in milk

AFM-BSA-AuNP conjugate

Buffer solution and milk

Zhang et al. (26)

Microcystin-LR

Side-by-side- 0.45 ng/mL; end-to-end- 5 pg/mL

Side-by side and end-to-end nanorod assembling

Microcystin-LR solution

Wang et al. (22)

Melamine

0.05 ppm

20 nm citrate AuNPs

Milk

Ma H. et al. (30)

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Target Analyte

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Table 4. List of Single and Double-Stranded DNAs and microRNAs Detected by NanoDLSay

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Target Analyte

Detection Limit

Gold nanoparticle Probe

Sample Source

Authors

DNA

1.0 pM

ssDNA probe functionalized onto 30 nm citrate stabilized AuNP

Buffered DNA solution

Dai et al. (8)

dsDNA

593 fM

AuNP modifies with oligonucleotides

Buffer DNA solution

Miao et al. (32)

DNA

1 nM

Gold nanoparticle probes

Buffer DNA solution

Zhang et al. (31)

let7 MicroRNA family

100 fmol

AuNP-ssDNA probe

Pure microRNA solution and mixture solutions

Seow et al. (36)

Sequence specific Nopaline synthase (NOS) gene

3.0 x 10-14 M

Citrate-capped AuNPs

DNA sequence in transgenic plants

Gao et al. (34)

cDNA

10.0 pM

Positively charged CTAB coated gold nanorods and nanospheres

A 21-mer ssDNA from the human immunodeficiency virus type 1 HIV-1 U5 long terminal repeat (LTR) sequence and a 23-mer ssDNA from the Bacillus anthracis cryptic protein and protective antigen precursor (pagA) genes were used as ssDNA models

Pylaev et al. (33)

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Table 5. A List of Proteins, Viruses, and Virus Antigens Detected by NanoDLSay Target analyte

Detection limit

Gold nanoparticle probe

Sample source

Authors

Alpha-fetoprotein

0.1 µg/ ml

Antibody functionalized AuNPs

Serum samples

Nietzold et al. (41)

Alpha-fetoprotein

0.01 ng/mL

Gold-coated iron oxide magnetic nanoclusters (Au-MNCs)

Buffered protein solution

Chun et al. (40)

f-PSA

0.1 ng/mL

AuNP conjugated with a detector antibody and gold nanorods (AuNRs) conjugated with a capture antibody for free-PSA

f-PSA standard solution prepared in calf serum

Liu at al. (7)

Carcinoembryonic antigen detection

35.6 pg/mL

Ag@Au core-shell nanoparticles (CSNPs)

Human serum

Miao et al. (43)

Influenza A

8.6 x 101 TCID50/mL

Antibody-conjugated AuNPs

Influenza A infected Samples

Driskell et al. (45)

Hepatitis B virus

0.005 IU/mL

Antibody-conjugated AuNPs

Buffer solution and hepatitis B virus infected human serum

Wang et al. (47)

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The arising of gold nanoparticles (AuNPs) has brought an excellent solution to solve the above problem. AuNPs are among the most intensely light scattering materials (5, 6). A AuNP scatters light 100s to 1000s times stronger than a polymer bead at similar size. The exceptional light scattering property of gold (or silver) nanoparticles is associated with its surface plasmon resonance phenomenon. By replacing the polymer beads with AuNP probes, the background light scattering from sample matrices can be effectively suppressed, and gold nanoparticle-enabled dynamic light scattering assay (NanoDLSay) becomes a reliable and powerful sensing and quantitative assay technique. Figure 1 is a schematic illustration of the assay. A chemical ligand or biomolecule that can specifically bind with a target analyte is conjugated to the AuNPs to form AuNP probes. Upon binding with the target analytes, the AuNPs are clustered together, leading to an average particle size increase of the assay solution. Quantitative analysis can be accomplished by developing a calibration curve using standard solutions. Following our initial reports on protein and DNA detection (7, 8), NanoDLSay has been applied for quantitative detection and analysis of a wide range of chemical and biological target analytes. Table 1 to 5 summarize the analytical performance of NanoDLSay for different target analytes. The following Literature Survey provides more details on each study. At the end of this review, we present a brief outlook on this emerging technique, and further research that needs to be conducted to move these applications from laboratories to real world settings.

Literature Survey Toxic Metal Ions Arsenic (As3+) Arsenic (As3+) detection is important due to the widespread contamination of arsenic species in drinking and ground water. This has led to a massive epidemic of arsenic poisoning around the world; mostly in Bangladesh. Kalluri et al. developed a test for the detection of inorganic arsenite (As3+) in drinking water using AuNPs modified with glutathione (GSH), dithiothreitol (DTT) and cysteine (CYS) coupled with dynamic light scattering (DLS) (9). The sulfur-containing ligands DTT, GSH, and CYS bind to the surface of the AuNP through Au-S bonds. It has been reported that As3+ can bind DTT through an As-S bond and GSH and CYS through As-O bonds. Using a spherical AuNP with a diameter of 110 nm, the detection limit of the GSH/DTT/CYS-modified AuNP was 3ppt for As3+ ions. The authors also tested other heavy metals (Hg2+, Zn2+, Pb2+, Cu2+, etc.) using the GSH/DTT/CYS-modified AuNP and it proved that the assay is specific for AsIII. Water samples from Bangladeshi wells that were contaminated with As3+ were collected and compared with bottled and water samples collected from a tap and well. NanoDLSay assay was used to measure the amount of arsenic species in 165 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

each type of water sample. The Bangladeshi well water contained 28 bbp of arsenic species and the tap and bottled water contained 380 ppt and 15 ppt of arsenic species respectively. NanoDLSay detected As3+ at levels about 3 orders of magnitude lower than the World health Organization’s (WHO) standard limit and is about 2 orders of magnitude more sensitive than the detection based on the colorimetric change of AuNP probes.

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Lead (Pb2+) Pb2+ is another highly toxic, environment hazardous ion. It is used in building construction and can be useful in ammunition for the armed forces and military. Pb2+ can be very poisonous to animals as well as humans by causing brain disorders and damage the nerve system if ingested. Pb2+ is also known to be a common water pollutant. Everyday items such as paint and plastic toys may also contain high levels of Pb2+. Beqa et al. reported on the use of a glutathione (GSH) modified gold nanoparticle probe (10). GSH is used as a chelating ligand and a stabilizer for the gold nanoparticle. Pb2+ is known to form a strong bond with GSH through the thiol (–SH) group of GSH. When GSH is mixed with AuNPs, the GSH is attached to the AuNPs through the thiol (–SH) group. While performing the experiment at pH 8.0, the NH2 group of GSH changes to NH3+ and COO- becomes the binding site of Pb2+. Thus, upon addition of Pb2+ to GSH-modified AuNPs, nanoparticle aggregation occurs. The detection limit of the NanoDLSay was 100 ppt for the detection of Pb2+. Due to the natural occurrence of other metals in drinking water, it was imperative to investigate whether the assay is selective for Pb2+. Experiments were performed on other heavy metal ions using NanoDLSay. The results show that the assay is highly selective for Pb2+. There are a few explanations for the selectivity of Pb2+: Pb2+ has a higher affinity for GSH carboxylate ions than any other heavy metal ion. Because the thiol (-SH) group is attached to the AuNP, Hg2+ and As3+ don’t bind strongly with GSH-modified AuNP. When the detection was carried out at pH 8, Fe2+, Zn2+ and Cd2+ ions bind only weakly to the AuNP. Pb2+ is capable of forming bigger aggregates because it can coordinate up to eight oxygen atoms. To demonstrate practicality, water, paint and plastic toy samples were tested. NanoDLSay produced results comparable to those produced by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The detection limit of the NanoDLSay was almost 2 orders of magnitude below the standard limit of the US Environmental Protection Agency (EPA), which is 10 ppb. In addition to gold, silver nanoparticles also scatter light intensely. Zhang et al. reports on the detection of lead using an aza-crown-ether (ACE)-modified silver nanoparticle (ACE-AgNPs) (11). ACE has been reported previously to have a high binding capacity for metal ions and can complex with many metal ions, therefore cannot selectively respond to Pb2+ in a colorimetric assay. However, the size increase of the nanoparticle by the metal ions is different and unique for each metal. Pb2+ demonstrates a more efficient aggregation level, which can be detected by DLS. This study showed Pb2+ had the largest size increase among 166 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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all the other metals tested (such as iron, copper and mercury). This suggestes that the ACE-AgNPs are more selective for Pb2+. The study reported a detection limit of 0.25 pM (1.03 ppt). Pb2+ was tested in environmental water samples. Water samples were taken from Yangtze River and East Lake in China. The water samples were treated and spiked with Pb2+ at concentrations over the range of 1.0 x 10-13 to 1.0 x 10-4 M. The detection limit of East Lake water was 0.20 pM and 0.22 pM for Yangtze River water. The results determined by NanoDLSay were very close to the results detected by ICP-MS and proves to be satisfactory. When drinking water was tested, the detection limit can reach 0.25 pM. All water samples tested using NanoDLSay had a detection limit much lower than the maximum contamination level (MCL) allowed by the US EPA, which is 72 nM. Durgadas et al. reports on the use of Pb2+ to disassemble glutathione (GSH)-mediated assembly of gold nanorods (AuNRs) for Pb2+ detection (12). Cetyl trimethylammomium bromide (CTAB) protected AuNRs had an average hydrodynamic diameter of 132 nm upon conjugation with GSH. When mixed with GSH, the average particle diameter increased to 258 nm and later to 393 nm due to the formation of nanorod chains in the presence of GSH. It was proposed that the GSH molecules were attached to the “end’ of the gold nanorods through Au-S bond formation, and the gold nanorods were assembled together into linear chains through hydrogen bond formation between the GSH molecules attached to the gold nanorods. When 0.1 mM of Pb2+ was added, the particle size decreased to 155 nm. This size reduction is caused by the dissembling of the nanorod chain upon binding of Pb2+ with GSH. To determine if the assay was selective for Pb2+, the authors tested the assembly and disassembly of AuNR in the presence of other ions including Cu2+, Cd2+, Hg2+, Zn2+, Mn2+, Co2+, Ni2+, Mg2+ and NH4+. Only Pb2+ was able to induce disassembly of AuNR chains. The dynamic range reported in this study for Pb2+ detection was between 0.1 and 0.025 mM, and the detection limit was around 0.025 mM. Miao et al. used unmodified AuNPs coupled with Pb2+ dependent DNAzyme to detect Pb2+ (13). When DNAzyme is double stranded, it cannot adsorb to the surface of the AuNPs. With the addition of Pb2+, the DNAzyme is cleaved into single stranded DNA (ssDNA) and the ssDNA can adsorb to the surface of the AuNPs, preventing AuNP aggregation in the presence of NaCl. The average diameter of the AuNP solution decreased with the increase in concentration of Pb2+. The detection limit reported by this study was 6.2 pM. To determine the selectivity of the assay, other metal ions were tested on their ability to cleave DNAzyme into single stranded fragments. Metal ions including Cr2+, Cu2+, Fe3+, Ca2+, etc., did not produce a significant average particle size change compared to Pb2+. The assay was used to determine the concentration of Pb2+ in groundwater. Even though Pb2+ could not be detected in the water samples, there was good recovery (95.12% to 104.76%) when Pb2+ was added to these water samples. Prior to this study, also based on the Pb2+ induced cleavage of DNAzyme, this group reported a slightly different version of the assay for Pb2+ detection, with a detection limit of 35 pM (14).

167 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Mercury (Hg2+) Mercury (Hg2+) is a toxic element and should be handled with care due to the possibility of it being absorbed through the skin and mucous membranes. This can cause severe damage to the body. For the detection of Hg2+, Xiong et al. used an oligonucleotide (oligo 1) that was designed to absorb to the AuNP surface and protect it from aggregation (15). Upon the addition of Hg2+, the oligo 1 strands unbind from the AuNP and complex with Hg2+. With the loss of the protection of oligonucleotide, AuNPs start to aggregate. Under optimal conditions, Hg2+ can be detected within a dynamic range from 0.75 nM to 25 nM, with a detection limit of 0.43 nM. Metal ions including Ca2+, Co2+, Cr2+, Pb2+, Cu2+, K+, Ba2+, Cd2+, Al3+, Zn2+ were tested to determine the selectivity of the assay for Hg2+. The addition of 50 nM Hg2+ resulted in much more significant increase of the average diameter of the AuNP solution compared to metal ions tested here. Three water samples (ZhuJiang river water and two pond water samples) in China were tested to explore the practicality of the test for the detection of Hg2+. After the water samples were filtered, Hg2+ was added at different concentrations to the water samples (2.5, 6.0, 12 nM). The results correlate with those obtained using ICP-MS. The detection limit was lower than the safety limit of drinking water standard set by the EPA, which is at a maximum contaminant level (MCL) of 0.002 mg/L. Ma et al. developed an Hg2+ aptamer DNA probe conjugated to AuNPs for mercury ion detection (16). Probe DNA was able to bind to the surface of the AuNP and maintain AuNP monodispersity. In the presence of Hg2+, probe DNA desorbs from the AuNP surface and results in AuNP aggregation. Under optimum conditions, the hydrodynamic diameter of the AuNP increased with the increase of Hg2+ concentration in the range from 1 nM to 5 µM with a detection limit of 0.1 nM. To determine selectivity of the assay, other ions Al3+, Ca2+ Cu2+, and Clwere evaluated. The change in the hydrodynamic diameter was much greater and significant in the presence of Hg2+. Water samples were tested for the presence of Hg2+ and the results were very similar to the results obtained by inductively coupled plasma optical electron spectroscopy (ICP-OES). The detection limit that was obtained using NanoDLSay was much lower than the standard amount of Hg2+ allowed in drinking water defined by the World Health Organization (WHO), 10 nM (2011 WHO standards), and the U.S. Environmental Protection Agency (EPA), 30 nM (or 6.0 ppb) published in 2011. The current maximum contaminant level (MCL) set by EPA is 0.002 mg/L.

Small Chemicals and Biomolecules 2,4,6-Trinitrotoluene (TNT) 2,4,6-Trinitrotoluene is an aromatic compound used in the armed forces as well as in weaponry for terrorist activities. TNT has also become a key component in polluted water. Due to the environmental and chemical hazards that TNT can cause, there has been an increasing need for the detection of TNT. Dasary et al. created an AuNP modified with para amino-thiophenol (p-ATP) to detect TNT 168 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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(17). p-ATP can form a covalent bond with gold through the thiol group and the amino group of p-ATP can form π bonds with TNT. In the presence of TNT, p-ATP-conjugated AuNPs undergo aggregation. Higher amounts of TNT lead to increased aggregate formation. The assay was able to detect TNT at 100 pM. In order to prove the assay was selective for TNT, other nitro containing compounds such as 2,4-dinitrotoluene (DNT), 2,4,6-trinitrophenol (TNP), picric acid (PA) and nitrophenol (NP) were also tested. All the compounds tested contained a nitro group, but TNT is the only nitro-containing compound that is able to make strong π-donor-acceptor interactions with p-ATP. Comparative studies show that the test is selective for TNT. This study also demonstrates that the NanoDLSay technique is five times more sensitive than the colorimetric methods based on AuNP aggregate formation. Lin et al. modified the surface of AuNPs with 1, 2-ethylenediamine (EDA) to detect TNT (18). The detection limit of the assay was 0.4 pM. For comparison purposes, the colorimetric detection limit was 400 pM. This assay was also found to be selective for TNT over other nitro compounds, including DNT, NP and PA.

Adenosine Adenosine is an important, small biomolecule that is involved in a number of important biochemical processes including energy transfer and signal transduction. Yang et al. introduced the NanoDLSay for the detection of adenosine (19). The authors have split adenosine aptamer into two fragments, both are ssDNA fragments. The ssDNA was conjugated to the AuNPs. In the presence of adenosine, the split aptamers would form a complex with adenosine, inducing AuNP aggregation. As the concentration of adenosine increased, the average particle size of the AuNP assay solution increased. The limit of detection is 7 nM. Compared to a colorimetric assay, the NanoDLSay is 5 orders of magnitude lower than the colorimetric assay, which is 0.25 mM. Adenosine analogues including uridine, cytidine, and guanosine were tested to determine the specificity of the assay for adenosine. The assay was found to be specific to adenosine.

Glucose Glucose plays a significant role in physiological processes such as metabolism, signal transduction as well as in the central nervous system. Miao et al. developed a NanoDLSay to detect glucose (20). AuNPs were functionalized with oligonucleotides to produce Oligo 1-AuNP and Oligo 2-AuNP. Both Oligo 1-AuNP and Oligo 2-AuNP can hybridize with Oligo 3 and induce aggregation of AuNPs. The addition of glucose resulted in the cleavage of Oligo 3 into DNA fragments and induce dispersal of the AuNPs. The increase in glucose concentration resulted in a decrease of the Oligo-AuNPs diameter over a range from 50 pM to 5.0 nM. The detection limit of the assay was 38 pM. Other sugars 169 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

(lactose, galactose, sucrose, maltose and dextrin) were tested to determine the selectivity of the assay. The decrease in the diameter of the Oligo-AuNP mixture was more than 280 nm after the addition of glucose and the samples that contained other sugars had an average diameter decrease of less than 40 nm.

Food and Environmental Toxins

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Microcystin-LR (MC-LR) Microcystin-LR (MC-LR) is an environmental toxin. Microcystin has been known to cause mass poisoning and prolonged exposure can lead to liver cancer (21). Wang et al. created a side-by-side and end-to-end nanorod assemblies for the detection of MC-LR toxin in the environment (22). Calibration curves generated from standard MC-LR solutions show that the two assembling motifs lead to different sensing parameters. The limit of detection using side-by-side nanorod assembling was 0.45 ng/mL and the end-to-end assembling was 5 pg/mL. The end-to-end motif also provides a broader detection range than the side-to-side motif. The test has no cross reactivity towards ochratoxin A. The detection limits for MC-LR using the nanorod assemblies surpass the required standard of drinking water by the World Health Organization (1 ng/mL) and the new test is substantially faster than existing methods.

Aflatoxin Aflatoxin M1 (AFM1) is a known carcinogen and immunosuppressant (23). Aflatoxins (AF) are mycotoxins and contaminate agricultural products such as milk, dried fruits and nuts. AF can be very toxic and dangerous, thus ready-made foods for retail can contain at the most 2 µg/kg. The most common methods used to quantify AFM1 are high performance liquid chromatography (HPLC) and enzyme linked immunosorbent assay (ELISA) (24, 25). Both methods are time consuming, labor intensive and require a generous amount of sample. Zhang et al. coupled DLS with gold nanoprobes and magnetic beads (26). AFM1 was conjugated to bovine serum albumin (BSA) to create an AFM1-BSA conjugate. The AFM1-BSA conjugate was then conjugated to the surface of spherical AuNPs; thus producing nanoprobes. Anti-AFM1 antibody was linked to the surface of the magnetic beads by coupling the AFM1 antibody to protein G. The combination of the magnetic beads, AFM1 antibody and protein G resulted in antibody dynabeads. To determine the concentration of AFM1, antibody dynabeads, nanoprobes and AFM1 were combined in solution and incubated. The AFM1 and nanoprobes would compete for binding sites on the antibody dynabeads. The solution is then separated using a magnet and the remaining solution is analyzed by DLS, where the DLS intensity of nanoprobe is directly proportional to the concentration of AFM1 in sample solution. The limit of detection of AFM1 in milk was 27.5 ng/L and 37.7 ng/L in buffer. 170 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Aflatoxin B1 (AFB1), like other aflatoxins, is produced from mold and fungi, specifically, Aspergillus flavus and A. parasiticus. It has been debated to be the most potent carcinogen known and has said to be twice as carcinogenic as an X-ray if given in equal dosages (27). Alfatoxin B can penetrate through the skin and can thus contribute to many health issues. Xu et al. created AFB1-BSA conjugates that were easily attached to gold nanorods (AuNRs) and resulted in AuNR-AFB1-BSA conjugates (28). Each AFB1-BSA conjugate contained about 8 to 12 AFB1 per BSA. The conjugates would then be combined with AFB1 antibody. This leads to the aggregation of nanorods due to the binding of AFB1 antibody and AFB1 on the AuNR surface. If AFB1 is present in the sample solution, AFB1 will bind with the AFB1 antibody in the assay solution, preventing the aggregation of AuNR probes. There would be more individual AuNRs in the assay solution. The concentration of the dispersed AuNRs is equal to the concentration of free AFB1 in solution. The limit of detection was found to be 0.16 ng/mL. The assay was also found to be selective for AFB1 over other mycotoxins including Ochratoxin A (OTA), T-2, Deoxynivalenol (DON), and Zearalenon (ZON). To demonstrate its practical applications, studies were conducted on peanut samples. The peanut samples were artificially contaminated with AFB1. The concentration of AFB1 in each sample was analyzed according to a calibration curve. The results show recoveries ranging from 94.2 % to 117.3 %, suggesting that the new assay is suitable for agricultural samples.

Melamine Melamine is an organic base and most of its mass is due to the copious amounts of nitrogen. Melamine is harmful if swallowed, toxic and causes bladder cancer if ingested (29). In recent years, melamine has been added to milk products illegally to increase the total amount of protein in China. Small amounts of melamine can cause renal failure and kidney stones. Ma et al. used NanoDLSay for melamine detection (30). The author formed the hypothesis that melamine can induce the aggregation of citrate ligand-capped AuNPs by simply forming AuNP-melamine complexes. The average particle size of the assay solution increased at increasing concentrations of melamine between 0 ppm to 0.5 ppm. In order to determine if the assay is selective for melamine, the authors analyzed a broad range of analytes including aminoacetic acid, L-alanine, L-valine, L-serine, L-threonine, L-isoleucine, triethylamine, and ethanediamine. The average size of the AuNPs in the presence of melamine was more than 75 nm and in the presence of the other analytes was 30 nm or less. Milk samples were spiked with melamine at different concentrations and analyzed by DLS. The hydrodynamic diameter of the AuNP increased as the concentration of melamine increased. The detection limit of melamine in milk samples was 0.05 ppm. Although the sensitivity of NanoDLSay was not as high as that of liquid chromatography-tandem mass spectrometry (10 ppb) and extractive electrospray (270 ppb for melamine in milk powder), this method is highly reproducible, low cost, and rapid compared to the liquid chromatography-tandem mass spectrometry and extractive electrospray. 171 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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DNAs and RNAs With the essential role of DNAs and RNAs in the biological functions, there is a continuous demand for more sensitive and lower cost methods for their detection. Dai et al. synthesized a 30 nm AuNP and conjugated it to two ssDNA creating two AuNP-DNA probes (DNA1-AuNP, DNA2-AuNP) (8). The two ssDNA probe are designed to bind with the two ends of the target DNA. To detect the target DNA, a 1:1 mixture of the two DNA-AuNP probes with a concentration of 100 pM was mixed with target DNA that ranged from 5 pM to 5 nM in concentration. The detection limit was about 1 pM. To determine selectivity of the assay, the authors showed single base mismatched DNAs can be differentiated from matched DNA targets. The DLS assay is more sensitive than absorption based methods such as UV-vis absorption spectroscopy, which has a sensitivity of 10 nM. Zhang et al. reported on the detection of DNA using NanoDLSay (31). In this simple assay, 50 µL of single stranded monothiol DNA with different concentrations (5 µM, 1 µM, 100 nM, 10 nM, 2 nM) was combined with 4µL of 1,4-dithio-DL-threitol (DTT) solution (800 µM). DTT is a redox agent, and the dithiol group of DTT causes aggregation of AuNPs. The monothiol DNA terminates and stabilizes the cluster formation. The negative charge of the DNA acts as a stabilizer preventing larger aggregate formation due to electrostatic repulsion. As the concentration of the DNA that was added into the AuNP solution increased, the hydrodynamic diameter of the AuNP decreased. This study found a detection limit of 1 nM for single stranded DNA. Miao and coworkers developed an assay using AuNP probes modified with oligonucleotides to detect sequence specific double stranded DNA (dsDNA) (32). Two oligonucleotides, oligonucleoties 1 (Oligo 1) and 2 (Oligo 2) probes, were synthesized and adsorb to AuNPs separately to create AuNP probes: AuNP-Oligo1 and AuNP-Oligo2. The target dsDNA was composed of two complementary oligonucleotides, Oligonucleotides 3 and 4. The dsDNA can hybridize with Oligo1 or Oligo2. Hybridization of Oligo 1 and Oligo 2 from the AuNP with the target dsDNA can result in AuNP aggregation. The average particle size of the assay solution is proportional to the concentration of the target dsDNA. The detection limit of the assay was found to be 593 fM and the dynamic range was from 593 fM to 40 pM. Selectivity studies showed that single and double base pair replacements of the target dsDNA resulted in a decrease in the average diameter. Pylaev et al. used CTAB-capped AuNPs for target DNA detection (33). ssDNA from HIV, Bacillus anthracis and protective antigen precursor (pagA) were used as models. The limit of detection using AuNPs was 10 pM. For specificity study, the authors introduced single- and three-base-pair mismatches and the assay was able to discriminate between those and perfectly matched target DNAs. The assay can also distinguish between degrees of hybridization. The group performed colorimetric tests based on AuNP aggregation, and have shown that the detection of the colorimetric assays were 10-100 pM. Gao et al. developed an ultrasensitive detection method for the detection of sequence specific Nopaline synthase gene from transgenic products using label free AuNPs (34). Citrate-capped AuNPs will aggregate in the presence of high 172 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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salt (NaCl) conditions. When single stranded ssDNA probes were mixed with the AuNPs, the ssDNAs attached to the surface of the AuNPs, preventing AuNPs from aggregation in the presence of NaCl. When a non-complementary DNA sequence was added to the AuNP solution, the AuNPs stayed well dispersed. When the target DNAs and ssDNA probes were added to the AuNP solution together, a hybridization event of the target and probe DNA occurs. The hybridized double stranded dsDNA cannot bind to the citrate AuNPs. In the presence of NaCl, the unprotected AuNPs started to form aggregates, indicating the presence of target DNAs. There was a linear increase of the AuNP’s hydrodynamic diameter with the increase of the target DNA concentration. The assay demonstrated a dynamic range from 1.0 x 10-15 M to 5.0 x 10-9 M with a detection limit of 3.0 x 10-14 M. As a comparison, the colorimetric method using AuNPs had a detection limit of 1 x 10-8 M. A fluorescence method using ZnS and CdSe quantum dots had a detection limit of 2 x 10-9 M. The NanoDLSay is several orders of magnitude more sensitive than the colorimetric and fluorescence method. RNA interference is a pathway activated by a double strand RNA molecule and RNA molecules are able to inhibit gene expression. Two types of small RNA molecules that are central to RNA interference are microRNA and siRNA. MicroRNAs were first discovered in Caenorhabditis elegans and are also found in humans (35). The functions of microRNA are still being investigated and its specific targets are not well known, but it is apparent that microRNAs play a key and fundamental role in gene expression. MicroRNAs can affect post-transcriptional regulation and can play a role as a tumor suppressor or an oncogene. MicroRNAs such as miR15 and miR16 are tumor suppressors that target the 3’UTR of the anti-apoptotic protein BCL2 for post-transcriptional repression. An oncogenic miRNA, miR21 has been implicated as an anti-apoptotic factor and is overexpressed in human glioblastoma tumors (35). let7 miRNAs were discovered in Caenorhabditis elegans and are highly conserved in vertebrates and invertebrates. Humans have twelve let-7 genes that encode nine miRNAs. let-7 genes have been studied extensively and their biological role has been implicated to be altered or deleted in a variety of cancers, thus acting as a tumor suppressor with targets such as Ras and AT-hook 2 (HMGA2). let-7 has become a well-known cancer biomarker. Seow et al. used AuNPs and dynamic light scattering for the detection of the let-7 family (36). Their focus was to monitor the formation of defined AuNP assemblies and not the aggregation-based approach. Different sequences of the let7 miRNA (let7-a, let7-f, let7-g) were used as the biomarker targets. All the let7 miRNAs used are 22 bases long. Two complementary probes, L7a-1 and L7a-2, were designed to bind to complementary target nucleic acid from both the 5’ and 3’ ends, respectively, with an AuNP located at the other end. This creates a sandwich structure. In the presence of let7-a, there was a formation of dimers and trimers. The detection limit of the assay was 100 fmol. let7 individual family members are implicated in many diseases and the selection of one member over the other is very important. In order to identify the selectivity of the AuNP probe, probes specific to let7-a miRNA were used with let7-f and let7-g. let7-a, let7-f and let7-g were added separately to wells containing L7a probe. let7-a had an average size 173 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

change of 60 nm, let7-f had an average size change of 30 nm and let7-g had an average size change of 20 nm. In a mixture containing all members of the miRNA family, the L7a probe was still able to detect the presence of let7-a even with the different miRNAs present.

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Protein Biomarkers A biomarker is used as an indicator of the onset of disease and can serve as a tool to assess risk, presence, prognosis as well as therapeutics that are appropriate in response to the disease. Molecular biomarkers take many forms and thus can be explored for the validation as a reliable indicator of a specific disease state. Proteins have been sought after as molecular biomarkers because they are most directly affected in disease. Prostate specific antigen (PSA) is produced by cells of the prostate gland. The blood level of PSA is often elevated in men with prostate cancer (37). Men with prostate cancer have a decreased ratio of free PSA (f-PSA) to total PSA (38). The risk of prostate cancer onset increases as the ratio of f-PSA to total PSA decreases. Using this as a potential molecular biomarker for prostate cancer has shown to be promising in eliminating unnecessary surgeries for men with PSA levels between 4 and 10 ng/mL. Liu et al. applied NanoDLSay for the detection f-PSA (7). Two probes were prepared in the study. Citrate-protected spherical AuNPs with an average diameter of 40 nm were conjugated with a detector antibody and the CTAB-protected gold nanorods were conjugated with the capture antibody. When the two nanoprobes were mixed and then added to a standard f-PSA solution with different concentrations, the binding of f-PSA with the nanoparticle probes led to particle aggregate formation. f-PSA in the concentration range from 0.1 to 10 ng/mL was detected using NanoDLSay. A control experiment was performed to examine the selectivity of the assay. The mixed nanoprobe solution was added to solutions containing CA125, another cancer biomarker. There was no change or difference in the average particle size of the assay solution when combined with CA125. Alpha-fetoprotein (AFP) is produced during fetal development in the yolk sac and liver. Clinically, AFP overexpression has been associated with tumors and carcinoma (39). Chun et al. developed gold-coated iron oxide magnetic nanoclusters (Au-MNCs) combined with DLS for AFP detection (40). AFP was added to the Au-MNC solution in concentrations ranging from 0.01 to 50 ng/mL. The addition of AFP resulted in aggregation of the nanoparticles. The detection limit of the AFP-mediated aggregation was 0.2 pM, which was better than the detection limits of conventional fluorescence (170 pM) and ELISA assays (100 pM). In order to determine specificity of the assay, C-reactive protein (CRP) was added to the anti-AFP-functionalized Au-MNCs. There was no significant change in the average diameter of the AuNP assay solution. Nietzold et al. also developed an antibody functionalized AuNP for the detection of AFP (41). The citrate on the AuNP surface was replaced by mercaptopropionic acid (MPA) and anti-AFP monoclonal antibody. A 60 nm AuNP was used and had a fixed concentration of 0.08 nM. In the presence of AFP, there was an increase in the average particle size. Aggregation was observed between 0.1 and 0.4 µg/ml. The detection limit of the assay was 0.1 174 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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µg/ml. The specificity of the assay was confirmed using other proteins such as ovalbumin, β-lactoglobulin A and carbonic anhydrase I. These proteins did not cause nanoparticle aggregate formation when added to the anti-AFP AuNP probe. AFP was also detected from human serum samples. The serum was mixed directly with the AuNP probe solution and AuNP aggregation was observed, especially with serum samples with higher AFP concentration. Carcinoembryonic antigen (CEA) is involved in cell adhesion and is produced in the gastrointestinal tissue during fetal development and stops before birth under normal conditions. CEA is present in the blood in low concentrations in healthy adults, but has shown an up-regulation in colon cancer (42). Miao et al. developed silver core gold shell nanoparticles (Ag@Au) in conjunction with DLS for the detection of CEA (43). Anti-CEA antibody was attached to the surface of the Ag@Au. The Ag@Au/anti-CEA probe was about 65.3 nm in size. The average particle size of the probe increased with the addition of CEA in increasing concentration, with a final size increase of 201.6 nm when 10.0 ng/mL of CEA was added to the probe. The detection limit of the assay was 35.6 pg/mL and the dynamic range was from 60 pg/mL to 50 ng/mL. Other human serum proteins were tested to evaluate the selectivity of the assay. The addition of CEA (1.0 ng/mL) to the serum samples led to a much greater particle size increase as opposed to the other proteins, including bovine serum albumin (BSA), cancer antigen 19-9 (CA19-9), cancer antigen 15-2 (CA15-3), alpha-fetoprotein (AFP) at a significantly higher concentration of 10 µg/mL. Six serum samples with different concentrations were evaluated and the test showed a recovery between 96.1 % and 104.3 %. The results of NanoDLSay were validated by ELISA.

Viruses and Virus Antigens Influenza A virus causes influenza in birds. Influenza A can be transmitted from birds to poultry, which can result in human influenza pandemics. H1N1 and H3N2 are influenza A subtypes most commonly found in people. In 2009, a new influenza A virus had risen and caused major illness in people (44). The 2009 H1N1 virus replaced the H1N1 virus that was previously causing illness in people. This new H1N1 virus caused a pandemic of the influenza A virus and has encouraged researchers to discover a way to diagnose this disease in a more rapid manner. The current diagnostic tests are Polymerase Chain Reaction (PCR), enzyme-linked immunosorbent assay (ELISA) and some immune-chromatographic assays which can be time consuming, costly, labor intensive and do not offer rapid results. Driskell et al. explores the use of AuNPs for influenza virus (PR8) detection (45). A monoclonal antibody, IC5-4F8 that binds to intact influenza virus A was conjugated to AuNPs with different sizes to form the AuNP immunoprobe. The NanoDLSay offers great improvements to detection limits compared to colorimetric based assays, which are not quantitative. The detection limit for the assay was 8.6 x 101 TCID50/mL and was calculated as equal to the concentration of PR8. This is orders of magnitude improved over the detection limits by FDA-approved commercial influenza test kits which are reported in a range of 2.5 x 103 to 1.0 x 104 TCID50/mL. 175 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Hepatitis B is caused by the hepatitis B virus and ultimately has negative effects on the liver (46). Hepatitis B can be acquired through sexual intercourse, IV drug use as well as exposure to bodily fluids. The disease can cause yellowing of the skin, fatigue, and dark urine, but most people are asymptomatic. People with a severe case of Hepatitis B can develop cirrhosis and liver cancer which will lead to death. Wang et al. developed an assay to effectively detect Hepatitis B virus (HBV) using Hepatitis B surface antigen (HBsAg) and AuNPs in a sandwich-type model (47). Two types of AuNPs with different size combinations were conjugated with two anti-HBsAg antibodies to form a pair of AuNP immunoprobes. The group first analyzed the detection limits of AuNP size combinations. Combinations using AuNPs with sizes 10 nm and 50 nm (10:50), and 100 nm with 50 nm (100:50) were compared by adding HBsAg to the AuNP probe solutions. The 100:50 probe pair exhibited a much higher sensitivity than the 10:50 immunoprobe pair with a detection limit of 0.005 IU/ml. The group further investigated the capability of the 100:50 nanoprobe pair in analyzing HBsAg in HBV-infected samples. The nanoprobe was able to distinguish between HBV-positive and HBV-negative samples (47). The mixed nanoprobes proved to be 80 times more sensitive than ELISA and twice as sensitive as the Surface Plasmon Resonance (SPR) detection system.

Future Outlook DLS was not traditionally used for quantitative analysis. However, combined with the exceptionally strong light scattering AuNP probes, DLS becomes an excellent analytical tool for quantitative detection and analysis. As demonstrated from published studies and examples presented in this review, NanoDLSay can be used for the detection of a wide range of chemical and biological target analytes, ranging from toxic metal ions, small chemicals, to large biomolecules including proteins, DNAs, and viruses. The assay shows excellent reproducibility and specificity in most studies. The sensitivity of NanoDLSay is either on par with or significantly better than most of the current techniques. NanoDLSay is a single step, homogeneous solution assay. No washing step is required for the assay. The assay process is extremely simple. Typically the test results are obtained within 30 min. DLS is a relatively simple and low cost technique. Combined with its simplicity, fast analysis, and low cost, NanoDLSay is well positioned for point-of-care applications. So far most applications of NanoDLSay have been demonstrated in laboratory settings. More work needs to be conducted to adapt this technique for field and clinical applications. There are two specific issues particularly necessary to be dealt with: one is the quality and stability of the AuNP probes; and the second one is how to manage the inhomogeneity, interference and background signal from various sample matrices. As to the first issue, the quality of the AuNP probe is the most critical element for the success of NanoDLSay. Quality, here mainly refers to the qualitative and quantitative binding activity of the AuNP probe with the target analytes. If the binding activity of the AuNP cannot be maintained at a constant level, standardizing the NanoDLSay test results will be difficult. 176 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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For field and real world applications, the AuNP probe needs to remain stable under reasonable storage conditions, and the binding activity of the AuNP probe cannot be compromised during storage. Another quality issue of AuNPs is the potential particle fusion or crosslinking due to decreased stability of the AuNP during storage. NanoDLSay specifically relies on the formation of target-induced AuNP aggregates for the detection. Non-specific particle fusion or crosslinking will interfere with the NanoDLSay tests. According to our literature survey, it appears that no systematic studies have been conducted to validate the long-term storage stability of the AuNP reagents used for each assay. It is important that the AuNP probe can be manufactured in large quantity with constant quality and long-term stability. More extensive optimization and long-term validation studies should be conducted for each assay being developed. The second challenge along the way towards the real world application development for NanoDLSay is how to manage the inhomogeneity and potential interference arising from the sample matrices. Real world samples, regardless of whether it is drinking water from wells, lakes and rivers, agriculture product, or biological fluids such as blood and urine, have different physical forms, and most of these samples contain micro- and nanoparticles that also scatter light intensely. For example, human blood is a thick and viscous liquid that contains millions of cells, tens of thousands of proteins and other biomolecules, as well as a large number of vesicles, exosomes, and other particulate species. These biomolecular species may present strong non-specific interactions with the AuNP probes, and the light scattering from these biomolecular species can have significant effect on the average particle size measurement of the assay solution. We have recently started to examine and address both issues. In one study, we studied the interactions between different types of biomolecules with citrate-capped AuNPs and discovered the existence of several interaction modes (48). This study helps to shed light on potential non-specific interactions between biomolecules in blood and AuNPs. In another study, we demonstrated that in order to adapt NanoDLSay for blood sample analysis, the size of the AuNP probe needs to reach approximately 100 nm (diameter) in order to overcome the background scattering from blood serum samples (49). Using the 100 nm citrate-capped AuNP, we developed a new blood test for early stage cancer detection and risk assessment based on the tumor-induced increased immune activity in cancer patients. Overall, NanoDLSay has shown promise as a powerful technique for chemical and biological target detection and analysis. Further research and development on this technique could lead to some commercially viable products for diagnostics, food safety, and environmental protection applications.

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Chapter 10

Conducting Polymeric Nano/Microstructures: From Fabrication to Sensing Applications Publication Date (Web): December 8, 2015 | doi: 10.1021/bk-2015-1215.ch010

Bin Dong* and Lifeng Chi Institute of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-based Functional Materials and Devices & Collaborative Innovation Center of Suzhou Nano Science and Technology, Soochow University, Box 33, Ren-ai Road, Suzhou Industrial Park, Suzhou 215123, People’s Republic of China *E-mail: [email protected].

By utilizing a variety of different lithographic methods (such as tip lithography, e-beam lithography and nanoimprinting lithography) on conducting polymers with various compositions (e.g., polypyrrole, polyaniline or copolymers) with surface active monomer, we will be able to fabricate conducting polymeric structures at nanoscale. The size of the resulting conducting polymer nanostructure can be controlled with a structural resolution higher than 100 nm. By carefully studying the properties of these nanostructures, we find that they can be utilized as an ammonia gas sensor with high sensitivity, fast response and good reversibility. In addition, a size-effect is observed for the nanosensor device, with the sensitivity being reverse proportional to its size.

Introduction With the fast growth of nanoscience and nanotechnology, researchers with multiple disciplinary backgrounds have been involved in this developing field, aiming to understand and get new insight on the nano-effect of the nano/microstructures (1–7). A variety of different nano size effects have been reported. For instance, the quantum dots are typical examples of the size effect, © 2015 American Chemical Society In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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for which the fluorescence properties are highly dependent on their size with a red shifted emission as a result of their increased size (8). Magnetic nanomaterials exhibit properties different from their bulk counterpart. For example, iron oxide nanoparticle shows size dependent magnetic properties. When its size shrinks below a critical value, it became superparamagnetic, i.e. there is no residual magnetism after the magnetic field is removed, which is in sharp contrast to the residual magnetism for the bulk ferromagnetic materials (9). Other examples include carbon materials, such as the activated carbon or carbon based nanomaterials, including C60 (10), carbon nanotube (11), graphene (12, 13), etc. Due to their high surface area to volume ratio, they have found wide applications in energy storage devices. Zinc oxide (14) and titanium oxide nanostructures (15), have also been reported to exhibit similar size dependent properties. On the other hand, polymer science is a rapid developing field, where a great many of new advances have been made. Other than the traditional polymers, such as the polyethylene terephthalate, polytetrafluoroethylene and polyphenelene terephthalamide, etc., functional polymers have also been widely utilized in a variety of different energetic devices, for example, polyphenylene vinylene used in polymer light emitting diode (16), poly (3-hexylthiophene) utilized in polymer solar cell (17) or Nafion proton exchange membrane utilized in direct methanol fuel cells (18, 19). By combining these two research fields, i.e. by utilizing the nanotechnology fabrication method to obtain polymeric nanomaterials, we will be able to systematically study the polymer structural properties at nanoscale. In particular, although nano-effects have been demonstrated for inorganic materials, the size effects in polymeric materials have seldom been reported. Through such study, not only can we gain new knowledge of the polymer material property at such a small scale, but also apply the new property in the construction of new devices, which may have great potential from both the science and technological point of views. Based on our previous works, we have found that there are also size effects for polymeric materials, when shrinking its size down to the nanometer scale (1–3).

Results and Discussion Figure 1 summarizes the research method. By utilizing the nanofabrication method, such as top down lithographic method, we will be able to obtain polymeric nanostructures with various shapes, such as circle, wire, etc. with at least one dimension reaching the nanometer scale. The polymeric materials utilized in current study are conducting polymers (20), such as polypyrrole (PPy) (21, 22) and polyaniline (23, 24). Our researches focus on the unique physical and chemical properties of the polymeric nanomaterials, and emphasize on the nano-size effect which the polymeric nanostructures possess. By carefully studying these unique properties, we will be able to construct functional nano devices. The potential applications of these structures in chemical sensors have been explored. 182 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 1. Schematic illustration showing the research method.

Nanosensors have attracted more and more attentions in recent years. The works in this field have been pioneered by the researchers in Stanford (25) and Havard (26) university, who reported the carbon nanotube and silicon nanowire based gas nanosensor, which exhibits a ultra-high sensitivity with the capability of realizing the detection of a single molecule. As compared to the conventional sensors, the nanosensors possess several advantages. For example, the nanosensor requires lower power consumption, exhibits high sensitivity and fast response time, while the conventional sensor normally operates at high temperature and exhibits low sensitivity. However, the nanosensor based on inorganic material suffers from several limitations. First, the inorganic material is not versatile for sensing applications. Second, the synthesis and fabrication are complicated which takes multiple time-consuming steps. Third, in order to realize the high sensitivity, the post-synthesis modification of the inorganic materials is often required, which is tedious. As compared to the inorganic materials, the utilization of conducting polymer inside a sensor may offer several advantages (27). They are versatile sensing materials, low cost and can be easily synthesized. The electrical and sensing properties of the conducting polymers can also be finely tailored. 183 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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In order to fabricate the conducting polymer nanostructures, we combined the atomic force microscopic (AFM) tip lithography with the lift off process. The fabrication process is illustrated in Figure 2 (1). A resist material (i.e. polymethyl methacrylate or PMMA) is first spin coated on the silicon substrate. Tip-lithography is then utilized to obtain pattern on the surface of PMMA coated silicon substrate. After exposing the substrate to the pyrrole terminated silane, a layer of ferric chloride (FeCl3) and sodium dodecyl sulfate (SDS) is spin coated on its surface, which is then exposed to the pyrrole atmosphere to obtain the PPy. The conducting polymer pattern is finally formed after the lift-off process.

Figure 2. Fabrication process of the conducting polymer nanostructures. Adapted with permission from reference (1). Copyright 2005 Wiley.

By utilizing this method, we will be able to fabricate the conducting PPy nanowires in between two electrodes. Figure 3a shows the AFM image indicating a single PPy nanowire bridging the gap between two metal electrodes. Furthermore, we have studied the electrical property of this structure. As can be seen from Figure 3b dashed line, before the deposition of the PPy nanowire, the two electrodes are not conductive at all. After the deposition of the PPy nanowire, the two electrodes are conductive, indicating the PPy nanowire has successfully bridged the gaps between the two electrodes, as shown in Figure 3b solid line. In addition, we have further explored the fabrication resolution of the current method. As illustrated in Figure 3c, the fabrication resolution is approximately 250 nm, indicating this method can be utilized to realize high resolution conducting polymer nanostructure fabrication. 184 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 3. (a) AFM image showing a single PPy nanowire bridging the gap between two gold electrodes (size: 10 μm × 10 μm). (b) I-V curve of the structure in the presence of the PPy nanowire (solid line) and in the absence of the PPy nanowire (dashed line). (c) A 250 nm wide line fabricated using this method (size: 5 μm × 5.6 μm). Reproduced with permission from reference (1). Copyright 2005 Wiley.

Since conducting polymers are p-type doping materials, the conductivity of the conducting polymer is highly dependent on the surrounding environment. When exposing the conducting polymers to atmosphere containing different gas molecules, the conductivity of the conducting polymer can be altered. For example, the exposure of the conducting polymer to an electron donating gas molecules, such as ammonia gas, the conductivity of the conducting polymer will decrease; on the other hand, when exposing the conducting polymer to an electron withdrawing gas molecules, such as nitrogen dioxide, the conductivity will increase accordingly. Based on this mechanism, the conducting polymers have been widely utilized for sensing applications. The synthesized PPy nanowire can be utilized as a gas sensor. Figure 4 shows the changes in the resistivity of the PPy nanowire before and after the exposure to 240 ppm ammonia gas. The arrow in Figure 4 indicates the time when the ammonia gas is added. Upon the ammonia gas exposure, the resistance of the PPy nanowire increases dramatically, indicating the PPy nanowire is very sensitive to ammonia gas. The sensitivity can be calculated by the resistance changes before and after the ammonia gas exposure to be around 0.8. Moreover, we have further studied the PPy thin film based sensor. As can be seen from Figure 4b, the PPy thin film is still sensitive to the ammonia gas. However, the resistance change is much less as compared to the PPy nanowire based sensor. The sensitivity for the PPy thin film based sensor is then calculated based on Figure 4b to be approximately 0.1, which about 1/8 of that of the PPy nanowire nanosensor. This result demonstrates the superiority of the nanowire based nanosensors, i.e. the high sensitivity during exposure to the same amount of the ammonia gas. 185 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 4. (a) Sensing behavior of the PPy nanowire based nanosensor. (b) The sensing behavior of the PPY thin film based sensor. Adapted with permission from reference (1). Copyright 2005 Wiley.

Although the above mentioned method can be utilized to construct conducting polymer nanostructures, the resolution of the conducting polymer nanostructure is relatively low. Since we have already demonstrated that the sensitivity of the conducing polymer based nanosensor is more sensitive when shrinking its size. We wonder whether we can further obtain conducting polymer nanosensors consisting of smaller sized nanowires. For this purpose, we developed a copolymer strategy by introducing a surface active comonomer to the backbone of the conducting polymer, which will further enhance its adhesion to the substrate so that the tiny structure can withstand the peel-off process. Figure 5 summarizes the fabrication process, which combines the standard peel-off process with the copolymer strategy and is generally applicable to both PPy and polyaniline (2). 186 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 5. The fabrication of the conducting polymer nanostructures based on copolymer strategy. Reproduced with permission from reference (2). Copyright 2005 Wiley.

By combining the copolymer strategy, e-beam lithography and the peel-off process, we will be capable of fabricating the conducting polymer nanostructures with sub-100nm resolutions. Figure 6a shows the typical AFM image of an 80 nm wide PPy conducting polymer nanowire fabricated by this strategy. By utilizing e-beam lithography, different structures can be easily defined, which allow us to fabricate a nanosensor exclusively based on conducting polymers, i.e. both the electrodes and active nanowire parts are made of conducting polymers. Figure 6b shows the 6 pairs of conducting polymer electrode shaped structure. All 6 pairs of structures are connected with a nanowire, which is approximately 100 nm in width. A typical AFM image shown in Figure 6c exemplifies one example of the nanowire bridging the gap between two conducting polymer electrodes. 187 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 6. (a) AFM image of an 80 nm wide PPy conducting polymer nanowire fabricated by copolymer strategy. (b) The optical microscopic images indicating the six pairs of electrode shaped structure consisting exclusively of PPy conducting polymers. The width of the electrode shaped structure is about 50 micron (size: 475 μm × 390 μm). (c) The enlarged AFM image showing a 100 nm wide PPy nanowire bridging the gap between the two PPy conducting polymer electrodes (size: 9 μm × 10 μm). Adapted with permission from reference (2). Copyright 2005 Wiley. The structures consisting exclusively of the conducting polymer electrodes and the nanowire can be directly utilized for sensing applications. We first examined the conductivity of this nanodevice. As can be seen from Figure 7a, the current potential characteristic indicates that the resulting structure is conducting and exhibits an ohmic behavior. By exposing this device to 240 ppm ammonia gas (40 s on and 40 s off, Figure 7b), it was shown that this device exhibits very fast response toward ammonia gas. The sensitivity of this device can be calculated to be around 0.8. Furthermore, by utilizing e-beam lithography and copolymer strategy, we will be capable of altering the width of the PPy conducting polymer nanowire and evaluating its performance accordingly. As illustrated in Figure 7c, the sensitivity exhibits a width dependent sensing behavior, i.e. the sensitivity of the sensor increases as the width of the PPy conducting polymer wire shrinks to the nanometer scale, which demonstrates the superiority of the nanosensor.

Figure 7. (a) The I-V curve of the 100 nm nanowire device consisting of PPy electrodes. (b) The sensing performance of this device when exposing it to 240 ppm ammonia gas. (c) Size dependent sensitivity of the nanosensor devices with different width. Adapted with permission from reference (2). Copyright 2005 Wiley. 188 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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This fabrication method is generally applicable and can be extended to the fabrication of other conducting polymers. Figure 8a shows the example of the polyaniline conducting polymer electrode shaped structures fabricated by this strategy. As can be seen from the enlarged AFM image shown in Figure 8b, the end of the polyaniline electrode shaped structures are connected by a tiny polyaniline nanowire which is about 200 nm in width. This structure is also electrically conductive, as shown in Figure 8c, indicating it is suitable for further device fabrication.

Figure 8. (a) Electrode shaped structures made of polyaniline (size: 350 μm × 200 μm). (b) AFM image (3.5 μm × 3.5 μm) of one enlarged structure shown in (a) indicating a 200 nm wide polyaniline nanowire has bridged the gap between the two polyaniline shaped electrodes. (c) I-V characteristic of the structure shown in (b). Reproduced with permission from reference (2). Copyright 2005 Wiley. 189 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Furthermore, by utilizing the same copolymer strategy, we will be able to fabricate the conducting polymer nanowire arrays when combined with the large area fabrication method, such as nanoimprinting lithography (28, 29). Figure 9 summarizes the construction process, which involves the fabrication of the resist structures enabled by nanoimprinting lithography followed by the deposition of the conducting polymer nanostructures based on copolymer strategy (3). The large area conducting polymer nanostructures are formed after the peel off process. This strategy can be utilized to obtain both the PPy and polyaninline nanostructures.

Figure 9. The fabrication process of conducting polymer nanowire arrays based on nanoimprinting and the copolymer strategy. Adapted with permission from reference (3). Copyright 2006 Wiley.

Figure 10 shows the SEM images during each fabrication step. The structures of the mold used for the nanoimprinting lithography are shown in Figure 10a-b, where the large area nanostructures are clearly visible. By imprinting this structure directly onto the surface of the polymeric resist, the structure on the mold can be successfully transferred to the surface of the resist (Figure 10 c-d). Since a residual layer is present, plasma etching is then exploited to remove it, after which the bare silicon surface is exposed, as illustrated in Figure 10 e-f. 190 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 10. (a-b) SEM images showing the fine structures of the mold utilized for nanoimprinting lithography. (c-d) SEM images indicating the resist structure after the imprinting, in which the structures are transferred to the resist. A residual layer is clearly visible in this case. The patterns are 900 nm and 450 nm for (c) and (d), respectively. (e-f) The SEM images showing the structure after plasma etching process, after which the residual layer is removed and the bare silicon surface is exposed. Reproduced with permission from reference (3). Copyright 2006 Wiley.

Based on the structures fabricated by nanoimprinting lithography, copolymer strategy is utilized to deposit conducting polymer structures directly onto the surface of this structure. After the peel-off process, the polymers deposited on the surface of the resist layer are removed while those bonded to surface silicon remain. Thus, the conducting polymer nanostructures are formed. The resulting PPy nanostructures can be made to cover a large area due to the large area patterns obtained by nanoimprinting lithography. As can be seen from Figure 11, areas as large as 30 by 30 microns can be easily constructed without any defects. By utilizing different molds with various trench structures, a variety of resist structures can be obtained, based on which the conducting polymer nanostructure with various spacing can be obtained. Figure 11 shows a series of AFM images of the different PPy conducting polymer nanostructures obtained, including 600 nm 191 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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wide, 300 nm wide and 150 wide structures. The separations between different conducting polymer nanostructures can also be tuned, which ranges from 540 nm, 300 nm to 150 nm (Figure 11). As can be seen from the AFM section analysis shown in Figure 11e, the fabricated conducting polymer nanostructures possess a rather uniform height. The controlled line width, separation, the uniform height and large area demonstrate the superiority of this fabrication method.

Figure 11. (a) AFM image of the large area, high density PPy conducting polymer nanowire arrays fabricated by the nanoimprinting lithography and copolymer strategy. The width of the PPy wire is 600 nm separated by 540 nm distance. (30 × 30 μm). Inset: AFM image of the enlarged area showing the details of the fabricated structure (size: 5 × 5 μm). (b) AFM of the PPy nanowires with a width and separation of 300 nm and 300 nm, respectively. (30 μm × 30 μm). Inset: the enlarged AFM image which is 5 μm × 5 μm by size. (c) AFM image of the conducting PPy nanowires with 150 nm width and 150 nm separation (size: 30 × 30 μm) (d) The enlarged AFM image of (c) showing a 5 × 5 μm sized area and (e) the corresponding AFM section analysis of the structure shown in (d). Adapted with permission from reference (3). Copyright 2006 Wiley. 192 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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The combination of the nanoimprinting lithography and the copolymer strategy renders the fabrication of a series of conducting polymer nanostructures in a controllable fashion. The resulting structures exhibit structural colors originating from the ordered pattern at nanoscale. Figure 12 shows the CCD camera image indicating the colors caused by the diffractions from the structures deposited on the surface of the silicon substrate. Depending on the spacing of the structure, the color can be altered. For instance, 100 nm wide structure with 150 nm spacing exhibits blue color, while the largest 350 nm wide structure separated by 400 nm spacing shows red color.

Figure 12. CCD camera image of the light diffraction from the structures containing high density PPy conducting polymer nanowire arrays. (1) PPy nanowire which is 100 nm wide with a separation of 150 nm. (2) 150 nm wide nanowire with a distance of 200 nm. (3) 250 nm wide structure separated by 300 nm distance. (4) 350 nm wide PPy nanowire with a separation of 400 nm. (5) 320 nm PPy nanowire which is 380 nm apart from each other. (6) 220 nm wide nanowires with a separation of 380 nm. Reproduced with permission from reference (3). Copyright 2006 Wiley.

Gold pads are deposited onto the PPy conducting polymer nanowire arrays by utilizing shadow mask evaporation method. As can be seen from Figure 13a, the AB and BC directions are perpendicular and parallel to the PPy nanowire directions, respectively. By studying the conductivity along these two directions (Figure 13b), we found that the BC direction, which is parallel to the nanowire direction, is conductive, while the AB direction, which is perpendicular to the nanowire structures, is not conductive. Furthermore, we have tested the sensing 193 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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performance of this device by exposing it to 240 ppm ammonia gas. As shown in Figure 13c, the device is reversibly sensitive to the repetitive exposure to the ammonia gas and air. The sensitivity of this device is determined to be around 0.5. In addition, we have compared its sensing performance with another device containing a 5 μm wide PPy wire array. As can be seen from Figure 13d, the sensitivity is approximately 0.25, which is about one half of that of the nanowire based nanosensor, demonstrating the superiority of the nanowire based sensor. Another advantage of the nanowire array based nanosensor is that it can tolerate error, i.e. one or two fabrication failures will not influence the device performance since the device consists of many nanowires. The current fabrication method can be easily made to produce large area enabled by nanoimprinting lithography, which thus provide a potentially effective method to fabricate the conducting polymer nanosensor at low cost.

Figure 13. (a) Gold pads deposited on the surface of the PPy conducting polymer nanowire arrays (size: 330 μm × 330 μm). The PPy nanowires are parallel to the BC direction and perpendicular to the AB direction. (b) The I-V characteristics of the measurement performed through BC and AB gold pads. (c) The sensing performance of the resulting PPy nanowire arrays when exposed to 240 ppm ammonia gas (40 s on and 40 s off). (d) Response of a sensor made of 5 μm wire arrays to 240 ppm ammonia gas (40 s on with 40 s off). Adapted with permission from reference (3). Copyright 2006 Wiley. 194 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Due to the versatility of the copolymer strategy and nanoimprinting lithography, the current method can be extended to the fabrication of different conducting polymers with a variety of different structures and shapes. Figure 14 shows the polyaniline structures fabricated by this method. Various structures and shapes, including circles, line, holes with a variety of different sizes can be successfully obtained, demonstrating its versatility.

Figure 14. AFM images showing the polyaniline nanostructure with various shapes and sizes fabricated by nanoimprinting lithography and copolymer strategy (size: 56 × 56 μm). (a) 800 nm diameter circular structure separated by 3 μm. (b) 800 nm diameter hole structures with a separation of 2 μm (size 50 × 48 μm). (c) 650 nm wide polyaniline wire separated by 400 nm (size 15 × 15 μm). Reproduced with permission from reference (3). Copyright 2006 Wiley.

Conclusion In summary, by utilizing the nanofabrication methods to construct polymeric nanostructures, we will be able to study the physical and chemical properties of the as-fabricated polymeric nanostructure and reveal the property differences between the nano-sized structure and its bulk counterpart. Furthermore, through such study, we will be capable of applying these polymeric nanostructures to the preparation of polymeric nanomaterials and fabrication of nano-devices. By utilizing top-down fabrication method widely utilized in nanotechnology, we have successfully fabricated a variety of different nanostructures based on functional polymeric materials, i.e. conducting polymers. In addition, we have researched the size-effect of the resulting polymer nanostructure and explored its influence on the sensing performance, which shows the way toward the fabrication of low cost and highly sensitive nanosensors. 195 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Chapter 11

Organic-Inorganic Supramolecular Gels and Contrast Agents for Magnetic Resonance Imaging Based on the Surfactant-Covered Polyanionic Clusters Bao Li and Lixin Wu* State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Qianjin Avenue 2699, Changchun 130012, Jilin, People’s Republic of China *E-mail: [email protected].

The progress of a recently developed supramolecular strategy for organizing nano-sized fine particles into soft material systems was reviewed and discussed. By covering inorganic polyanionic clusters bearing uniform topologic architecture and well-defined chemical composition with cationic surfactants through electrostatic interactions, a type of ionic complex was prepared for the amphiphilic building blocks of self-assemblies in soft materials systems. Because of the core-shell composite structure, these complexes can be regarded as a supramolecular reversed micelle or supramolecular surfactant, depending on the phase separation capability, and thus be used for the fabrication of new hybrid organic-inorganic self-assembled structures and bio-applications. These inorganic clusters introduced in these complexes not only play a role as a structural unit, but also as a functional group for the functionalization of obtained soft materials. In the present chapter, we summarized synergistic interactions and correlations between two incompatible components in these complexes during the self-assembly for responsive supramolecular gels and magnetic resonance imaging contrast agents.

© 2015 American Chemical Society In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Introduction The close cross connection with material science, nano science, biology, medicine, and so forth has been one of the attractive aspects in colloids and interface chemistry in recent years, and thereby creates new exciting crossing borders among those areas. The combination of classic surfactants with fine particles and corresponding physicochemical behaviors are of the fundamental interests from among multidisciplinary fields. Polyoxometalates (POMs) belong to an interesting family of metal-oxygen polyanionic clusters bearing well-defined topologic architectures that are usually comprised of early transition metals such as vanadium, niobium, molybdenum, and tungsten (1–5). POMs can be prepared as an isolated dispersion, or endless linear and network crystalline solids, in which discrete single clusters are in uniform sizes from less than one to several nanometers. Depending on the composition and framework, POMs possess diverse behaviors in solid acidity, redox activities, catalysis, optics, and magnetism, and they have been applied for clinical studies due to their antibacterial and antiviral activities (6–9). Although there are inspiring merits for these self-assemblies due to their beautiful topological architectures, POMs still have not yet become common building blocks in solution systems in the past decades, toward soft materials. Recently some giant polyanions were found to form fantastic hollow spherical aggregations called “blackberries” (10), but the dynamic time scale of their ordered organization and their structural instability requires improvements to facilitate the POMs’ use in further convenient applications in colloids and interface chemistry. Main problems lying in this area are the cluster’s stiffness, the difficulty of chemical modification by organic groups, the lack of necessary components (not for crystals), and the multiple surface charge-induced repulsion between clusters in solution and at interfaces. Some effective methods have been developed to tune these POMs’ colloidal behaviors, including covalent modifications of organic groups (11, 12), surface charge controlling, as well as the selection of solvents (13). However, most of the known strategies were designed for specific clusters and a universal strategy suitable for water soluble POMs is highly desired. On the other hand, all POMs have multiple surface charges and can be used as the binding force bewteen organic components bearing countercharges via electrostatic interactions. Some early efforts in transferring POMs into organic phases for catalysis demonstrated the feasibility of this route by simply replacing the surface counterions with cationic surfactants. Further progress to spread cationic surfactant-covered giant POMs on the water surface provided typical examples of POMs supported by organic components which can be compared with results obtained from in-situ two phase mixing methods (14, 15). Through detailed evaluations for the composition of organic cation/POM complexes, these hybrid building blocks were extended from air/water interface to solution systems, and the regularity in getting diverse self-assembly behaviors targeting the functional applications of POMs in soft materials was discovered. Herein, we would like to summarize representative achievements in the research of gelation behavior and magnetic resonance imaging capability of this kind of POM complexes prepared following the strategy. 200 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Experimental Section Preparation of Surfactant-Covered POM Complexes

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The preparation of electrostatically binding complexes undergoes simple but complete procedures depending on the hydrophilicity of organic components. For the two phase mixing route, the detailed preparation includes following process: mixing the aqueous solution of polyanion cluster and the weakly polar organic solution of surfactant together, stirring the incompatible mixture for full charge interaction, then transferring the POM component into the organic phase, and finally separating the product from the organic phase and carrying out a purification (16). Elemental analysis, TGA, NMR and IR spectroscopy, and Mass spectrometry are necessary tools for the determination of the chemical composition. Preparation of Supramolecular Gels The preparation of supramolecular gels is similar to the general route for the preparation of organic gels. In most cases, solids of complexes were dissolved in organic solvents. For the mixture solvents, the complexes were usually dissolved first in an easily soluble solvent to which the other one was added. Gels often form quickly, but in some cases, solutions need to be heated and then cooled to room temperature slowly. Relaxivity Measurement Sample solutions at the investigated concentration were prepared through sonication of the complex in water for a short period of time. Then the solution was left to stand for 24 h at room temperature before the following characterization. For the preparation of sample Mn-12-C18 mixing with C18EO10, a stable emulsion was obtained by sonicating the mixture of Mn-12-C18 in n-hexane and C18EO10 in water in a suitable ratio. The organic solvent was evaporated during vigorous stirring, yielding the C18EO10 encapsulated Mn-12-C18 aggregates. The prepared Mn-12-C18/C18EO10 aggregates were well-dispersed in aqueous solution and underwent a centrifugation to remove insoluble residues. The relaxivity measurements were carried out at 25 °C on a Bruker Ultrashield 500 MHz spectrometer. For each sample, longitudinal 1H relaxation time (T1) was measured using the inversion recovery method. Slopes of plots of longitudinal relaxation rate r1 (1/T1) versus the concentration of Gd-POM complexes were used to calculate r1 values.

Results and Discussion Fundamental Properties of Surfactant-Covered POM Complexes After the charge exchange and coupling, the formed POM complexes no longer dissolve in water, but become soluble in weakly polar organic media instead because the cationic hydrophilic head of surfactant molecules have anchored on the POM surface while the hydrophobic tail faces toward the outside 201 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

environment. After this process, initial counterions of both POMs and surfactants have been removed upon the separation of the aqueous phase. Therefore, in these complexes, the cationic surfactant and the polyanionic cluster become counterions of each other. The negative charges are delocalized on the surface of the POM, facilitating the formation of ion-pairs where surfactant cations are available.

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Supramolecular Gels of POM-Cored Complexes In comparison to polymeric gels, in which the three dimensional network comprised of the fibrous structures of polymer gelators stabilizes the solvent molecules in the aqueous solution, supramolecular gels are derived from the bicontinuous self-assembled structure of small molecules due to intermolecular interactions. Therefore, in principle, most low molecular weight molecular components or composites in solutions that stabilize solvent molecules by generating bicontinuous self-assemblies may be used as the gelator. Because of rich self-assembled properties of surfactant-covered POMs, it is also possible to utilize these amphiphilic complexes to prepare supramolecular gels. We evaluated the basic synergistic effect of organic and inorganic components in hybrid complexes as gelator candidates. Both parts have been demonstrated to be crucial for the formation of the self-assembly structure. The length of the alkyl chain and the number of organic cations covering POMs (17, 18), the terminal modification (19, 20), the shape and the surface charge density of POMs (21) were found to be important in the modulation of interactions between alkyl chains and even between complexes in the assemblies. These factors are decisive in subsequently fabricating various aggregation architectures under selected conditions (22). To understand the effects of alkyl chain density and the shape of POMs on the gelation property, we first employed the cationic surfactant, dioctadecyldimethylammonium bromide (DODA·Br) to wrap up POMs bearing different charges. To make a convenient comparison among the complex gels, we investigated three types of complexes according to the structure of POMs and amount of the surface charge (Figure 1). Complex-1 (a, b), and -2 (a, b, c) correspond to complexes bearing Keggin-type clusters ([XW12O40]n−, X = P, Si and n = 3, 4; a: PW12O403− and b: SiW12O404−) and lacunary Keggin-type POMs ([XW11O39]n−, X = P, Si, B, and n = 7, 8, 9; a: PW11O397−, b: SiW11O398−; c: BW11O399−), respectively, while complex-3 (a, b, c) afford cashew-shaped POMs comprising two lacunary Keggin-type POMs linked by an europium ion ([Eu(XW11O39)2]n−, X = P, Si, B, and n = 11, 13, 15; a: Eu(PW11O39)211−; b: Eu(SiW11O39)213−; c: Eu(XW11O39)215−). The gelation behavior of these complexes was examined in common organic solvents at the same concentration (3 wt%). The collected results revealed that weakly polar solvents were in favor of the fabrication of supramolecular gels. Complexes of less charged POMs were hard to dissolve in weakly polar solvents even upon heating, due to their deficiency in binding cationic surfacants. On the contrary, complexes comprising POMs with appropriate charges dissolved better in weakly polar solvents such as n-hexane, cyclohexane, isooctane, and nonpolar carbon tetrachloride due to the increased amount of organic components. The formation of gels could be 202 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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then observed after slow cooling of the heated solutions to room temperature. However, in benzene the gelation of Complex-2 series was not observed. For complexes of POMs with relatively large numbers of charges, the situation became complicated. On one side, the increased organic groups still could not provide enough hydrophobicity for complexes to dissolve in n-hexane, while Complex-3 (b, c) yielded typical gels in cyclohexane and isooctane, in which it is insoluble. In carbon tetrachloride and benzene, Complex-3 (b, c) existed as a solution and a jelly respectively, while the existing state of Complex-3a was just the opposite in those two solvents. In comparison to non-polar solvents, all these complexes dissolved well in weakly polar solvents such as toluene, dichloromethane, chloroform and strong polar solvents such as ethanol and methanol, but no obvious gel formation has been observed.

Figure 1. The chemical composition of cationic surfactant with different chain lengths, polyanionic clusters bearing different charges, and the formed electrostatic complexes.

To evaluate the role of alkyl chains on the fabrication of hybrid supramolecular gels, beside DODA, we covered the inorganic core, (K9BW11O39)9−, with three other surfactants bearing shorter alkyl chains: dihexadecyldimethylammonium bromide (DHDA· Br), ditetradecyldimethylammonium bromide (DTDA·Br), and didodecyldimethylammonium bromide (DDDA·Br). Among these three prepared complexes, DTDA- and DDDA-hybridized complexes could not form supramolecular gels in general solvents due to the lowered self-assembly capability. Similar to the DODA covered POM complex, the gelation of DHDA-grafted complex took place in n-hexane, cyclohexane, and isooctane due to the relatively higher hydrophobicity and interaction between alkyl chains. However, the gels became no longer stable over time, and the gel state could only be kept at low temperature. The worsened gel behaviors can be well attributed to the weakened van der Waals interaction between shortened alkyl chains (16). 203 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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The observed gels are not transparent, especially when the concentration of gelator complexes was high. Polarizing optical microscopy demonstrated that the phenomenon was closely related to the aggregation’s morphologies and sizes in the gel phase. SEM images of xerogels of Complex-2 series in n-hexane and cyclohexane indicated the entangled and tape-like network structure (23). On the contrary, xerogels of Complex-3 series in cyclohexane adopted network structure, which could be attributed to the partial fusion of spherical aggregations into larger ones. Apparently, the enhancement in the size scale of stripe and sphere aggregations increased the turbidity of gels. Those long, thin stripe structures occupied more overlapping areas and cross-linked together stably comparing to dispersed spherical aggregation structures of stable supramolecular gels. XRD diffractions of those xerogels suggested lamellar structures of complexes. As confirmed in previous publications (24), the charge neutralization between organic and inorganic components lead to the attachment of the cationic head of surfactants to the surface of polyanionic clusters at a very close distance instead of being fixed at a certain site in a potential field, due to the delocalization of surface charges on POMs. Because of the charge delocalization and the larger surface area on POM surface, it was proposed that the surfactant cations would move and rearrange in the surface electrostatic field around POMs. Driven by the interfacial energy in the solvent environment, the phase separation of complexes propelled surfactant cations to accumulate into a nonspherically symmetric state. Thus, considering the smaller lateral size of the cationic head, the polarity of the polyanionic core and its tight electrostatic interaction with surfactants, the self-assembly of these complexes into the reverse bilayer structure in organic media became a favorable state for tight packing density and low interfacial energy. Independent investigations have confirmed that the reversed bilayer structure in which the inorganic component located in the middle could induce different morphologies accompanied by the change of solvent polarity (25, 26).

Responsive Gels of Organically Grafted POM Complexes with Additives In comparing to the lateral combination between complexes derived from the van der Waals interaction, by introducing additional agents to control the lateral force between complexes, we realized the response of the supramolecular gels to the external condition. Hasenkernopf and his coworkers reported the initial example of organically modified POM complexes that could form supramolecular gels through coordination with metal ions (27). Via the organic modification of pyridyl groups on both sides of an Anderson-type disc-like cluster, [MnMo6O24H6]3−, we built hybrid supramolecular gels comprising three components (28). By mixing with dicarboxylic acids in acetonitrile, the main gelator component of pyridyl-substituted POM cluster (Py–MnMo6) bearing tetrabutylammonium counterions formed gels immediately. The hydrogen bonding between pyridyl groups and the carboxylic acid additives leads to the generation of supramolecular polymer chains, and then the primary fibrils self-assemble into fibrous bundles, which further entangled with each other to yield cross-linking network structures. The distance between adjacent POMs 204 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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was confirmed to be an important factor for the formation of gels, which was dominated by the interfacial energy of the polymer chains. Only those additives with a proper molecular length supported the construction of gels. Though quantitative estimation on the interfacial energy seemed difficult in the present stage, these influence factors referring to the synergy among components provided useful traces for the design of other POM hybrid gels. Due to the hydrogen bonding feature of the polymer chain, the hybrid gels displayed a quick response to the addition of organic bases due to the competitive disruption of the hydrogen bond. In principle, any actions capable of breaking supramolecular polymer networks could induce the response of the gels (Figure 2).

Figure 2. Schematic drawing of the gelation of TBA-Py-MnMo6 grating pyridyl groups under the existence of dicarboxylic acid through hydrogen bonding and the response to the organic base 4-dimethylaminopyridine (DMAP). 205 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Following the approach of lateral modification, the suitably modified POMs could afford more gelators for hybrid gels. A pair of Anderson clusters [MnMo6O24H6]3− that have been organically grafted with adenine and thymine base groups on both sides, respectively, were synthesized (Figure 3). When the base pair-modified clusters were mixed together, they linked to each other linearly through complementary hydrogen bonds, yielding an alternative POM supramolecular polymer, which enhanced processing properties such as mold casting and electrospinning (29). Though the counterions of the POM had been changed to tetrakis(decyl)ammonium (TDA), the formed aggregation could not afford gelation in solution. As mentioned above, the modification and the substitution of cation changed the amphiphilicity. However, the polyanionic cluster in the main polymer chains also provided electrostatical binding sites for crosslinking, because there is no saturation for electrostatic interaction. After the addition of a bola-form cationic surfactant into the hybrid supramolecular polymer in chloroform solution, a quick gelation was found. The combination of POM cluster in the main chain with cationic additives provided an additional adjustment for the lateral intermolecular interaction when controlling aggregation structures. One could envision that this kind of organic-inorganic hybrid gels can provide another effective method to accommodate inorganic clusters in soft materials systems.

Figure 3. The schematic illustration of base-pair modified Anderson type POMs, the formation of complementary hydrogen bonds and the supramolecular chain cross-linking via electrostatic interaction with a bola-form cationic cross-linker. 206 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Self-Assembly and Alterable Relaxivity of Gadolinium-Incorporated POM Complexes While the encapsulation of cationic surfactants to POMs through electrostatic interaction changed the surface properties, basic physical and chemical characteristics of these clusters in their free state were maintained (30, 31). Thus, the structural stability and the biocompatibility of POMs could be greatly improved. More importantly, functional properties of POM clusters could be optimized in hybrid organizations due to the synergy of components in aggregation structures. Appealing biomedical applications of some POMs as antiviral, antitumor, and antibiotic agents have been carefully studied (32). Considering these features, it is of significance to combine them with probes or labelings of biomolecules, because phosphotungstic acid and its derivatives are often used as dyes of biomolecules for TEM measurements. The paramagnetic property of Gd(III)-Sandwiched POM complexes as bio-imaging enhancement materials such as contrast agents (CAs) for magnetic resonance imaging (MRI) has received interest recently (33). Compared to normal organic ligands, lacunary POMs as inorganic multidentate ligands serve for the preparation of stable paramagnetic polyanions. The POM’s stiff architecture and high molecular weight could also provide longer rotational correlation time leading to reinforced longitudinal relaxivity (r1) (34). However, naked polyanions often bind strongly to the positively charged biological molecules such as proteins, and may yield unexpected cluster disintegration in the physiological environment (35), thereby inducing toxicity. Therefore, it is important to wrap up Gd-substituted POMs with neutral organic components with the covering layer PROVIDING both protection of the cluster and binding sites for specific recognition to target groups when organic modification is necessary. To meet these purposes, an amphiphilic molecule (EO12BphC10NC12) possessing a poly(ethylene oxide) (PEO) terminal on one end of a double-chain quaternary ammonium connected to a biphenyl group was designed to enwrap the paramagnetic POM, K13[Gd(β-SiW11O39)2]·27H2O (Gd-POM) (Figure 4). The PEO chain locating at periphery of hydrophobic shell provided hydrophilicity, while the hydrophobic part in the middle sustained the electrostatic interaction of the amphiphilic molecule with POM. Thus, the formed Gd-POM complex was not only soluble in aqueous solution, but also spontaneously generated a phase-separation and aggregated into larger assemblies (36). The magnetic moment of the Gd-POM remained after the electrostatic encapsulation, and those assemblies introduced an additional modulating approach for T1 value of water protons as the complex existed in different states with the change of concentrations. For example, at high concentration, relaxivity was lower than that of the free POM, while at lower concentration the relaxivity of the complex was enhanced by several folds to the naked POM. Apparently, aggregation is unfavorable for the MRI quality due to the congestion of water molecule exchange, which was not hindered by the covering layer of the isolated complex. Importantly, the increased molecular weight corresponding to the naked POMs promoted the relaxation time, which increased the relaxivity r1. 207 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 4. Representative drawing for the preparation and aggregation of Gd-POM complex for magnetic resonance imaging contrast agent. Reproduced with permission from reference (36). Copyright 2015 Royal Society of Chemistry.

Manganese Cluster Single-Molecular Magnets as MRI CAs Mn12O12(OOCCH3)16 (Mn-12) clusters are known as a type of single molecular magnets (SMMs) due to their large ground spin (S) state and magnetic anisotropy derived from the parallel array of partial Mn (III) ions in the Jahn–Teller axis (37). The Mn-12 SMM possesses a precise chemical composition and electronic configuration, and it is susceptible for chemical modifications on both the framework and periphery. Based on the particle size and the paramagnetic property, Mn-12 clusters were hopefully to be developed into a CA system in connection to paramagnetic and superparamagnetic nanomaterials. However, the instability of Mn-12 SMMs in aqueous solution became one of the major drawbacks of applications. In a highly concentrated acetic acid solution, Mn-12 retained its structure and performed as a negative CA. By anchoring onto the carboxylic acid surface of polystyrene spheres, Mn-12 was found to have an improved stability to some extent, and the effect of negative contrast was observed (38). However, more convenient methods to stabilize clusters and a much closer environment to the physiological condition are still required when attempting to apply the clusters as CAs. Under the illumination of the paramagnetic POM complex, we proposed a quite mild approach for transferring and stabilizing stearic acid (C18)-coordinated Mn-12 (Mn-12-C18) cluster in an aqueous solution for CAs through an emulsion-supported method without obvious structure decompositions (39). A nonionic surfactant, C18H37(OCH2CH2)10OH (C18EO10) was used as the emulsifier. By mixing the Mn-12-C18 cluster in n-hexane and the nonionic surfactant in water, we prepared an emulsion. Controlling the evaporation speed of organic solvent gave the surfactant-encapsulated cluster complex in the microenulsion aggregates in which the Mn-12 clusters were located in the center (Figure 5). Due to the protection of nonionic surfactant that covered the surface via the van der Waals interaction between alkyl chains, the Mn-12 cluster could be dispersed in water, and its magnetization hysteresis loop at 2 K exhibited characteristic SMM performance. Within the multicomponent system, the alkyl chains of carboxylic acid dispersing on the surface of Mn-12-C18 accommodated hydrophobic carbon chains of C18EO10, which greatly improved the structural stability of the inorganic cluster aggregates. In the meantime, the PEO chain 208 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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locating outside of the cluster aggregates promoted the water solubility and especially the biocompatibility. It should be noted that, while improving the application of Mn-12 derivatives as negative CAs, the intermediate alkyl chain layer between Mn-12-C18 and C18EO10 blocked the water proton relaxation by the “inner-sphere” mechanism (40). Apparently, a further modification on terminal groups of the carboxylic acid ligand is promising to simplify the system so that the nonionic surfactant becomes no longer necessary.

Figure 5. Formation process of Mn-12-C18/C18EO10 aggregates through emulsion-assisted self-assembly at room temperature in aqueous solution. Reproduced with permission from reference (39). Copyright 2015 Royal Society of Chemistry.

Concluding Remarks and Perspectives The electrostatic combination of cationic surfactant with inorganic polyanionic cluster yields new type of amphiphilic complexes. Being different from general surfactants, the interfacial energy-propelled polarity separation keeps the hydrophilic part and hydrophobic part of complex away from each other and self-assembles into diverse aggregation structures, relying on the occupation of the organic component on the surface of each inorganic cluster. The selection of suitable solvents not only modulated the aggregation morphology and structure, but also triggered the gelation of the complexes in solution. The introduction of additives and the interaction control between components yielded the sensitive response of supramolecular gels. On the other side, electrostatic covering could greatly improve the surface property of inorganic clusters and thereby directed the enhancement of the stability and biocompatibility, toward the application of POMs as the CAs. The present method can be apparently applied in the functionalization of other charged nanoparticle systems. 209 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

As a general approach, the collection of surfactant molecules by POM cluster provides a possibility to build single complex reverse micelles. The electrostatic interaction increased the stability of the small complex aggregation, so that we can use it to fabricate more complicated hybrid assemblies. The present method could be also used for other similar systems and the realizations of functions that regular surfactants and micelles do not possess, for example, the simple phase transfer of graphene from an aqueous solution to the organic phase, the large accommodation of guest insolubles and so forth.

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Acknowledgments The authors acknowledge financial support from the National Basic Research Program (2013CB834503) and the National Natural Science Foundation of China (NSFC) (51203059, 91227110, 21221063) and MOE (20120061110047).

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

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Chapter 12

Diverse Near-Infrared Resonant Gold Nanostructures for Biomedical Applications Publication Date (Web): December 8, 2015 | doi: 10.1021/bk-2015-1215.ch012

Jianfeng Huang and Yu Han* Advanced Membranes and Porous Materials Center, Physical Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Kingdom of Saudi Arabia *E-mail: [email protected].

The ability of near-infrared (NIR) light to penetrate tissues deeply and to target malignant sites with high specificity via precise temporal and spatial control of light illumination makes it useful for diagnosing and treating diseases. Owing to their unique biocompatibility, surface chemistry and optical properties, gold nanostructures offer advantages as in vivo NIR photosensitizers. This chapter describes the recent progress in the varied use of NIR-resonant gold nanostructures for NIR-light-mediated diagnostic and therapeutic applications. We begin by describing the unique biological, chemical and physical properties of gold nanostructures that make them excellent candidates for biomedical applications. From here, we make an account of the basic principles involved in the diagnostic and therapeutic applications where gold nanostructures have set foot. Finally, we review recent developments in the fabrication and use of diverse NIR-resonant gold nanostructures for cancer imaging and cancer therapy.

Introduction In recent years we have seen tremendous advances in modern medical diagnostics and therapeutics. Nevertheless, there exists many opportunities for advancement in early detection and treatment of various diseases (e.g., cancer). Conventional imaging modalities, such as ultrasound imaging (UI), X-ray © 2015 American Chemical Society In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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computed tomography (CT), magnetic resonance imaging (MRI) and positron emission tomography (PET) usually suffer from poor contrast, small dynamic ranges, low sensitivity or low spatiotemporal resolution (1, 2). Meanwhile, the nonspecificity of popular therapeutic strategies, like chemotherapy and radiotherapy, bring many pernicious side effects. In addition, theranostic (diagnostic and therapeutic) agents, such as Gd3+ for MRI enhancement, radiolabeled molecules or chemotherapy drugs are often limited by short blood circulation time and biodistribution (3). Consequently, it is imperative to develop improved theranostic strategies for simpler and more effective treatments. In recent years, the appealing biological, chemical and physical properties of gold nanostructures are causing them to emerge as an important class of theranostic agents for biomedical applications including imaging, therapy and drug delivery (4). Biologically, they exhibit exceptional cell compatibility, without significant adverse effects on cell viability and function (e.g., proliferation and differentiation) due to the inherent low cytotoxicity of elemental gold (5, 6). Chemically, they are stable and readily functionalized via surface modifications for either passive or active targeting to selective sites, which enhances specificity and thus, effectively eliminates or mitigates nonspecific damage to surrounding healthy tissues (7). In addition, a number of state-of-the-art synthetic methods have been developed that offer precise control over their physicochemical parameters (e.g., size, shape and aggregation state). Physically, gold nanostructures possess rich and intriguing optical properties arising from an excitation of localized surface plasmon resonance (LSPR). LSPR is an electromagnetic resonance under which conduction electrons oscillate collectively with the incident light at the interface between metallic nanostructures and their surrounding dielectric media (Figure 1a) (8). Reflected in the extinction spectrum, there are one or more peaks positioned at different wavelengths, depending on the size, shape, composition, the surrounding medium, etc. For example, for Au nanospheres, there is generally one LSPR band at around 525 nm. Upon such a resonance, gold nanostructures can effectively trap the incident light and render an intense electromagnetic field in the vicinity of the particle surface at the resonant wavelength. Optical applications, such as surface-enhanced Raman scattering (SERS) and surface-enhanced fluorescence (SEF), benefit greatly from the enhanced electromagnetic field (9). Following LSPR excitation, localized surface plasmons decay on the timescale of femtoseconds, either radiatively through resonant light scattering or nonradiatively via creation of hot electrons (Figure 1b) (10). While scattered light can be used for nanoparticle-enhanced optical bioimaging, the generated hot electrons can either be captured by external species, for example, molecular oxygen to generate reactive oxygen species (ROS) for photodynamic therapy (11), or be cooled down through electron–phonon collisions at a time scale of 1−100 ps (12), ultimately leading to a rise in the lattice temperature. Thereafter, the thermal energy dissipates into the surroundings, which plays an essential role in broad biomedical applications, such as photoacoustic imaging, photothermal therapy and light-controlled drug release. In the case of non-radiative decay, either intraband excitations within the conduction band or interband excitations between other bands (e.g., d-bands) and the conduction band may take place. For noble-metal nanostructures, interband 214 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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excitations also account for luminescence that can be used for fluorescence imaging. Figure 1b summarizes localized surface-plasmon decay routes and their potential biomedical applications.

Figure 1. Excitation and decay of localized surface plasmons. (a) Schematic of plasmon oscillation for a metal sphere. (b) Schematic representation of radiative and nonradiative decay of localized surface plasmons in noble-metal nanoparticles and their potential biomedical applications. e−—electron, h+— electron hole, ROS—reactive oxygen species, PDT—photodynamic therapy, PTT—photothermal therapy, PAI—photoacoustic imaging and LCR—light-controlled release.

To harness the unique optical properties of gold nanostructures for in vivo applications, it is critically important to tailor the LSPR band into biological optical windows I (650–900 nm) and II (1000–1350 nm), where light attenuation, including absorption and scattering, from oxygenated blood, deoxygenated blood, skin and fatty tissue is lowest (Figure 2) (13, 14). Gold nanostructures present themselves as an extremely attractive nanomedical agent for exploiting this spectral regime, because their LSPR can be finely adjusted by changing their size, shape composition and aggregation states. This broad degree of tuning makes gold nanostructures resonant in near infrared (NIR) conditions, and thus very useful for various clinical diagnostic and therapeutic applications. By the same token, NIR resonant among miscellaneous gold nanostructures will be discussed in this chapter, with particular emphasis on their up-to-date biomedical applications.

Biomedical Applications: Imaging and Therapy This section briefly discusses the underlying physical principles of the imaging and therapy modalities for which Au nanostructures are exploited. Imaging applications include scattering- and luminescence-based optical imaging, surface-enhanced spectroscopy-based imaging, and photoacoustic imaging. Therapeutic applications include photodynamic therapy, photothermal therapy, drug delivery and light-controlled drug release. 215 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 2. Optical windows in biological tissues. These plots of effective attenuation coefficient (on a log scale) versus wavelength show that absorption and scattering from oxygenated blood, deoxygenated blood, skin and fatty tissue is lowest in either the first or second near-infrared window. Reproduced with permission from reference (13). Copyright 2009 Nature Publishing Group.

Scattering- and Luminescence-Based Optical Imaging When LSPR in Au nanostructures undergoes a radiative decay, strong light scattering occurs at the LSPR frequency. Under optical microscopy, the scattered light is collected, and the location of the Au nanostructures can be imaged. Serving as imaging contrast agents, gold nanostructures that are accumulated in specific cells via either passive or active targeting enable the differentiation of target cells from surrounding cells. Two optical techniques, reflectance confocal microscopy (15) and optical coherence tomography (16) have been widely used. Moreover, gold nanostructures have been found to generate luminescence (17). Single-photon luminescence is identified as a three-step process: (i) creation of electron-hole pairs via one-photon excitation of electrons from the d-band to the sp-band, (ii) scattering of the excited electrons and holes by phonons with partial energy transferred and (iii) recombination of electron–hole resulting in photon emission (18). Two-photon luminescence is considered to follow a similar mechanism, with the exception of two sequential one-photon absorption events in the first step (19, 20). Two-photon luminescence is relatively weak, but can be greatly intensified by the strong electric field produced during LSPR. For elemental Au, as the d-sp band transition takes place below ~600 nm, two-photon luminescence excited by NIR light and amplified by NIR-resonant Au nanostructures is particularly desirable for biomedical imaging. Compared with fluorescent probes used in optical image, gold nanostructures do not suffer from 216 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

photobleaching or photoblinking, thus encouraging their wide use as contrast agents for light-scattering or two-photon luminescence bioimaging.

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Surface-Enhanced Spectroscopy-Based Imaging The term surface-enhanced spectroscopy generally encompasses surfaceenhanced Raman scattering (SERS) and surface-enhanced florescence (SEF). When probes, such as characteristic biomolecules of cells, tissues or biomarkers, are situated in the vicinity of Au nanostructures, their “fingerprint” Raman signals can be greatly amplified by the strong electric field resulting from the LSPR (21). In addition, NIR light can be reliably exploited, as Raman shifts are excitation wavelength independent. Thanks to these merits, SERS has become a powerful technique for in vivo detection and imaging applications (vide infra). In SEF, the fluorescence enhancement is jointly attributed to an enhanced absorption of fluorophores by the strong electric field, an improved radiative decay rate of fluorophores (for example, owing to the localized density of photonic states of plasmonic nanocrystals), and an increased emission via coupling of the fluorescence emission to the far field (3). Unlike in SERS, however, the fluorophores in SEF should not be in close proximity to the Au nanostructure’s surface (< ~4 nm). Otherwise, fluorescence will be significantly quenched due to the damping of molecular oscillators by electron tunneling/transfer between the metal and the fluorophore, and/or the fluorophore’s own field which is reflected by the metal and out of phase with the directly emitted field of the fluorophore (22). SEF with optimally placed fluorophores near the surface of Au nanostructures has been widely employed for in vitro and in vivo imaging. Photoacoustic Imaging (PAI) Photoacoustic imaging (PAI), also known as optoacoustic imaging, combines light (typically NIR) and ultrasound to produce an image with a greater spatial resolution than ultrasound techniques and deeper depth profiles than purely optical techniques. Briefly, when short laser pulses are absorbed and then dissipated to local heats, a rapid thermoelastic expansion of surrounding tissues will take place, leading to the generation of an ultrasound wave. The ultrasound wave is then collected and converted to electric signals with a transducer and finally processed to produce an image. The photoacoustic effect can be approximated by a simplified equation (23):

Where, P is the pressure rise of the generated acoustic wave, Γ is the Grueneisen parameter, µa is the absorption coefficient, F is the laser fluence, β is the thermal coefficient of volume expansion, c is the speed of sound and Cp is the heat capacity at constant pressure. From this equation, it is evident that the PA signal is directly related to temperature, as both β and c in the Grueneisen parameter are positively correlated with temperature. Au nanostructures, capable 217 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

of effectively absorbing and transforming light energy into thermal energy at the LSPR frequency, have been widely applied as contrast agents for this imaging modality.

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Photodynamic Therapy (PDT) Photodynamic therapy (PDT), also known as photochemotherapy, involves cell death induced by reactive oxygen species (ROS, e.g., singlet oxygen 1O2, superoxide radical anion •O2ˉ and hydroxyl radical •OH) through the destruction of cellular components (e.g., DNA, RNA and proteins). ROS are generally produced as a consequence of energy or electron transport in photochemical and photobiological processes that are initiated by the reaction of organic photosensitizer chromophores with tissue oxygen under the irradiation of a specific wavelength of light. PDT is particularly promising for its site-specific treatment and dark non-toxicity (24). A major drawback of PDT is caused by the long retention of photosensitizer drugs in the body, which renders the patient highly sensitive to light (7). In addition, many organic photosensitizers have their excitations in the UV-visible range, limiting their in vivo applications for deep-tissue-buried tumors. Moreover, common photosensitizers, such as porphyrins and phthalocyanines, are often too hydrophobic to be used without chemical modifications, which poses considerable challenges for targeting them in tumor sites (25). In recent years, a series of inorganic nanomaterials have also been proven capable of generating ROS under irradiation including typical semiconductor nanomaterials (TiO2, quantum dots) (26, 27) and carbon nanomaterials (nanotubes, C60) (28, 29). In particular, some studies found that 1O2 can be produced from noble-metal nanostructures through an energy transfer mode when they are illuminated with a continuous wave (CW) or pulsed laser source (30–32). During Au nanoparticle (AuNP) irradiation, two pathways of 1O2 production have been proposed: a plasmon-activated pathway via interactions of plasmons and hot electrons with molecular oxygen, and an indirect photothermal pathway that induces extreme heat development leading to particle fragmentation and thermionic electron emission (30). The later pathway is more pronounced in the case of AuNP irradiation with pulsed laser sources. The role that Au nanostructures play in PDT is generally considered as either a carrier for organic molecular photosensitizers or on their own as inorganic photosensitizers. Photothermal Therapy (PTT) Photothermal therapy (PTT) generally refers to a hyperthermic treatment, including low-temperature hyperthermia (41-45 °C) and high-temperature thermal ablation (46-56 °C), which uses light as the heating source to damage abnormal cells. Hyperthermic effects range from the induction of cell apoptosis by denaturalizing intracellular proteins that are related to cellular survival and proliferation to cell destruction and tissue ablation via direct cell necrosis (33, 34). The necrotic destruction of cancer cells involving high temperatures usually 218 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

causes collateral damage to healthy cells and undesirably reshapes nanostructures (34). Because cancer tissues do not have a sufficient blood supply and vascular structures to dissipate heat, they have a lower temperature tolerance limit than do healthy tissues; therefore, they can be selectively damaged at temperatures between 41 and 45 °C (35). For less invasive cancer cell death, it is therefore advantageous to use a low-temperature-based (41-45 °C) PTT strategy. Au nanostructures hold particular promise as PTT-photosensitizers due to their large photon absorption and efficient photothermal conversion at LSPR.

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Drug Delivery and Light-Controlled Drug Release Many clinically used drugs are either highly hydrophobic or low molecularweight compounds that diffuse readily into healthy tissues. As a consequence, little if any of the drug reaches the target sites (36). It is essential to improve the region-specific delivery and release of drugs that can greatly increase the efficacy of therapies. A promising strategy is to use Au nanostructures as a “nanocarrier” to transport drugs to target sites and then liberate them under light irradiation. Compared to conventional drug delivery systems, Au nanostructures serving as a delivery vehicle have at least three advantages: decreased biodegradation of drugs, improved solubility of hydrophobic drugs and reduced immunogenicity (37). In particular, instead of a direct use for hyperthermal killing of malignant cells, the heat generated from the photothermal conversion of Au nanostructures can be harnessed to spatially and temporally control drug release. Currently, three major photothermo-responsive drug release schemes have been reported (38): breakage of polymer or liposome structures where drugs are encapsulated, rupture of a linker molecule through which drugs are tethered to the Au nanostructures via a covalent bond, and diffusion of drugs from thermosensitive hydrogels, silica matrices or polymers.

Diverse NIR-Resonant Gold Nanostructures Although gold nanoparticles have a long history of applications in clinical treatments, the use of NIR-responsive gold nanostructures only began this century. To date, a variety of NIR-resonant gold nanostructures have been developed and thoroughly explored as either imaging or therapy agents. Most recently, research on these structures has shifted to the design and application of their integration with other materials to construct a versatile platform that can accommodate multiple theranostic modalities within a single nanoscale complex. The currently available NIR-resonant gold nanostructures can be roughly categorized into five classes: Au Nanorods, Au Nanoprisms, and Au Nanoplates These three structures feature an anisotropic shape as opposed to the isotropic shape of spherical AuNPs (Figure 3). Reduced symmetry means that these structures possess more than one surface SPR band. In particular, one well-defined band can be tuned into the NIR range via changing the size parameter. For 219 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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example, Au nanorods (AuNRs) exhibit two SPR bands, one weak transverse band in the visible range (~520 nm) and another strong longitudinal band in the NIR range, provided that the aspect ratio (the ratio of length to diameter) is larger than ~3.5 (41). Seed-mediated growth is the most exploited method for preparing AuNRs (42, 43); this involves the addition of citrate-capped Au nanoparticle seeds into a growth solution containing the Au precursor (HAuCl4) and a reducing agent (ascorbic acid) together with cetrimonium bromide and Ag+. The aspect ratio can be finely controlled by varying the ratio of seed to Au precursors as well as the time delay between synthesizing steps (44). For Au nanoplates and nanoprisms, the in-plane modes dominate the spectra while out-of-plane excitations are only important for small, thick nanoprisms (45). In-plane modes can be tuned into NIR via changing the ratio of the edge length to the thickness. To date, the pioneering solution-phase light-mediated syntheses and various thermal techniques are reliable for producing nanoprisms/nanoplates with a high yield (46). A commonality shared by most of these syntheses is mediated reduction of metal ions onto pre-synthesized nanoparticle seeds with twin planes. Therefore, all experimental details influencing either the crystallographic structure of the seeds or the redox chemistry of the second step can have a drastic effect on the final structures and thus on the LSPR bands.

Figure 3. (a, c) Transmission electron microscopy (TEM) images of (a) Au nanorods and (c) Au nanoprisms. (b) Scanning electron microscopy (SEM) image of Au nanoplates. Figure 3b adapted with permission from reference (39). Copyright 2005 American Chemical Society. Figure 3c adapted with permission from reference (40). Copyright 2008 American Chemical Society.

The ease of large-scale and high-yield synthesis together with the superior optical properties of AuNRs have made them one of the most exploited NIRresonant gold nanostructures for various biomedical applications as discussed in Section 2. Recent research efforts are improving the previous theranostic modality and developing more effective thermo-chemotherapy (47). Some progress toward these directions is highlighted below. Signal generation by metal nanostructures during PAI mainly relies on the conversion of light to heat, the transfer of the heat to the environment and the 220 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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resultant production of pressure transients. Previous focus has primarily been on the conversation of light to heat by developing efficient light-to-heat conversion structures. Recently, it was demonstrated that improvements to the heat transfer process can strongly amplify photoacoustic (PA) performance. For example, Emelianov et al. reported that silica-coated AuNRs could produce three-fold higher PA signals than uncoated AuNRs of the same optical density (48). The results of a series of control experiments suggested that the enhancement was caused by a reduction in the interfacial thermal resistance between AuNRs and the surrounding solvent as a consequence of the silica coating. Lim et al. found that PA performance could be further improved through coating AuNRs with reduced graphene oxide (RGO) (49). Simulations found that the electromagnetic fields of the AuNR@RGO were 1.5 and 4 times stronger than those of the AuNR@SiO2 and bare AuNRs, respectively. Moreover, AuNR@RGO also showed a heat transfer rate 2.5 and 10 times higher than the AuNR@SiO2 and bare AuNRs, respectively, due to the excellent thermal conductivity of RGOs. Thus, these results revealed that the RGO coating contributed to both light absorption and heat transfer, improving PA performance. A challenge to PTT lies in adequately heating an entire tumor mass, while avoiding unnecessary collateral damage to the surrounding healthy tissue. Recently, Berlin et al. (50) identified an innovative method to improve the intratumoral distribution of AuNRs, thereby increasing the efficacy of PTT by conjugating tumor-tropic neural stem cells (NSCs) with AuNRs. Results show that after loading AuNRs, NSCs were unimpaired in their viability, yet they retained AuNRs long enough to migrate throughout tumors. In a mouse model, intratumoral injections of Au nanorod-loaded NSCs were more efficacious than free Au nanorod injections, as evidenced by a reduced recurrence rate of triple-negative breast cancer (MDA-MB-231) xenografts following NIR exposure. This work highlights the advantage of combining cellular therapies and nanotechnology to generate more effective cancer treatments. Meanwhile, previous reports were largely based on organic photosensitizermediated PDT, usually in combination with AuNR-mediated PTT to achieve synergistic PTT and PDT effects to kill cancer cells. Xu et al. (51) recently demonstrated that owing to their large two-photon absorption cross-sections, AuNRs can effectively generate ROS singlet oxygen (¹O2) under two-photon excitation, which is significantly higher than traditional organic photosensitizers such as Rose Bengal and Indocyanine Green. Guided by AuNRs’ two-photon fluorescence imaging, the two-photon PDT effect was demonstrated on HeLa cells in vitro. Hwang et al. (52) later demonstrated that AuNRs alone can sensitize the formation of ROS ¹O2 and exert PDT effects on the destruction of mice tumors under very low LED/laser doses of single-photon NIR excitation (915 nm, < 130 mW/cm²). They found that AuNR-mediated phototherapeutic effects could be switched from PDT to PTT or a combination of both, depending on the NIR light excitation wavelengths. In particular, in vivo mice experiments revealed that the PDT effect via irradiation of AuNRs from 915 nm could destroy the B16F0 melanoma tumor in mice far more effectively than chemotherapy using the anticancer drug doxorubicin (DOX) and PTT under 780 nm light irradiation. 221 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 4. DNA assembly of a targeted, NIR-responsive delivery platform and disassembly under NIR irradiation. Reproduced with permission from reference (53). Copyright 2012 John Wiley and Sons. In the field of light-induced drug delivery, targeting is always important for theranostic agents to reduce side effects induced by a lack of specificity. Farokhzad et al. (53) designed a targeted NIR light-responsive delivery platform through DNA self-assembly. As shown in Figure 4, the platform comprises three functional components: complementary DNA strands, the AuNR and a polyethylene glycol (PEG) layer. The DNA strands provide loading sites for doxorubicin (DOX) via the intercalation of DOX with the strands’ GC base pairs. In addition, one of the two strands is thiolated for AuNR capture, and the other is pre-conjugated with ligands for cell-specific targeting. AuNRs serve as the NIR light-to-heat transducer for PTT and for disassembling the DNA double strands under NIR irradiation, which leads to the triggered release of loaded drugs at target chemotherapy sites. The PEG layer facilitates the nanoparticles to evade recognition by the immune system and prolongs the circulation of the nanoparticles. Both in vitro and in vivo results demonstrated that this platform selectively delivered DOX to target cells, released them upon NIR irradiation and effectively inhibited tumor growth through thermo-chemotherapy. One limitation of this study lies in the local delivery strategy of intratumoral injection, which cannot provide as rich biodistribution information of the particles as does intravenous injection; for example, Qian et al. (54) co-loaded AuNRs and DOX in polymersomes (P-AuNRs-DOX) to facilitate co-therapy of photothermal and chemotherapies. Under NIR, laser irradiation induced local hyperthermic heating of AuNRs such that polymersomes were corrupted and released DOX. Ablation of tumor cells in vitro and in vivo showed that co-therapy offered significantly improved therapeutic efficacy over chemotherapy or PPT alone. However, biodistribution analysis after intravenous injection showed that AuNRs accumulated primarily in reticuloendothelial systems (RES) with a tumor uptake of 7.94 % ID/g at 24 h, implying that further efforts could be devoted to improve 222 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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targeting (e.g., by conjugation with tumor cell surface receptor-ligands). Chen et al. (55) developed a novel NIR laser-induced anticancer targeting strategy with facile control and practical efficacy, but without using active targeting ligands (Figure 5). It involved AuNR-PNIPAM nanocomposites that contain AuNRs encapsulated in a thermoresponsive polymer, poly (N-isopropylacrylamide) (PNIPAM). This polymer undergoes a reversible phase transition in aqueous solution from an extended hydrophilic chain to a condensed hydrophobic globule when the temperature rises above 32 °C. As reduced size favored extravasation of nanocomposites from the pore-enlarged vasculature system to the tumor tissue at elevated temperatures, when the tumor (mouse murine 4T1 breast tumor on the right hind leg) was irradiated with a NIR laser (760 nm) for 20 min immediately after the intravenous administration of the nanocomposites, a significantly enhanced accumulation (7.6-fold) of nanocomposites was observed in the tumor. This enhanced accumulation of nanocomposites provided a prerequisite for their effective therapeutic application. For example, it further induced sufficient temperature increase for PTT due to the photothermal conversion of AuNRs under NIR irradiation. Moreover, when they were loaded with the anticancer drug DOX, effective heat-induced release of doxorubicin to the tumor was realized. This thermo-chemotherapy almost completely inhibited tumor growth and lung metastasis.

Figure 5. NIR laser-induced targeted thermo-chemotherapy using the Au nanorod-PNIPAM nanocomposites. Reproduced with permission from reference (55). Copyright 2014 American Chemical Society.

Although to a lesser extent than AuNRs, Au nanoplates/nanoprisms have also been explored for biomedical applications. For example, Okamoto et al. (56) demonstrated that triangular nanoplates could be used as two-photon-induced photoluminescence imaging agents for cell imaging. When Au nanoplates were conjugated to yeast cells which were either dead in air or alive in water, a visible two-photon excited luminescence could be detected by two-photon laser scanning microscopy using an NIR 810 nm laser as an excitation. Cui et al. (57) used Au nanoprisms as signal amplifiers in multispectral optoacoustic tomography 223 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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to visualize gastrointestinal cancer. Au nanoprisms were first PEGylated to increase their biocompatibility then injected into mice for the visualization of tumor angiogenesis in gastrointestinal cancer cells. The results demonstrated the capacity of PEGylated Au nanoprisms to penetrate tumors and provide a high-resolution signal amplifier for optoacoustic imaging. Tortiglione et al. (58) explored photothermal cell ablation by using Au nanoprisms in an invertebrate model organism. Living polyps (Hydra vulgaris) were first treated with Au nanoprisms and then NIR irradiated. The results showed that Au nanoprisms could be well internalized into living specimens, with no sign of toxicity. Moreover, they induced efficient cell ablation throughout the body and the overexpression of the hsp70 gene under NIR irradiation. The results showed that different cells/tissues responded differently, initiating either necrosis or a defense response. Therefore, this work not only demonstrated that gold nanoprisms could be employed as efficient heat mediators in model organisms, but also suggested NIR-triggered cell ablation as a tool to study cell function. In addition to their application for singular imaging or therapy techniques, Au nanoplates have also been demonstrated to be able to perform multiple functionalities as theranostic agents. In a recent study by Zhen et al. (59), Au nanoplates synthesized via an epitaxial growth of Au on palladium nanosheets and then modified with SH-PEG, were found to be an effective multifunctional platform for both PA and CT imaging and photothermal cancer therapy. The PEGylated Au nanoplates showed a rather high accumulation in the tumor site after an intravenous injection. Besides the enhanced permeability and retention (EPR) effect due to the tortuous and leaky nature of tumor vasculature and the surface PEGylation prolonged circulation time in the blood, the unique two-dimensional (2D) structural feature of the nanoplates was believed to be another critical contributing factor to such high accumulation in tumors. Moreover, obvious enhancement of CT value and four-fold enhanced PA signals after 24 h injection of the PEGylated Au nanoplates were observed. Imaging-guided PTT was also achieved using an 808-nm laser with a low power density of 0.5 W/cm2, much lower than that for most reported photothermal agents. These results thus, demonstrated the superiority of 2D nanostructures for in vivo biomedical applications. Au Nanostars, Au Nanopopcorns, Au Nanoflowers, Au Nanoechinus Protruding tips from a solid core geometrically characterize these four structures. Depending on the sharpness (length divided by width), the tips are roughly classified as branches, bumps, petals or spikes (Figure 6). The plasmonic modes of these structures arise from the hybridization of the individual plasmons from the core and the tips. The tips localize the low-energy plasmon mode at their apexes, which results in an LSPR band in the NIR region (60, 64). Synthesis of these structures typically involves kinetically controlled growth of polycrystalline gold nanoparticle seeds. Under the fast reduction rate, preferential growth occurs along certain crystalline facets of the starting seeds and thus, multiple tips form on solid cores. The molar ratio of the Au precursors to the seed is a crucial factor in determining the morphology and tip plasmon resonance wavelength (65). 224 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 6. (a, d) SEM images of (a) Au nanostars and (d) Au nanoechinus. (b, c) TEM images of (b) Au nanopopcorns and (c) Au nanoflowers. Figure 6a adapted with permission from reference (60). Copyright 2006 American Chemical Society. Figure 6b adapted with permission from reference (61). Copyright 2010 American Chemical Society. Figure 6c adapted with permission from reference (62). Copyright 2014 the Royal Society of Chemistry. Figure 6d adapted with permission from reference (63). Copyright 2014 John Wiley and Sons.

Through appropriate surface modifications, these structures have found wide biomedical applications. Lu et al. (66) demonstrated that Au nanostars, when modified with amine-terminated (positively charged in acidic condition) and carboxyl-terminated (negatively charged in basic condition) polyethylene glycol (PEG), could be endowed with a sensitive response in cellular uptake and PTT efficacy to the extracellular pH (pHe) gradient between normal tissues and tumors (Figure 7). Specifically, by optimizing the composition ratio of amine-/carboxyl-terminated PEG to be 4, the resulting structure (GNS-N/C 4) exhibited high cell affinity and therapeutic efficacy at pH 6.4 for Hela cells, but low affinity and almost “zero” damage to cells at pH 7.4. Mice models with an intravenous injection of GNS-N/C 4 further revealed that a significantly increased tumor accumulation and complete ablation of orthotopic breast cancer xenograft under NIR-laser (808 nm) irradiation. 225 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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226 Figure 7. Schematic illustration of the preparation of PEGylated mixed-charge Au nanostars (GNSs) and their pH-reversible cell affinity and photothermal therapeutic efficacy. Reproduced with permission from reference (66). Copyright 2015 John Wiley and Sons.

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Ray et al. (61) reported that the modification of Au nanopopcorns with Rh6G-labeled aptamers and an anti-PSMA antibody for targeted diagnosis, nanotherapy treatment and in situ monitoring of the PTT response of prostate cancer LNCaP cells using SERS. Since the PSMA level in cancer cells is usually much higher than in normal tissues, the anti-PSMA antibody conjugated Au nanopopcorns would selectively accumulate and then aggregate on the prostate cancer LNCaP cells. Once aggregated, these Au nanopopcorn aggregates exhibit an extremely high SERS intensity for probe molecule Rh6G-modified aptamers (Enhancement Factor: 2.5 × 109) due to the formation of hot spots inside aggregates. Meanwhile, localized heating of PTT under NIR irradiation caused irreparable cellular damage to the prostate cancer cells. After PTT, breakage of Rh6G-labeled aptamers from morphologically changed Au nanopopcorns resulted in a greatly reduced SERS signal. Therefore, an in situ time-dependent SERS assay could be used to monitor the photothermal-nanotherapy response during the therapy process. Xu et al. (67) functionalized Au nanoflowers with folic acid for targeting the human hepatocellular carcinoma cancer cell line (HepG2) and 4-mercptopyridine as a Raman probe. By taking advantage of the intraparticle hot-spot-induced superior SERS performance of individual Au nanoflowers (Enhancement Factor: ~2.1 × 108), they managed to perform in vitro SERS imaging of living cells. Provided that an appropriate coating was exerted on the Au nanoflowers as a spacer, such superior intraparticle hot spot property of Au nanoflowers can also be used for enhancing the fluorescence efficiency of quantum dots (QDs). In a recent study by Zhu et al. (62), Au nanoflower@SiO2@CdTe/CdS/ZnS (QD) composite structures were synthesized. After linking them with antibody (AT) molecules, the resultant Au nanoflower@SiO2@QD-AT composites well targeted the membrane of MCF-7 and MDA-MB-231 breast cancer cells. As a result, due to the enhanced local electric field and the large absorption cross section of Au nanoflowers, an enhanced florescence emission was observed from the cancer cells and an efficient PTT-induced cancer cell death was found under NIR-laser irradiation. Au nanoechinus was only fabricated most recently by Hwang et al. (63) A Au nanoechinus has an average particle size of ~350 nm and many well-defined tips protruding from the particle surface (aspect ratio: ~ 9). An intriguing property of this structure is that it exhibits an extended NIR absorption of up to 1700 nm, which covers both the NIR-I (650–950 nm) and NIR-II biological windows (1000–1300 nm). The extinction coefficients, ~ 0.69 × 1012 M−1cm−1 at 915 nm (NIR I) and 0.74 × 1012 M−1cm−1 at 1064 nm (NIR II), are the largest values ever reported for nanomaterial, which are 7-9 orders and 3-4 orders higher than those of conventional organic dyes and gold nanoparticles, respectively. Impressively, this structure was demonstrated to be able to sensitize the formation of ROS 1O2. After coating with lipid molecules, resultant lipid-coated Au nanoechinus performed effectively in vivo PDT and PTT in both the first- and the second-NIR biological windows for complete destruction of tumors in mice. Upon 915-nm (NIR I) and 1064-nm (NIR II) light exposure, cellular deaths were mainly induced by PDT, while PTT contributed around one-fifth and one-fourth for 915 nm and 1064 nm, respectively. This structure represents the first example where gold 227 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

nanostructures work as dual modal PDT and PTT reagents for the complete destruction of mice tumors in both the first and the second biological windows.

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Au Nanocages, Au Hollow Nanospheres, Au Nanotubes, Au Nanorod-in-Shell This set of nanostructures is all produced via a galvanic replacement reaction between HAuCl4 and sacrificial Ag nanoparticles with a certain shape, although in the case of Au hollow nanospheres, Co nanoparticles are more commonly used than their Ag counterparts. Specifically, the sacrificial nanoparticles employed for these four structures are Ag nanocube (68, 72), Co nanosphere (69, 73), Ag nanorod (70), and Ag-coated Au nanorod (71). The resultant nanostructures generally still maintain their overall geometric shapes, but a hollow interior with porous walls or shells are newly formed (Figure 8). The SPR bands of these structures undergo a considerable bathochromic shift relative to the original sacrificial templates, and can be readily tuned into NIR by tuning the inner size, outer wall thickness and the ratio of Au to Ag (68).

Figure 8. (a) SEM image of Au nanocages. (b-d) TEM images of (b) Au hollow nanospheres, (c) Au nanotubes, and (d) Au nanorod-in-shell. Figure 8a adapted with permission from reference (68). Copyright 2004 American Chemical Society. Figure 8b adapted with permission from reference (69). Copyright 2014 American Chemical Society. Figure 8c adapted with permission from reference (70). Copyright 2015 John Wiley and Sons. Figure 8d adapted with permission from reference (71). Copyright 2013 American Chemical Society. 228 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Au nanocages (AuNCs) with hollow interiors and tunable SPR peaks in the NIR region have been used recently for orthogonally triggered release by choosing the right laser according to the AuNCs’ SPR. In a study by Qu et al. (74), two types of AuNCs were prepared with two different LSPRs and preloaded with two types of effectors in the hollow interiors before being covered with a smart polymer shell (Figure 9). When exposed to a laser beam with a wavelength matching the absorption peak of the AuNCs, this polymer collapsed due to the high local temperature, thus exposing the pores on the nanocages and thereby releasing the pre-loaded effectors. When the laser was turned off, the polymer chains would relax back to the extended conformation and terminate the release. As a result, when enzyme and substrate were chosen as the two effectors and selectively released from two different AuNCs, enzymatic reactions between enzyme and substrate occurred only after the successful opening of both types of AuNC capsules. The system acts as an “AND” logic gate. However, when the AuNCs are preloaded with isoenzyme or enzyme inhibitor, an “OR” or “INHIBIT” logic gate operation is established. This study represents a good example of NIR light-encoded, logically controlled, intracellular release systems. AuNCs were also used as a nanocarrier for a PDT molecular photosensitizer. Pandey et al. (75) demonstrated that when conjugated with a PDT photosensitizer, AuNCs enabled dual image-guided delivery of the photosensitizer and significantly improved the efficacy of PDT in a murine model. The photosensitizer, 3-devinyl-3-(1’-hexyloxyethyl)pyropheophorbide (HPPH), was non-covalently entrapped in a poly(ethylene glycol) monolayer that was coated on the surface of AuNCs. Such entrapped HPPH can be delivered more effectively to the tumor as compared to free HPPH. In addition, the presence of the AuNCs enhanced the 1O2 generation and the phototoxicity of the HPPH in vitro. As a result, the growth of the tumor in vivo was suppressed due to the combination of the effective delivery and the enhanced phototoxicity of the AuNC-HPPH conjugates. In the meanwhile, fluorescence and photoacoustic imaging provided information aiding the monitoring of the progression of delivery and tumor treatment following PDT. AuNCs themselves can directly work as a PDT photosensitizer. They were selected as representative plasmonic metal nanostructures, owing to their strong one-/two-photon absorption in the NIR region, to study the relevant mechanism of photo-induced tumor cell death, which involved hyperthermia and PDT effects. In a recent study by Gao et al. (76), NIR-light-excited hot electrons were found to generate either ROS (including 1O2, •O2ˉ, •OH) or hyperthermia (Figure 10). Compared with one-photon irradiation, two-photon irradiation brought about much more ROS. In addition, in vitro experiments disclosed that ROS-triggered mitochondrial depolarization and caspase protein up-regulation, which resulted in tumor cell apoptosis under two-photon irradiation, while hyperthermia mainly induced tumor cell necrosis. These findings suggest a regulation of plasmon-mediated ROS and hyperthermia for optimized anticancer phototherapy. Despite being a promising phototherapy agent, AuNCs have a short blood circulation lifetime, which limits their tumor uptake and thus in vivo applications. This limitation was overcome recently by cloaking AuNCs with red blood cell (RBC) membranes that act as a natural stealth coating. Yang et al. (77) found that while the fusion of RBC membranes over AuNCs preserved the unique 229 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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porous and hollow structures of AuNCs, the resulting RBC-membrane coated AuNCs (RBC-AuNCs) were further rendered good colloidal stability. NIR laser irradiation experiments demonstrated that the RBC-AuNCs possessed in vitro photothermal effects and selectively ablated cancerous cells as did the pristine biopolymer-stealth-coated AuNCs, but they further exhibited greatly enhanced in vivo blood retention and circulation lifetime in a mouse model. As a result, tumor uptake of RBC-AuNCs increased, and mice that received PTT cancer treatment modulated by RBC-AuNCs achieved a 100% survival rate over 45 days. These results show that applying a stealth coating is effective in prolonging circulating RBC-AuNCs for in vivo applications and thus improves PTT efficacy.

Figure 9. Schematic representation of a NIR light-encoded logic gate for controlled release based on the AuNC copolymer. Adapted with permission from reference (74). Copyright 2014 John Wiley and Sons.

In a recent study by Drezek et al. (78), a novel magnetic hollow Au nanosphere complex that incorporates iron oxide nanoparticles (IONPs) in the hollow interior was designed. This complex was synthesized by conjugating IONPs (~10 nm) onto silver cores (~35 nm) using 3mercaptopropyltrimethoxysilane (MPTMS) followed by a second layer of silver enwrapping the IONPs. After gold salt was added, a thin gold layer was formed while etching away the silver in a similar fashion to traditional hollow Au nanospheres. Compared to previous hollow Au nanospheres or Au nanoshell structures, two advantages of this complex are obvious. First, multiple IONPs were incorporated in this way, improving the overall magnetic properties, and thus the particle’s capability as a MRI contrast agent. Second, the resultant complexes are ~60 nm, which is optimal for cellular retention and tumor accumulation of nanoparticles, while a plasmonic peak was still maintained in the NIR range (~800 nm). As a result, the complexes performed well as MRI T2 contrast agents and also debulked tumors and improved survival with effective PTT. 230 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 10. Schematic illustration of AuNCs as intrinsic inorganic photosensitizers mediating the generation of ROS (1O2, •O2ˉ, •OH) and hyperthermia under NIR one-/two-photon irradiation. Depending on the irradiation power intensity, AuNC-PEG-mediated phototherapy could effectively affect tumor cells by two different pathways. ROS played a leading role in apoptosis at low power, and hyperthermia mainly resulted in necrosis at high power. Reproduced with permission from reference (76). Copyright 2014 American Chemical Society.

Evans et al. reported the first in vitro and in vivo study of gold nanotubes (AuNTs) most recently (70). The sacrificial Ag nanorods were 300–700 nm long with ~50nm diameters. These resultant Au NTs with ~6 nm-thick walls showed strong LSPR absorption in the NIR region that could be tuned by varying the length of the starting AgNRs or by adjusting the Au precursor to Ag nanorod ratio. After being functionalized with poly (sodium 4-styrenesulfonate) (PSS), the PSS-AuNTs possessed high colloid stability and low cytotoxicity, and could be internalized by cancer cells (SW480 cells) and macrophages (RAW 264.7) as revealed by the in vitro dark-field optical imaging. In vivo experiments showed that PSS–Au NTs accumulated at the SW620 tumor site but had a hepatobiliary clearance within 72 h. In addition, the AuNTs rendered excellent photoacoustic signals and photothermal ablation performance. Unlike the nanotube structures fabricated from pure Ag nanorods, the AuNR-in-shell structure synthesized using Ag-coated Au nanorods as a sacrificial substrate were responsive to both the first and second NIR windows, despite their small dimensions below 100 nm (71). Specifically, the structure (length: ~53 nm, 231 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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width: ~26 nm) containing an Au NR (length: ~40 nm, width: ~10 nm) in a cavity of a AuAg nanoshell showed an intriguing attribute, i.e., a broad absorbance band across 300–1350 nm with two NIR SPR bands located at approximately 1100 and 1280 nm. Very few, if any, nano-sized light absorbers in the second NIR region were reported prior to this work. With this nanostructure, the first in vitro and in vivo photothermal cancer therapy in the second NIR window was demonstrated. Using a continuous wave of 808 nm (first NIR window) or a 1064 nm (second NIR window) diode laser, large cell-damaged area beyond the laser-irradiated area was observed, indicating a high efficacy of the NIR photothermal destruction of cancer cells.

Au Nanoshells The Au nanoshell (AuNS) nanostructure, first generated by Halas’ group, is comprised of a dielectric silica core surrounded by an ultrathin gold shell (SiO2@AuNSs) (79). Its synthesis includes an attachment of Au seeds (~2 nm) on SiO2 spheres followed by an iterated growth of gold layers until a continuous shell with a desired thickness is formed (Figure 11). The plasmon hybridization between the shell’s inner and outer surfaces dictates the SPR properties of this structure. By varying the core size and shell thickness, a single SPR band can be generated in the NIR window. This SiO2@AuNS is sometimes referred to as the first generation of AuNSs. Since its invention, a variety of the so-called the second generation of AuNSs structures, including magnetic-cored AuNSs (e.g., Fe3O4@AuNSs and FePt@AuNSs) (80–82), polymer-cored AuNSs (e.g., poly (lactic-co-glycolic acid, PLGA@AuNSs) (83), quantum dot-cored AuNSs (e.g., CdSe/ZnS@AuNSs) (84), and liposome-cored AuNSs (e.g., poly-L-histidine@AuNSs) (85), have been synthesized by replacing the SiO2 core with other materials.

Figure 11. TEM images showing the formation of SiO2@AuNSs via iterated growth of gold layers on Au seeds attached SiO2 sphere. Adapted with permission from reference (79). Copyright 1999 American Institute of Physics. 232 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Recent studies on AuNSs have been extensively focused on the development of a multi-modality image-guided theranostic platform. For example, gadolinium was conjugated to SiO2@AuNSs for multimodal diagnostic imaging and photothermal cancer therapy. West et al. (86) found that after conjugating gadolinium, the resulting conjugates were rather versatile in affording contrast enhancement for a wide range of diagnostic modalities, including MRI, X-ray, optical coherence tomography, reflectance confocal microscopy, and two-photon luminescence, with resolutions spanning anatomic to sub-cellular length scales, thus facilitating the application in image-guided PTT enabled by AuNSs. Melancon et al. (87) reported the fabrication of super-paramagnetic iron-oxide-containing AuNSs (SPIO@AuNSs), which are capable of simultaneous photoacoustic (PA) and magnetic-resonance- (MR) guided photothermal ablation (PTA) therapy. Because of the intrinsically high near-infrared optical absorbance and strong magnetic property of SPIO@AuNSs, in vivo dual-modality PA-MR imaging-guided monitoring of therapeutic effects after PTT for mouse tumors have been demonstrated. In addition, a much clearer structure of the tumor blood vasculature was visualized using the PA technique after an intravenous administration of SPIO@AuNSs. Dai et al. (88) developed an AuNS-based multifunctional theranostic nanoparticle (DOX@PLA@AuNS-PEG-MnP) by growing AuNSs around poly(lactic acid) (PLA) nanoparticles encapsulating DOX, followed by tethering a Mn-porphyrin derivative (MnP) on the AuNS surface through the linker molecule polyethylene glycol (PEG). The biodegradable PLA served as a drug carrier, while AuNSs worked as the NIR photo absorber to perform PTT and trigger instant drug release. PEG prolonged the circulation time in vivo, whereas a Mn-porphyrin derivative effectively enhanced the MRI contrast. As a result, a greatly improved longitudinal relaxivity was realized, as demonstrated in both in vitro and in vivo experiments, which provided accurate information regarding the location and detailed structure of the tumor through MRI. Under the irradiation of an NIR laser, the combined light-triggered release of DOX and photothermal treatment was more cytotoxic than either treatment alone in both cellular experiments and tumor-bearing nude mice models.

Au Nanoparticle Ensembles The aforementioned four families of NIR-responsive gold nanostructures were all developed within a single particle’s regime. Their NIR absorption arises either from particular shape-induced plasmon mode or from coupling between plasmon modes within the particle. A novel type of NIR-active nanostructures (Au nanoparticle ensembles) was also designed in recent years, based on the aggregation/assembly of a collection of individual AuNPs (Figure 12). The NIR–responsive property originates from the aggregation/assembly induced interparticle plasmon coupling, and thus can be delicately tailored by controlling the properties (e.g., size and shape) of the individual building particles. Currently, two common strategies have been developed to achieve such nanoparticle ensembles: pH-induced aggregation (89, 91–95) and amphiphilicity-driven self-assembly (90, 96, 97). 233 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 12. (a) TEM images showing pH-induced aggregation of individual gold nanoparticles (top) into aggregates (bottom). (b) SEM image of plasmonic gold nanovesicles self-assembled from Au nanoparticles. Figure 12a adapted with permission from reference (89). Copyright 2009 American Chemical Society. Figure 12b adapted with permission from reference (90). Copyright 2013 American Chemical Society.

Kim et al. (89) pioneered the use of pH to induce the aggregation of small AuNPs (~10 nm) into larger aggregates for PTT. They functionalized the AuNPs with a “smart” surface molecule citraconic amide. This molecule is negatively charged and stable in neutral or basic conditions, but it hydrolyzes to a positively charged protonated amine at a pH < 7.0. When AuNPs are internalized into the mildly acid intracellular environment of a cancerous cell, hydrolysis of the citraconic amide rendered nanoparticle surfaces with mixed charges, which resulted in an aggregation of small AuNPs via electrostatic interactions. The aggregates accumulated because their increased sizes caused the block of exocytosis. In particular, the absorption of the initial AuNPs in the visible range shifted to the far-red and NIR spectra. This absorption shift was then exploited for in vitro photothermal cancer therapy with a 660 nm laser. Using B16 F10 mouse melanoma, NIH 3T3 mouse embryonic fibroblast cells, and HeLa cells, the photothermal efficacy of aggregates was greatly improved compared to those of two control groups using citrate capped AuNPs and without using any AuNPs. 234 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 13. Schematic illustration of the working mechanism of “smart” AuNP-DOX conjugates. AuNP-DOX conjugates consist of AuNPs modified with smart surface ligands and covalently conjugated DOX. The AuNP-DOX releases DOX by pH-triggered linker cleavage under the mild acidic conditions typical of a tumor. Simultaneously, AuNP-DOX converts the surface charge from negative to a mixture of negative and positive charges, which induces a rapid aggregation among the nanoparticles via electrostatic interactions. This spatiotemporally concerted release from AuNP-DOX was exploited for chemo- and photothermal combination cancer therapy. SANDC—Smart Au Nanoparticles-Dox Conjugates. Adapted with permission from reference (94). Copyright 2014 American Chemical Society.

Based on a similar principle, other pH-sensitive bifunctional platforms that combine PTT and chemo-therapy (94) or PTT and SERS imaging (95) were developed by functionalizing the small AuNPs with the anticancer drug DOX or Raman probes, respectively. For example, they grafted DOX to the terminals of 235 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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the smart surface molecule citraconic amide via a carbodiimide coupling between the –NH2 group of DOX and the terminal –COOH group of citraconic amide (Figure 13). Again, when the individual small AuNPs with surfaces functionalized with both citraconic amide and citraconic amide-doxorubicin conjugates, were internalized into cancerous cells, the acidic intracellular environment induced not only an aggregation of small particles, but a release of doxorubicins due to hydrolysis of the carbodiimide bond. Such a doubly pH-responsive (aggregation and DOX release) therapy system showed nearly an order of magnitude enhanced cytotoxicity in vitro when compared with two sequential independent treatments. The advantage of this synergistic effect was also confirmed by an in vivo animal model, where a significant suppression of tumor growth and no noticeable damage to other organs were detected. In the case of SERS imaging, 4-mercaptobenzoic acid was introduced to the AuNP surface as a Raman probe. The pH-induced aggregation of AuNPs provided hot spots for SERS with the enhancement factor reaching 1.3 × 104, thus enabling a concomitant SERS imaging in addition to PTT. In addition to irregular macroscopic aggregates, self-assembly of plasmonic nanoparticles into more well-defined, discrete ensembles has also been developed for biomedical applications. Duan et al. (96) developed plasmonic vesicles assembled from SERS-active amphiphilic AuNRs for cancer-targeted drug delivery that allowed for simultaneous SERS detection and synergistic chemo-PTT of specific cancer cells (Figure 14). To synthesize the SERS-active amphiphilic AuNRs, they coated initially cetrimonium bromide (CTAB)-stabilized AuNRs with a Raman probe 2-naphthalenethiol (NPT) and mixed polymer brushes of hydrophilic poly (ethylene glycol) (PEG) and hydrophobic polylactide (PLA). After that, the plasmonic vesicle was generated by self-assembly of the amphiphilic Au nanorods via a film rehydration method. The interparticle plasmonic coupling of the resultant plasmonic vesicles provided a large electric field for SERS detection and induced a significant red-shift of the SPR band into the NIR range; for example, 808 nm. In addition, the plasmonic vesicle offered a hydrophobic PLA shell and large aqueous cavity for loading of anticancer drugs. In this way, plasmonic vesicles were shown to be capable of specifically targeting EpCAM-positive cancer cells, leading to ultrasensitive spectroscopic detection of cancer cells. Moreover, the combination of the photothermal effect of AuNRs and the large loading capacity of the vesicles showed higher efficiency in killing targeted cancer cells than either single therapeutic modality, due to the localized synergistic PTT and photothermal-triggered chemotherapy. Finally, LSPR peaks have been engineered to the NIR region by self-assembly of spherical AuNPs. For example, Nie et al. (97) reported a dialysis-guided assembly of amphiphilic block copolymer, poly (ethylene glycol)-b-poly (e-caprolactone) (PEG-b-PCL), tethered AuNPs (~26 nm) into Au nanovesicles (AuNVs) in a THF/water system (Figure 15). The resultant AuNVs exhibited broad extinction spectra with major peaks positioned in the NIR range and tunable by changing the initial AuNP concentration. The strong NIR absorption and high photothermal conversion efficiency (~ 37%) enabled simultaneous PAI and enhanced PTT efficacy. Moreover, after the completion of PTT, the AuNVs dissociated into discrete AuNPs, which improved particle clearance. 236 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 14. Schematic illustration of the synthesis of SERS-active amphiphilic AuNRs (AuNR@NPT/PEG/PLA) with Raman probes NPT and mixed polymer brushes PEG and PLA, and self-assembly of AuNR@NPT/PEG/PLA into plasmonic vesicles (Top). The plasmonic vesicles exhibited a unique combination of SERS, PTT, and laser-induced drug release (Bottom). Adapted with permission from reference (96). Copyright 2013 American Chemical Society.

The advantage of the hollow interior space of the vesicles could further be taken to load active compounds for constructing a multifunctional theranostic platform. For example, when photosensitizer Ce6 molecules were encapsulated in the plasmonic vesicles, a unique trimodality NIR fluorescence/thermal/photoacoustic imaging-guided synergistic PTT/PDT with improved efficacy was demonstrated by the same group (90). The AuNVs that were prepared by rehydration-triggered self-assembly of polyethylene oxide-b-polystyrene (PEO-b-PS)-tethered AuNPs had a strong absorbance in the NIR range of 650–800 nm, as a result of the plasmonic coupling between neighboring AuNPs in the vesicular membranes. This enabled the use of 671-nm laser irradiation to simultaneously excite AuNVs to generate heat for PTT and 237 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Ce6 to produce ROS for PDT, killing cancer cells. Meanwhile, the efficacy of such a treatment could be monitored by visualizing the tumor tissues with the aid of the fluorescence, thermal and PA signals arising from AuNVs-Ce6 in tumor cells. Both in vitro and in vivo results showed that the therapeutic efficacy of AuNVs-Ce6 was enhanced compared to either PTT or PDT alone, or even the sum of PTT/PDT, indicating a synergistic effect. These results, together with other advantages, such as high Ce6 loading efficiency (~18.4 wt. %) and enhanced Ce6 delivery into cells, make AuNVs-Ce6 a potential theranostic platform for imaging-guided synergistic PTT/PDT of tumors in vivo.

Figure 15. Self-assembly of biodegradable Au vesicles composed of poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-b-PCL)-tethered AuNPs through the dot–line–plane–vesicle mode during the dialysis process. Au vesicles with an ultrastrong plasmonic coupling effect were superior PAI and PTT agents with improved clearance after the dissociation of the assemblies. The PA signal and PTT efficiency of Au vesicles increased as the distance (d) between adjacent AuNPs decreased. GNP—Au nanoparticles. Reproduced with permission from reference (97). Copyright 2013 John Wiley and Sons. 238 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Summary and Outlook The inherent properties of gold nanostructures, such as their good biocompatibility, ease of surface functionalization and shape-dependent LSPR, make them well suited as NIR photosensitizers in biomedical applications. We describe how a variety of recent advances use NIR-resonant gold nanostructures (nanorods, nanoprisms, nanoplates, nanostars, nanoflowers, nanoechinus, nanopopcorns, nanocages, hollow nanospheres, nanotubes, nanorod-in-shell, nanoshells and nanoparticle ensembles) for efficient imaging (scattering- and luminescence-based optical imaging, surface-enhanced spectroscopy-based imaging and photoacoustic imaging) and therapeutic (photodynamic therapy, photothermal therapy, drug delivery and light-controlled release) agents for in vitro and in vivo theranostic applications. Despite their great potentials, some contradictions associated with the in vivo toxicity and theranostic efficacy of gold nanostructures in a complex body environment need to be well compromised before they can be translated to clinical practice. (i) Shape features such as corners, tips, edges and pores are essential structural origins of the intriguing LSPR properties of NIR-resonant gold nanostructures. However, in many cases these features have small dimensions (e.g., < 5 nm) and/or are enclosed with high-index facets. As a result, although gold nanoparticles larger than 5 nm are generally inert and considered to be nontoxic and biocompatible, gold nanostructures may be more reactive than bulk gold and thus present certain toxicity. (ii) Surface modifications represent a good strategy to improve the colloidal stability and blood circulation time or to increase the accumulation in target sites, thus enhancing theranostic efficacy. Nevertheless, surface ligands introduced for these specific functionalities (e.g., colloidal stability and targeting) may either directly impart the gold cores with toxicity (for example, the toxicity of CTAB-stabilized AuNRs was found to stem from CTAB) or polarize the gold cores into a more reactive oxidation state (for example, a Au-S bond induces Auδ+-Sδ-). (iii) It appears that gold nanostructures in size of 10-100 nm are ideal because they are small enough to have sufficient diffusion in the extracellular space and resistance to the phagocyte system but also large enough to avoid being rapidly cleared during circulation through extravasation or renal clearance. Currently, some of the gold nanostructures described in this chapter (e.g., nanoechinus, nanotubes, nanoshells and nanoparticle ensembles) are much larger than 100 nm, which could affect their uptake and thus limit the full exploitation of their theranostic potentials. In addition, these structures are too big to be efficiently eliminated from the body, for example via renal clearance, and they would, therefore, redistribute and pose chronic hazards to other healthy tissues or organs. Ideal NIR-resonant gold nanostructure-based theranostic agents for clinical applications should possess strong NIR-responsivity, low inherent toxicity, good site-specific targeting ability and effective clearance from the body after use.

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Chapter 13

Bioanalysis within Microfluidics: A Review

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Wenwen Jing1,3 and Guodong Sui*,1,2 1Shanghai

Key Laboratory of Atmospheric Particle Pollution and Prevention (LAP3), Department of Environmental Science and Engineering, Fudan University, 220 Handan Road, Shanghai, 200433, People’s Republic of China 2Institute of Biomedical Science, Fudan University, Shanghai, 200032, People’s Republic of China 3Department of Medical Microbiology and Parasitology, School of Basic Medical Sciences, Fudan University, Shanghai, 200032, People’s Republic of China *E-mail: [email protected].

Rapid analysis of bioaerosol, including airborne pathogens plays a critical role in bioaerosol study as well as in the early warning of infectious diseases. Related research is essential not only to the scientific society, but also for public health and disease prevention. We present herein the current situation of bioaerosol detection, enumerate the main existing problems, put forward countermeasures and suggestions, and report a series of integrated microfluidic chips and systems that can execute airborne microbe capture, enrichment and continuous-flow high-throughput bioanalysis. Six microbes have been used to validate the capture and analysis efficiency of the system. Our experimental results showed that capture efficiency and detection limit had been greatly improved by microfluidics compared to traditional methods. The capture efficiency of microfluidic chip reaches almost 100%, and the detection limits down to approximately 118 cells were achieved toward Escherichia coli (E. coli), without the DNA purification process. It can collect enough bacteria from low concentration bioaerosol (as low as 100 cfu/m3) for the downstream direct protein/nucleic acid analysis. The whole operation is simple

© 2015 American Chemical Society In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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and feasible, suitable for on-site application, e.g. at airports, subway stations and hospitals, showing potential application in environmental monitoring and public health protection.

Airborne pathogens are generally defined as pathogens that exist independently or attached to liquid or solid particles suspended in a gas (1–3). For the past few decades, human beings faced threats from all kinds of emerging infectious diseases and old infectious diseases (4–10). In addition to the familiar Tuberculosis (caused by Mycobacterium tuberculosis [TB]), Severe Acute Respiratory Syndromes (SARS), and Avian Influenza (avian flu) (caused by viruses H1N1, H5N1 and H7N9), there are many other kinds of infectious diseases that are caused by various airborne pathogens (11). These airborne pathogens are divided into the following types. The type bacteria includes Bacillus anthracis, Yersinia pestis, Vibrio cholera, Bacillus maller, Tularemia coli, etc. The type viruses includeds Yellow fever virus, Venezuelan equine encephalitis virus (VEEV), Variola virus, Semliki forest encephalitis virus, Dengue fever virus, Lassa fever virus, Rift valley fever virus (RVFV), etc. The type rickettsiae includes Typhus group rickettsiae and Q fever rickettsia (QFR), etc. The type chlamydia includes Chlamydia psittaci. The type fungi includes Aspergillus flavus, Aspergillus niger, etc. (12, 13) Infectious diseases are difficult to control and prevent worldwide because their pathogens can spread fast among people through the air as the medium (14). When exposed to air polluted by pathogenic bacteria, the human body could be invaded through the skin or mucous membranes or respiratory tract and become infected (15). Human health is threatened and damaged frequently, not to mention the public panic and economic losses accompanying airborne pathogens, which are gaining increasing interests in recent years. Methodology for rapid or on-line detection of airborne pathogens is essential for disease control and prevention. In this review, we will provide an overview of detection of airborne pathogens, the challenges, and the future perspective.

Challenges in Airborne Pathogen Analysis So far, identification of airborne pathogens still relies on the clinical observation and follows precise molecular and immunological diagnosis of blood samples from potentially infected patients (16, 17). This process normally takes days, which is too slow for prompt disease control and prevention. The rapid and direct analysis of airborne pathogens helps disease control and surveillance resources for pandemic diseases, especially for highly populated metropolitan areas (18, 19). However, there are still technical obstacles even with great development in molecular biology in the past couple decades. The obstacles come from two aspects of airborne pathogen analysis: initial airborne pathogen collection techniques and downstream bioanalytical techniques for identification of collected pathogens. 246 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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The sampling procedure is a key to get a reliable observation in the application of airborne pathogen analysis (20, 21). The selection of sampling equipment is an important issue in this procedure to ensure the collection efficiency is directly related to the accuracy in the evaluation of final analysis results. According to practical requirements of sampling, sensitivity, stability, reliability, portability, and ease of operation, maintenance and analysis are issues that need to be considered. The first airborne pathogen natural sedimentation method was first developed by Koch in 1881, where airborne pathogens were settled by gravity on nutrient agar media (22). It is the simplest and most economical sampling method in which the results roughly reflect the structure and amount of airborne microorganisms. However, the defects of natural sedimentation methods are obvious. The sampling conditions are hard to control and may induce great measuring error because of interferences such as wind force, electric force, magnetic force, thermal force, buoyancy force, and diffusive force. Besides the airborne pathogen natural sedimentation method, there are machine sampling methods for airborne pathogens (23). For example:

Anderson Sampler The Anderson sampler is the widely used airborne pathogen sampler which can not only measure the approximate size of the pathogens, but also their concentration (24). The sampler size ranges from 0.2~20 µm which is able to satisfy most pathogen sampling requirements except for those pathogens that could not be cultured on various media. However, operation is complex and needs a large number of culture plates. According to the different growth time of various pathogens, the culture time ranges from several days to several months before the concentration of pathogens fulfills the detection requirement (25). If pathogens are sensitive to air pressure, it is difficult to keep these microorganisms alive.

All-Glass Impinge (AGI) Sampler The AGI sampler is a glass collection device that is designed to collect airborne pathogens with a high concentration (26). It is operated by drawing airborne pathogens through an inlet tube and subsequently passing through a jet into a liquid medium (27). Take AGI-30 for an example, the jet is positioned 30 millimeters (mm) above the bottom of the glass impinger, and consists of a short piece of capillary tube. The medium of this sampler could protect the pathogens from losing their biological activity (28). All airborne pathogens drawn into the AGI-30 sampler could be contained efficiently (29). This sampler is only suitable for airborne pathogens with a high concentration. When the concentration of pathogens is low or when the temperature is low, it is hard to detect airborne pathogens successfully. 247 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Filter Sampler

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The Filter sampler is an air extracting instrument into which airborne pathogens could be intercepted by a filter membrane when bioaerosol passes through the membrane under negative pressure (30). The core component of this equipment is the filter membrane. According to different pore size and materials of the filter membrane, various airborne pathogens could be collected on the membrane with a high collection efficiency (31). However, this instrument is not suitable for airborne pathogens which are intolerant to dry conditions. The efficiency of the sampler may also be influenced by the particle elution process.

Static Sampler Vaporized bacteria carry electric charges, which could be used for bacterial aerosol collection, according to the electrostatic adsorption principle, in the static sampler (32, 33). This equipment contains a high voltage power supply, discharge electrodes, collecting electrodes and air extracting device. The sampling progress of this instrument has advantages such as retained bioactivity of aerosol bacteria due to its big sampling volume and high concentration ratio. However, ultraviolet light, ozone and nitrogen oxides produced during the sampling process will degrade the microorganisms (34). The big and complex instrument has some disadvantages such as inconvenience of installation and maintenance, and carries safety risks, which detract from practical application. Although many different types of samplers are promoted for the development of air microbiology, so far, fast and accurate sampling of the biological aerosol is still a difficult technical issue (35, 36). Almost no sampling technique could ensure that the collected microbial specimen reflects its original state and can be directly used in bioanalysis (37). Pathogen culturing is generally the necessary step before analysis, mainly because the concentration of collected pathogens is too low for direct bioanalysis for the methods mentioned above. Before the inventions in molecular biology, physical and chemical, airborne pathogens were mainly studied through cultivation methods on selective and nonselective media. These methods have been used in the study of aerosol analysis with a long history and will still be used as useful analysis tools in microbial aerosol particles analysis.

Cultivation Methods Cultivation methods can only detect certain viable airborne microorganisms in the air such as some living bacteria, fungi and algae. However, after death, these bacteria, fungi and algae, as well as their fragments, such as cell walls or cytoplasm materials are incapable of being detected or collected by cultivation methods. Furthermore, cultivation methods are only suitable for detecting and collecting those microorganisms that can grow on culture medium (38). However, studies of the cell viability of environmental microorganisms have found that the vast majority of viable microorganisms that exist in ambient environment are in 248 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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viable but non-culturable state (39). Only about 17% of known fungi species can be cultivated on the culture medium (40). When it comes to bacteria, only less than 10% of the total number of bacteria can be cultivated, which can be observed in the range of about 0.01% to 75%, with about 1% as its average values (41). Therefore, studies based on microbial cultivation methods often largely underestimated the microbial diversity and concentration of microorganisms. In addition, when microorganisms are collected by conventional sampling methods, this can cause a great damage on a number of viable cells, because the suction pressure generated in the sampling progress will lead to some viable cells death (42). The culturability of living microorganisms also decreases rapidly through the traditional sampling methods (43). Finally, the number of microorganisms are likely to be greatly underestimated, because one single aerosol particle may represent at least one kind of a microorganism’s clone, whereas the traditional sampling methods and cultivation methods will only form one colony on the culture media (10, 44, 45). Nonetheless, there are limitations, cultivation methods have the advantage that they are cheaper than molecular biology detection methods, and cultivation methods can give an indication of the quantity of viable microorganisms cells in the air, whereas molecular biology methods can only prove the existence of these microorganisms but are unable to distinguish whether these organisms are alive or dead (46–49). Cultivation methods are particularly suitable for detecting certain groups of microorganisms or targeting individual species, and collecting microorganism cells for culture collections (50–53). Culturing normally takes from 24 hours to several days to more than ten days, which is too slow for rapid analysis, especially for the early disease warning and prevention situation (54). Some bacteria were in the state of viable but non-culturable (VBNC) in natural environments (55). Most pathogens are sub micron in size, and the dimensions of the current samplers are at millimeter or centimeter level. Because of the size difference, the water used to rinse/wash the samplers is generally excessive, compared with the small amount of the pathogens collected in the samplers. Consequently, the concentration of the collected pathogens in the aqueous media is too low for direct bioanalysis.

Microscopy Techniques As extremely valuable research tools, microscopy techniques have played very important role in the analysis and detection of microbial aerosol particles (56). Various types of optical microscopy were applied in the classification and characteristics description aspects of the collected microbial aerosol particles (57). However, it has to be taken into consideration that when the particle size is smaller than 2 microns, it only appears as a dot, and its characteristics size, shape, and detailed microscopic structures could not be distinguished and analyzed clearly under optical microscope (58). Therefore, such kind of aerosol particles cannot be classified and analyzed by an optical microscope. The direct number counting and strain identification of microbial aerosol particles are both very tedious processes. It will be influenced by some subjective factors when counting the microbial aerosol particles with naked eye. For the species diversity 249 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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analysis of microorganisms, the credibility of microscopic analyses results is usually not high. In the identification of strains, some of the strains are similar in morphological characteristics, which causes difficulty in species differentiation. Some microorganisms can only be identified as belonging to broad categories or a specific family (59). Fluorescence microscopy is often used to observe microbial aerosol particles which are capable of autofluorescence (60, 61). Microbial aerosol particles also could be observed by fluorescence microscopy after labeling them with fluorescent dye (62, 63). The most commonly used traditional methods for counting the total number of environmental microbes are by direct counting under fluorescence microscopy. This counting is usually achieved by detecting the autofluorescence of certain biological compounds or by detecting the fluorescence of microorganisms that are treated with some commonly used fluorescent dye, such as 4.6-diamidino-2-phenyl (DAPI) and acridine orange (64, 65). Recently, more and more automated analysis techniques such as computer analysis of microscopic images and fluorescence spectroscopy have been applied in microorganisms detection fields combined with fluorescence microscopy (66–70). However, sometimes fluorescence microscopy is unable to clearly distinguish different types of biological particles, such as fungal spores and bacteria (71).

Chemical Tracers Chemical tracers, such as sugar alcohol, mannitol and arabitol are used as chemical tracers for assessing diversity of not only microbial aerosol but also other kinds of aerosol particles (72–74). These chemical tracers can be combined with multiple bio-analytical techniques including liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometer (GC-MS), ultraviolet spectrophotometry, fluorescent spectrophotometry, immunoassay, and dyes analysis for sample testing (75–77). The merit of chemical tracer analysis is quantification of information although biodiversity or identity of microbial aerosol particles at species level is generally not available (78).

Nucleic Acid Sampling and Extraction from Bioaerosols Microbial aerosol particles are gathered and extracted from samples including solid medium, liquid medium, water, biological aerosol particle collectors or air filters before analysis of microbial aerosol with tools of molecular genetics. Successful microbial DNA extraction is the basis of analysis with molecular biology method (79–82). Microbial protein is denatured and mixed with liquid, pigment, fragments of cell wall and organelles in the process of DNA extraction (83). DNA of microorganisms with thick membrane or cell wall is extracted insufficiently with above-mentioned protocol and the quantity of such microorganisms is underestimated in environment samples with diversified biological materials (84–88). 250 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Amplification of Genomic DNA The DNA of the target microorganism is supposed to be gathered so as to make the quantity of it much higher than that of other ones for the recognition of some single genera or species (89). DNA characteristic regions of one or several species of microorganisms are effectively amplified and analyzed with polymerase chain reactions (PCR) in which DNA double strand is denatured by heat and a corresponding single strand in target genome is paired with short DNA sequences called primers according to the base complementation pairing rule and finally a new complementary strand is assembled with mononucleotide in target region by DNA polymerase within one heating cycle (90). The amplified sequences in PCR are useful for research on species identifying, specific genes or phylogenesis (91). As it is, genomic DNA amplification is considered to be important in investigating constitution and diversity of microbial aerosol although not all DNA of every single species in atmosphere could be amplified equally with above-mentioned method (92). DNA polymerase is capable of amplifying sequences from even a single copy of some strand theoretically so as to be highly sensitive in detecting trace quantity of environmental microorganisms although practical sensitivity depends on several factors including specificity of primers and integrity of DNA samples (93, 94). So as to say, one copy of a single-strand target gene is enough to start amplifying with highly specific primers while one hundred times of that is enough with not-so-sensitive primers (39, 95, 96). Specificity is extremely important for precise amplifying and high signal-to-noise ratio. Although primers designed for given DNA sequences are supposed to attach exclusively to the specific region in the target genome, they are still possibly combined with non-target sequences because of several times and even more quantity of competing genes (96). Although DNA molecules are relatively stable in cool, dark and dry condition and are possible to be conserved for even up to thousand years, the degradation process starts as soon as life ends (97–99). DNA molecules are reduced into smaller fragments and chemically modified and the process is accelerated by ultraviolet light, ozone and freezing-thawing. Degradation of DNA and loss of genetic information is caused by the exposure of biological materials to air for a long time (100, 101).

Restriction Fragment Length Polymorphism Techniques (RFLP) Restriction endonuclease is a kind of enzyme which is capable of recognizing and cutting specific sequences of a molecule at special sites producing restrictive fragments (102, 103). This is the theoretical basis of the technique (104–107). Genomes of different species are distinct from each other. Difference between genomes lying in a cleavage site of a restriction endonuclease leads to imparity in recognition of enzyme and causes diverse characteristics of digested fragments in length and quantity (108). Polymorphism of these fragments is analyzed with a whole set of techniques including electrophoresis, trans-membrane, denaturation, hybridization with labeled probe and washing membrane (109–113). This protocol is supposed to be used after colony PCR (make sure the plasmid has been 251 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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inserted into bacterial genome) in order to collect as many colonies as possible for sequencing (114–118). T-RFLP is used to investigate microbial community and estimate diversity and relative abundance of aerosol samples roughly (119, 120). First, the target sequence is attached with fluorescence-labeled primers and amplified in PCR. Then PCR product is digested with restriction enzyme at target site producing shorter labeled fragments. Fragments differ in lengths because of diverse positions of target sites in bacterial genomes from different species. Fluorescence-labeled fragments are separated with electrophoresis and lengths and fluorescence intensity of them are measured. Fluorescence of a DNA strand with a given length reveals the concentration of the corresponding bacterial strain in original samples. Sequencing Methods Product of PCR is usually cloned and sequenced to identify genomes in atmosphere aerosol samples. The sequences are then compared with known sequences from on-line database in order to determine the species of microorganisms in samples (121–124). If a new species is found (for example, a new bacterial strain), genome sequences of its nearest relative could be found from on-line database. Generally accepted interspecies difference in bacteria is similarity of 97-99% and intergeneric difference is similarity of 95-97% (42, 43). Chain termination is used in conventional sequencing techniques. The quenching is similar to PCR steps only in which the number of primer is one rather than that of two in PCR (125–128). Therefore it is almost impossible to identify microorganisms in biological aerosol particles at species level with current high through-put techniques (129, 130). Microarray Technology Microarray technology is applied to investigate features of microorganism aerosol particles (131–133). Specific probe for species or population is fixed on a glass chip (134–137). Fluorescence-labeled DNA in atmosphere samples is supposed to hybridize with complementary DNA sequences on the chip if the sequences exist (138, 139). Gene sequences are determined by the position of fluorescence signal on the chip (140, 141). On-Line Auto Fluorescence Methods This technique is currently applied to the determination of 16S rRNA in bacteria from atmosphere aerosol samples. It is helpful to determination if biological material contains fluorescent substance. Ultraviolet Aerodynamic Particle Sizer (UV-APS) is the first commercial instrument that is capable of analyzing biological aerosol samples on line based on fluorescence (142). The aerodynamic diameter and lateral scattering parameter (similar to optical diameter) of particles is measured by testing flight time (633nm) of particles between two laser beams and by fluorescence with the wave length range of 420-575nm excited by pulsed UV laser (Nd: YAG, 355 nm). Wide Issue 252 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Bioaerosol Spectrometer (WIBS) is also produced in a limited run providing not only similar information with UV-APS but also a rough estimation of the sphere factor (143). The size of incident particles is determined by measuring with a scattering laser at first (144). Fluorescence emission spectrum at 310-400 nm (excitement wave length of 280nm) and 420-650 nm (excitement wave length of 280 and 370nm) is recorded with every particle excited by UV pulse by a xenon flash lamp of 280 and 370 nm (145). Report on environment monitoring with this technique is still rare although development of it has been published in military report and peer review (146).

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Flow Cytometry Flow cytometry has always been an important tool in research on environmental microorganism aerosol particles (147–151). In reports of testing biological aerosol on line using Flow cytometry successfully, there are many data about research on features of atmosphere microorganism using Fluorescent in situ hybridization (FISH) flow cytometry and about research on features of aerosolized by-product indicating the existence of bacteria spores and fungi (152). Light Detection and Ranging (LIDAR) and Remote Sensing The LIDAR technique, also called microwave radar, is a technology that measures distance by illuminating a target with a laser (including ultraviolet, visible, or near infrared light) and analyzing the reflected light (153–155). It has been utilized to quickly and remotely monitor the presence of microbe aerosol particles over a larger spatial range. A LIDAR system was operated to determine its sensitivity to aerosolized Bacillus subtilis spores (156, 157). Mass Spectrometry (MS) The Mass spectrometry technique has been applied to many areas in physics and biology in the past decades providing detailed chemical composition information (158, 159). Many different MS techniques have been applied to research on characteristics of microbial aerosol particles (20, 160, 161). Matrix-Assisted Laser Desorption Ionization Time-of-Flight (MALDI-TOF) has been applied to multiple researches on microbial aerosol successfully (162–166). Laser-Induced Breakdown Spectroscopy Laser-induced breakdown spectroscopy (LIBS) is one type of atomic emission spectroscopy which uses a highly energetic laser pulse as the excitation source. Many groups have also utilized LIBS to identify elemental composition as means of biological aerosol detection and analysis, although these have not been as widely applied to ambient measurements (167–170). LIBS technique has been applied to research on characteristics of diverse microbial aerosol particles including pollen, bacterial spores and fungi (167–170). Other form of elemental analysis including spark-induced breakdown spectroscopy (SIBS), particle-induced X-ray emission 253 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

(PIXE) and combustion analysis have been applied to experiment research on biological aerosol (171–175).

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Microfluidics for Airborne Pathogen Analysis and Remaining Technical Challenges Microelectronic chip and micro total analysis system were the most profound technology in the 20th century history (176–179). The microelectronic chip began to act as the heart of computer and appliance 40 years ago (180, 181). Micro total analysis systems (µTAS) which is also called Lab on a chip or Microfluidic chip was started in the 1990s and is based on micro electromechanical systems (182, 183). Microfluidics, a new emerging technique dealing with samples in micron size and in nano liter volume, is widely applied in protein analysis and nucleic acid analysis (184, 185). It can provide rapid downstream bioanalytical methods as well as initial airborne pathogen capture methods, which fits perfectly for airborne pathogen analysis (186, 187). There are a variety of reports about the microfluidic pathogens analyzed in aqueous medium, including viruses, bacteria and fungi. Characteristic antigen (protein) immune analyses as well as nucleic acid analysis by hybridization or PCR were both successfully performed in microfluidic chips (188). Recently, the capture and enrichment of the airborne bacteria by microfluidic chip was also reported (189, 190). By converting the laminar flow to twisted air flow inside the microfluidic chip to increase the contact opportunity between the channel wall and the bacteria in the airflow, the microfluidic chip can collect hundreds of bacteria within a couple microliters (μL) of aqueous media, sufficient concentration and amount for direct immunoanalysis or nucleic acid analysis. As a new technology emerging in recent years, microfluidic chips which have become an integral part of science and technology and are undergoing a fast development stage, will continue to play an important role in the process of technological development. As an interdisciplinary and cutting-edge science, microfluidic chip technology is widely involved in different fields and techniques (191). It is involved in methods and technology belonging to chemistry, biology, physics and so on (192, 193). In order to achieve the functionalization, techniques from those disciplines were applied alone or fused to the development of microfluidic chips (194, 195). Microfluidic immunoassays. Immunoassays have been used widely in varieties of applications (196). For example, they have been used in environmental, food safety testing, pharmaceutical analysis, medical diagnostics and fields of basic scientific investigation, because they are very sensitive, simple and specific (197). Antibodies (Abs) are proteins produced in animals and human bodies by immune responses. Antigens (Ags) are some kinds of invasive foreign substances to human and animals bodies (198). In nature, these Ags have a highly specific affinity for their cooresponding antibodies. In lock-and-key mechanism, each Ab has a unique structure recognized by a corresponding Ag. There are a variety of formats of these immunoassays. All formats are making use of the sensitivity and specificity of that Ab-Ag interaction (198). The specific Ab-Ag 254 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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interaction allows for the quantification and monitoring of drugs and metabolites and other small molecules such as large proteins, nucleic acids and even whole pathogens (198). In recent years, a promising platform has been extensively explored for the combination of immunoassays and microfluidics as microfluidic immunoassays (199). Most immunoassays include a series of processes, such as washing, mixing, and incubation steps. All those processes are time consuming and need high labor intensity (200). These processes often take several hours. Sometimes one single assay even needs about two days to perform (201). The reason that immunoassays require a long time is mostly attributed to the incubation time, which is inefficient, as well as mass transport process of immune agents from solution to solution surface where the conjugation occurs, but the immune reaction itself is relatively rapid (202). Besides, the immune agents used in immunoassays are very expensive. If the system is miniaturized, the consumption of the immune agents will be reduced largely. Therefore, microfluidic immunoassays which are automated and miniaturized are in great demand. They have simplified the procedures, reduced the assay time as well as the sample and regent consumption, and at the same time enhanced the reaction efficiency. Recently, extensive investigations using microfluidics in the immunoassay field have been reported (203, 204). Isothermal Nucleic Acid Amplification in Microfluidic Platforms. Isothermal amplification methods use the enzymes involved in the synthesis of DNA/RNA in vitro in a thermostatic reaction. Many isothermal nucleic acid assays have been developed with different types and different numbers of enzymes and primers, amplification times, incubation temperatures and detection methods (205). Isothermal nucleic acid amplification assays such as loop-mediated isothermal amplification (LAMP), multiple displacement amplification (MDA), helicase dependent amplification (HDA), recombinase polymerase amplification and nucleic acid sequence-based amplification (NASbA) have also been translated on microfluidic platforms (206). LAMP is different from PCR. The polymerase extension rate is the limiting factor in the isothermal nucleic acid amplification assays. The detection times of these assays in conventional detection formats are considerably high, ranging from 30 to 90 min (207, 208). The detection time was reduced to less than 30 min by several factors, such as the optimization of primer design and reaction, the application of highly fluorescent dye and the improvement in detector sensitivity. Microfluidic based isothermal nucleic acid amplification assays are simpler and less expensive because they don’t require any sample or temperature circulation. LAMP is first reported in 2000 as a relatively new gene amplification technique. Appealing features of LAMP include high sensitivity, high specificity, speed, and high product yield. LAMP is performed at a moderate incubation temperature between 60 °C and 65°C. Major reaction components are as follows, four primers and an enzyme (Bst polymerase) with strand displacement activity. The addition of two more primers called loop primers further enhances the sensitivity and speed of LAMP reactions. Amplification times of tube-based LAMP assays typically vary between 30 and 90 min, depending on the starting DNA template. Endpoint and real-time LAMP assays employed with turbidity or fluorescence-based detection schemes 255 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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have also been translated on microchips made of silicon and polymer. Positive endpoint microLAMP is confirmed either visually or by fluorescence microscopy. Fluorescence or turbidity increment during real-time microLAMP is commonly monitored by photodiodes. MicroLAMP is further quantified by measuring the threshold time (Tt) for a selected cutoff of signal, where the signal is above the background (209, 210). Although microfluidic techniques solved many of the problems for the conventional airborne pathogen analysis from sampling to the downstream bioanalysis, however, many challenges remain. Pathogens larger in size, such as bacteria and fungi can be easily trapped inside the microfluidic chip; there is still no report about the collection efficiency of the virus from air, possibly because of its nanometer size, which is much smaller than bacteria and fungi. Since many common respiratory infectious diseases, such as all kinds of flu, are caused by virus, there is great technical need for methods capable of high capturing efficiency of virus in microfluidic chips (14, 211). In addition, the proper salt concentration, pH values as well as relative environmental cleanliness are all essential for successful downstream immunoanalysis or nucleic acid analysis. Most airborne pathogens exist within particles and constitute only a tiny part of the particles (2). The major components of the airborne particles are dusts, salts and some organic compounds. The various species and ratios of these components are determined by the individual environments of the particles (211). The aqueous media used in the direct collection of the pathogen from the surrounding air sometimes is not suitable for direct bioanalysis. It is necessary to develop microfluidic methods capable of separating pathogens from particles without greatly changing the total volume. Furthermore, because of the many types of airborne pathogens to screen in the case of a cautionary situation, high throughput downstream analytical techniques within microfluidic chips are in great need (212). However there is no available system for direct accurate airborne pathogen analysis, there are still parts missing to connect the microfluidic sampler with microfluidic chip for bioanalysis. The connection between them as well as the systematic automation will be another challenge to be conquered before any commercial instrument is developed. Herein our group has reported series of integrated microfluidic chips and systems that can execute airborne microbe capture, enrichment and continuous-flow high-throughput bioanalysis. Our group has established a simple, cheap polydimethylsiloxane (PDMS) microfluidic device which is capable of rapid and efficient airborne bacteria capture and enrichment. The device consists of a two layer PDMS microfluidic chip with two plates of polymethyl methacrylate (PMMA) bonded to the upper and lower surfaces of the microfluidic chip. This microfluidic chip consists of an inlet, an outlet, and the inside enrichment channel. The core capture channel is 17.4 cm long, 600 μm wide, and 40μm high. The capture channel is designed as an s-shaped zone and chaotic flow zone. A staggered herringbone mixer (SHM) structure has been used in airborne bacteria enrichment on a microfluidic chip for the first time. The SHM structure is designed to create chaotic flow and increase the contact chances between the bacteria in the aerosol and the inner walls of the microchannels inside the microchip. This device was validated with 256 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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E. coli and Mycobacterium smegmatis. The capture efficiency of the microchip reaches almost 100% in 9 minutes. The capture limit is lower than the plate sedimentation method. This device can collect enough airborne bacteria for a direct test. The whole system is perfect for field application especially in some airborne microorganisms’ high-risk environment. It has the potential to become a crucial platform for aerosol microorganisms detection (213). Based on this work, we developed a rapid capture, enrichment, and direct bacteriological diagnosis method for airborne Mycobacterium tuberculosis bacteria with Enzyme-linked Immuno Sorbent Assay (ELISA) double sandwich method based on microfluidic chip. The microfluidic immunoassay chip was made of two layers of PDMS containing a fluidic layer and control layer. Five immune-reaction columns were designed to analyze five different samples independently at the same time. The operations of the columns could be performed either in sequential or parallel manner according to the experiment requirements. The microchannels were 25 μm high, 200μm wide. The fluid was controlled by regular valves and siece valves. The key component for the assay is the microcolumn filled with tiny microspheres in 9 μm diameter. The reaction volume for each reaction is only approximately 15 nL. This immunoassay system could successfully identify antigen protein Ag85B, and is very convenient to operate, with little reagent consumption (only needs 1 or 2 µL of each reagent). The detection time was about 35 minutes which is far less than the traditional ELISA reaction on 96-well plates. Only 1 µL to 5 µL sample containing about 100 to 500 cells is needed for the detection of positive results. Compared with the previously reported method of on-chip capture and off-chip analysis, their is no need to wash out the bacteria from the enrichment chip to perform analysis. This device could also be applied for the detection of airborne pathogens and provide a possibility for the early warning of the spreading of airborne infectious diseases and for the automation platform with this analysis system (214). We also presented a simple and low cost continuous-flow PCR device by using microfluidics and molecular biology technology. The device is capable of fast detection of environmental pathogens in field. Compared with the conventional PCR method, the required reagent and sample has decreased to 10% by using the presented method. Furthermore, a two-step PCR decreased the processing time by 1/3, and the structure of the chip is simpler. The device is more portable and is easier to operate, which makes it a promising platform for environmental bacteria identification. A high-throughput continuous-flow PCR chip was also developed for airborne bacteria detection. The integrated microfluidic device can perform airborne pathogen capture, enrichment and gene analysis in 2 hours. Six frequently encountered bacteria, including Klebsiella pneumoniae, Citrobacter koseri, Staphyloccocus aureus, E. coli, Pseudomonas aeruginosa, Enterococcus faecalis and Mycobacterium smegmatis, have been used to validate the capture and analysis efficiency of the system, using only 0.13 µL sample for each bacteria analysis. To the best of our knowledge, this is the first report of integrated on-chip airborne bacteria capture and molecular identification. This device can not only be utilized for analysis of multiple samples, but also for multiple analyses of a single sample. The reported device shows great potential towards applications in environmental analysis fields. The whole operation is simple and feasible, suitable 257 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

for on-site application, e.g. at airports, subway stations and hospitals, showing potential application in environmental monitoring and public health protection (215, 216).

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Future Perspective It must be mentioned, that microfluidics is not a technique capable of detection of airborne pathogens by itself, and instead, it provides a platform in micron size to facilitate sampling and downstream bioanalysis. Integrating with other new techniques is the key to deal with the challenges. For the efficient downstream characteristic antigen analysis, the protein microarray may hold potential for high throughput immune analysis if the necessary antibodies are available. Rather than protein analysis, new PCR technique, such as LAMP are attractive techniques with high sensitivity and selectivity while requiring less assay time. More importantly, the LAMP assay amplification is carried out under isothermal conditions, and the results can be displayed as ultraviolet-visible (UV-VIS) absorption or fluorescence without the need of conventional hybridization or electrophoresis. The LAMP assay can be easily adapted to a microfluidic chip and various pathogens were already analyzed in the microfluidic LAMP assay (217). Besides the antigen analysis of immune response and detection of target nucleic acid by analysis of pathogens, MS might be another promising technique which can provide rapid response from as little as a drop of medium, perfectly matching the volume of output from a microfluidic chip. Although current MS systems for aerosol analysis are mainly focused on inorganic and organic fragment analysis, the integration of microfluidic sampling and MS will be a very promising platform for airborne pathogen analysis, because of its advantages such as rigid structure, less bio-reagent requirements, temperature control, rapid response and easy automation. Another technical issue for MS is that most MS analysis results can be affected by salt in the buffered medium and it has difficulty in analyzing sample mixtures (218). This problem could be solved by adding microfluidic chromatography after the microfluidic sampler and before loading into the MS ionizer for the airborne pathogen analysis. The most instrumental progress is driven by great need from society or commercial markets. Airborne pathogen analytical systems are directly related to public health and have huge potential applications in disease control, clinical settings, and national security. The airborne pathogen analytical instrument based on microfluidic techniques might make great contribution in this field because of its unique properties compared with traditional techniques. Nowadays, life science, environmental science and medical science are going through the process of development from macro to micro. Biological and chemical equipments were miniaturized and integrated, a series of their functions were transferred from the analysis laboratory to a Lab on a chip. This miniaturization and integration could reduce the dosage of sample and chemical regents, as well as the costs, and energy consumption. Meanwhile it could increase the sensitivity of detection, shorten the test time, and achieve high-throughput. Microfluidic chip 258 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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techniques have integrated with traditional detection methods such as ELISA, PCR and immunofluorescence assay for proanalysis. In the near future, microfluidic chips will be applied to more fields than scientific research, and will also be transformed into products with more subdivisible functions as the development of science and technology. Analytical chemistry would be liberated from the dependence on large equipment in the laboratory and become implemented in common tools used in our daily life in the future. It is believed that microfluidics will become a popular detection-method because of their low cost and ease of application. In the environmental field as well as all kinds of point of care closely related to the safety and quality of our lives, such as water quality, pesticide residues on fruits and vegetables, infectious diseases, pathogenic microorganisms and air quality shall see improvements in the detection of airborne pathogens. We human beings would live a better life with the help of microfluidic chips.

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182. Lin, S. L.; Bai, H. Y.; Lin, T. Y.; Fuh, M. R. Electrophoresis 2012, 33, 635–643. 183. Lee, J. B.; Sung, J. H. Biotechnol. J. 2013, 8, 1258–1266. 184. Park, S.; Zhang, Y.; Lin, S.; Wang, T. H.; Yang, S. Biotechnol. Adv. 2011, 29, 830–839. 185. Li, Y. B.; Su, X. L. J. Rapid Methods Autom. Microbiol. 2006, 14, 96–109. 186. Costello, D. A.; Millet, J. K.; Hsia, C. Y.; Whittaker, G. R.; Daniel, S. Biomaterials 2013, 34, 7895–7904. 187. Liu, W. M.; Li, L.; Ren, L.; Wang, J. C.; Tu, Q.; Wang, X. Q.; Wang, J. Y. Chin. J. Anal. Chem. 2012, 40, 24–31. 188. Kempisty, B.; Walczak, R.; Antosik, P.; Szczepanska, P.; Wozna, M.; Bukowska, D.; Zaorska, K.; Dziuban, J.; Jaskowski, J. M. Med. Weter. 2011, 67, 25–28. 189. Jung, W. E.; Han, J.; Choi, J. W.; Ahn, C. H. Microelectron. Eng. 2015, 132, 46–57. 190. Jiang, B.; Zheng, W. F.; Zhang, W.; Jiang, X. Y. Sci. China: Chem. 2014, 57, 356–364. 191. de la Rica, R.; Pejoux, C.; Fernandez-Sanchez, C.; Baldi, A.; Matsui, H. Small 2010, 6, 1092–1095. 192. Arata, H.; Hosokawa, K.; Maeda, M. Anal. Sci. 2014, 30, 129–135. 193. Caplin, J. D.; Granados, N. G.; James, M. R.; Montazami, R.; Hashemi, N. Adv. Healthcare Mater. 2015, 4, 1426–1450. 194. Lu, X. Q.; Li, L.; Chen, H. M.; Chen, P.; Liu, J. Prog. Biochem. Biophys. 2015, 42, 301–312. 195. Chen, L.; Zheng, X. L.; Hu, N.; Yang, J.; Luo, H. Y.; Jiang, F.; Liao, Y. J. Chin. J. Anal. Chem. 2015, 43, 300–308. 196. Zhao, L. X.; Sun, L.; Chu, X. G. TrAC, Trend Anal. Chem. 2009, 28, 404–415. 197. Xu, Z. L.; Shen, Y. D.; Beier, R. C.; Yang, J. Y.; Lei, H. T.; Wang, H.; Sun, Y. M. Anal. Chim. Acta 2009, 647, 125–136. 198. Gan, S. D.; Patel, K. R. J. Invest. Dermatol. 2013, 133, E10–E12. 199. Zhao, W.; Zhang, L.; Jing, W.; Liu, S.; Tachibana, H.; Cheng, X.; Sui, G. Biomicrofluidics 2013, 7, 011101. 200. Liu, C. C.; Qiu, X. B.; Ongagna, S.; Chen, D. F.; Chen, Z. Y.; Abrams, W. R.; Malamud, D.; Corstjens, P.; Bau, H. H. Lab Chip 2009, 9, 768–776. 201. Ikami, M.; Kawakami, A.; Kakuta, M.; Okamoto, Y.; Kaji, N.; Tokeshi, M.; Baba, Y. Lab Chip 2010, 10, 3335–3340. 202. Kong, J.; Jiang, L.; Su, X. O.; Qin, J. H.; Du, Y. G.; Lin, B. C. Lab Chip 2009, 9, 1541–1547. 203. Ko, J. H.; Cheong, W. J. Bull. Korean Chem. Soc. 2001, 22, 123–126. 204. Ko, J. H.; Huang, H. Z.; Kang, G. W.; Cheong, W. J. Bull. Korean Chem. Soc. 2005, 26, 1533–1538. 205. Asiello, P. J.; Baeumner, A. J. Lab Chip. 2011, 11, 1420–1430. 206. Shen, F.; Davydova, E. K.; Du, W. B.; Kreutz, J. E.; Piepenburg, O.; Ismagilov, R. F. Anal Chem. 2011, 83, 3533–3540. 207. Rane, T. D.; Chen, L. B.; Zec, H. C.; Wang, T. H. Lab Chip 2015, 15, 776–782. 267 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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208. Zhou, Q. J.; Wang, L.; Chen, J.; Wang, R. N.; Shi, Y. H.; Li, C. H.; Zhang, D. M.; Yan, X. J.; Zhang, Y. J. J. Microbiol. Methods 2014, 104, 26–35. 209. Ahmad, F.; Hashsham, S. A. Anal. Chim. Acta 2012, 733, 1–15. 210. Deverell, J. A.; Rodemann, T.; Smith, J. A.; Canty, A. J.; Guijt, R. M. Sens. Actuators B 2011, 155, 388–396. 211. Zucker, B. A.; Trojan, S.; Muller, W. J. Vet. Med., Ser. B 2000, 47, 37–46. 212. Mitchell, B. W.; Buhr, R. J.; Berrang, M. E.; Bailey, J. S.; Cox, N. A. Poult. Sci. 2002, 81, 49–55. 213. Jing, W.; Zhao, W.; Liu, S.; Li, L.; Tsai, C.; Fan, X.; Wu, W.; Li, J.; Yang, X.; Sui, G. Anal Chem. 2013, 85, 5255–5262. 214. Jing, W.; Jiang, X.; Zhao, W.; Liu, S.; Cheng, X.; Sui, G. Anal. Chem. 2014, 86, 5815–5821. 215. Jiang, X.; Jing W., ; Zheng, L.; Liu, S.; Wu, W.; Sui, G. Lab Chip 2014, 14, 671–676. 216. Jiang, X.; Shao, N.; Jing, W.; Tao, S.; Liu, S.; Sui, G. Talanta 2014, 122, 246–250. 217. Mori, Y.; Notomi, T. J. Infect. Chemother. 2009, 15, 62–69. 218. Ho, Y. P.; Reddy, P. M. Mass Spectrom. Rev. 2011, 30, 1203–1224.

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Chapter 14

Plasmonics in Analytical Spectroscopy

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Pedro H. B. Aoki,1,2 Carlos J. L. Constantino,1 Osvaldo N. Oliveira Jr,*,2 and Ricardo F. Aroca1,2 1Faculdade

de Ciências e Tecnologia, UNESP Univ Estadual Paulista, Presidente Prudente, 19060-080, SP, Brazil 2São Carlos Institute of Physics, University of São Paulo, CP 369, 13560-970 São Carlos, SP, Brazil *E-mail: [email protected].

Surface plasmon resonances (SPR) can be excited in thin metal films and in metal nanoparticles as localized surface plasmon resonances (LSPR). The surface plasmon is extremely sensitive to the refractive index of the environment surrounding the metal film or metal nanoparticle. This is why refractive index sensing has been the source for the development of an array of techniques harnessing the power of both SPR and LSPR. In addition, LSPR is at the center of plasmon enhanced spectroscopy with a myriad of analytical applications. Here we examine the basic physical model of plasmon enhancement, with the intention of facilitating the design of plasmonic nanostructures and experiments, taking advantage of these emerging techniques. In particular, we discuss the plasmon enhanced work based on shell-isolated nanoparticles (SHINs) in Raman scattering (SHINERS) and in fluorescence (SHINEF). Typical examples have been selected to illustrate the physical interpretation of observations.

Introduction The central element in plasmonic sensing is the surface-plasmon polariton at a single interface, which is today part of the rapidly expanding field of plasmonics (1, 2). As pointed out in Maier’s book (3), the surface plasmon resonances can be conveniently classified into two groups - surface plasmon polaritons (SPP) and the localized surface plasmon resonances: “However, history has shown that © 2015 American Chemical Society In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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despite the fact that the two main ingredients of plasmonics - surface plasmon polaritons and localized surface plasmons - have been clearly described as early as 1900, it is often far from trivial to appreciate the interlinked nature of many of the phenomena and applications of this field. This is compounded by the fact that throughout the 20th century, surface plasmon polaritons have been rediscovered in a variety of different contexts” (3). The fundamental difference is that SPP are nonradiative surface plasmon excitations, while LSPR are radiative, and both will give rise to analytical spectroscopic techniques (4). Undoubtedly, the present interest in surface plasmons has come from recent advances in the investigation of the electromagnetic properties of nanostructured materials. Surface plasmons are usually generated by the electric field component of the electromagnetic radiation and the optical response of the materials is the main source of interest for applications. However, it is also possible to excite a surface plasmon with high-energy electrons. In particular, unique optical properties of metal nanoparticles arise from the large density and susceptibility of their free electrons, and the particle plasmon mode strongly interacts with optical waves. Analytical spectroscopy harnesses the properties of surface plasmon resonances (collective charge density fluctuations) that can be excited optically for a broad range of applications. The framework for the discussion in this Chapter is provided by the classical electromagnetic (EM) theory (5). The central material property for SPP and LSPR is the dielectric function , which determines the relationship between the and the electric field (5, 6): electric displacement

The property of a material is a second rank tensor, although for an isotropic medium it reduces to a scalar (7). The frequency dependence or optical dispersion of .

makes the refractive index

also frequency dependent:

When the medium is represented by a negative refractive index,

it is called metamaterial (8). The bulk value of can be used not only to study the properties of surface plasmon resonances, but also to describe, with some corrections, the properties of LSPR in metallic nanoparticles (3, 9). Surface plasmons can be excited in metals throughout the visible region of the electromagnetic spectrum, being highly sensitive to changes in the refractive index at the interface. The latter feature makes them amenable to a wide scope of applications, especially in bio-science (10), and promotes development of new instrumentation (11). Similarly, LSPR can be excited in noble metal nanoparticles in the visible, and give rise to techniques based on refractive index sensing (4, 12). In addition, the coupling of LSPR with molecular spectroscopy has spurred development of linear and non-linear plasmon enhanced analytical techniques. The most prominent is the surface enhanced Raman scattering (SERS) (13, 14), a powerful vibrational identification technique that can achieve the limit of single molecule detection (SMD) (15). 270 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Surface enhanced fluorescence (SEF) (16, 17) is a successful analytical technique, also referred to as metal enhanced fluorescence (MEF) (18). The literature on surface plasmon resonances is overwhelming and a search in the Web of Science® leads to more than 30 thousand hits for SPR, as seen in Figure 1, while the most dynamic analytical techniques are SERS and SEF or MEF. Figure 1 is given to illustrate activity in the SPR field and the main analytical techniques. There are many excellent review articles, monographs, and the original research papers on the surface plasmon field, and the choice of contributions for this present chapter is obviously subjective and incomplete. We do apologize to colleagues whose work is not cited here; further information can be found in review articles and references cited therein. For example, Pitarke, Silkin, Chulkov, Echenique (19) and Wang, Plummer, Kempa (1) discussed the physics of ongoing research, Kawata (20) reviewed spectroscopic and imaging applications, while a discussion of LSPR sensing is offered by Anker, Hall, Lyandres (4), and Mayer, Hafner (12).

Figure 1. Retrieved results from the Web of Science for the following topics: Surface plasmon resonance (SPR); Surface plasmon polaritons (SPP); Localized surface plasmon resonance (LSPR); Surface plasmon resonance sensing (SPR sensing); Localized surface plasmon resonance sensing (LSPR sensing); Surface enhanced Raman scattering (SERS); Surface enhanced fluorescence (SEF); Metal enhanced fluorescence (MEF); Plasmonics. 271 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Surface Plasmon Resonances The Bulk Plasmon

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In metals, the electron gas can be treated as a continuous fluid with electron in Amperes/m2. density , velocity and electrical current density The dielectric function represents the linear response of the electron gas, and the electric field is the driving force, which can be assumed to obey

where the term introduces friction or damping. Equation [2] can be rewritten in terms of the current:

The plasma frequency for the electron gas is defined as Assuming an harmonic time dependence current, the solution for the equation of motion is

.

for E and for the induced

with the current density being

Using Ampere’s law, i.e. , neglecting polarization and taking the time derivative and eliminating H, the wave equation is:

272 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Using a plane wave solution

,

conducting medium is

. Now we write the expression for

for the free electrons in a metal using

the dielectric function the plasma frequency of the metal:

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the dispersion for the

The dielectric function without damping is simply the Drude formula:

, and the common limiting forms are:

In addition, since the propagation equation for the transverse mode is , then , or , which is the dispersion relation for the transverse bulk plasmon shown in Figure 2. The following asymptotic forms are derived from the propagation equation:

. Most importantly, the surface mode can be found in the frequency region . The surface plasmon resonance is localized at a planar interface between two media with frequency dependent dielectric functions . Surface plasmons from planar surfaces as well as bulk plasmons and plasmon modes of metal nanoparticles and metal films can be probed using photons or electrons. Electron energy loss spectroscopy (EELS) is used to experimentally distinguish between bulk volume plasmons, surface plasmons on flat metal films, and localized surface plasmon modes in metal nanoparticles (21). Taking advantage of the atomic-resolution imaging through scanning transmission electron microscopy (STEM) coupled with high resolution EELS, one may now obtain simultaneous morphological and spectral analysis of individual metal nanoparticles (22, 23). Probing with electrons surpasses the commonly used 273 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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method of scanning near-field optical microscope (SNOM or NSOM) to achieve spatially resolved surface plasmon detection with detailed mapping of the near field properties in individual or array of nanostructures (24).

Figure 2. Diagram for the dispersion of the transverse bulk plasmon. The straight line is the photon line in vacuum.

The complete derivation of surface plasmons (25) leads to the following important points: -

for the surface plasmon to exist, signs:

and

must have opposite

.

In particular, the frequency of the nonradiative surface plasmon at the metalvacuum interface of smooth metal surfaces is related to the wave vector by (26):

Since the right hand side of (8) must be positive, then:

274 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

The relations (8) and (9) determine the interval in which the surface plasmon resonance occurs. For large wave vectors, the surface plasmon approaches asymptotically a constant frequency , which for simple metal-vacuum interface . This relationship has been confirmed experimentally by measuring is the characteristic electron energy loss spectrum of aluminium (27). The peak corresponding to the excitation of the bulk plasmon was found at the energy

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. The lower peak, corresponding to the excited surface plasmon, . However, the optical excitation and surface was detected at plasmon resonance have a different wave vector k, and in order to transform the metal from a reflector into a reservoir of photons, an external structure is needed to provide the wave vector matching. In other words, SPR cannot be excited directly by light if the surface is perfectly smooth. “However, any surface roughness permits the surface to impart some additional momentum to the SPR so that it can couple to the radiating electromagnetic field”, as was demonstrated by Teng and Stern in their report on plasma radiation from metal grating surfaces (28). In addition, prism coupling can be used to match the momenta, as demonstrated by Otto (29) and by Kretchmann and Raether (30). Since then, this so-called attenuated total reflection (ATR) method is the basis for the traditional SPR spectroscopy, an analytical technique used in biosensing, which measures the absorption of light at resonance via total internal reflection (TIR) excitation of SPR. Typically, it uses a monochromatic laser source for illumination of a metallic gold film on a glass substrate at a specific angle of incidence, where the momentum of incident light matches that of the SPR. A broadband/white light source can also be used (11). SPR spectroscopy has a broad range of applications to monitor biological interactions (2), in sensing heavy metal ions (31), in SPR imaging (32) and detecting virus (33). Although SPR cannot be excited by direct illumination on optically flat metal surfaces, a minor enhancement could be observed for light scattering of molecules located on it, and a clear physical model for this phenomenon can be found in the work of Efrima and Metiu (34). Moreover, without surface roughness there is no SERS enhancement (14, 35). Notwithstanding the fact that metal-molecule interactions may lead to substantial spectral changes in the vibrational spectrum. However, there are genuine attempts to use SPR, excited with the Kretschmann configuration, to measure plasmon enhanced spectra. A brief review of these efforts can be found in the work of Meyer, Le Ru, and Etchegoin (36).

Properties of Localized Surface Plasmon Resonances Plasmons in Nanoparticles A clear picture of the properties of LSPR can provide a guide for the synthesis of specific nanostructures (37–39), or development of nanostructured substrates for analytical applications (40–42). It is now possible to carry detailed 275 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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studies of isolated single nanoparticles (43), though most commonly ensemble measurements are obtained with averaging over a distribution of sizes and shapes. The optical response of the nanoparticle (NP) is a function of its size, shape (geometry) and the environment (9). The same geometrical parameters govern the optical response of NP aggregates and of NP ensemble: size, shape, environment, and, at this time, the gap between NP becomes extremely important. The spectral response of LSPR can be characterized in the near-field with properties, such as the intensity and spatial distribution of the electromagnetic field enhancement, closely related to the sensing capabilities of a nanostructure, and with far-field quantities such as absorption, scattering, and extinction (44). The light interaction with arrays of nanoparticles has also been investigated (45), as new coupling resonances might make it possible to design plasmonic NPs with unexpected optical properties (46). In addition, the excitation wavelength and associated electric field polarization also play a determining role. At the origin of LSPR is the problem of a metal sphere interacting with an oscillating electromagnetic field. Gustav Mie in 1908 reported the optical by solving the Maxwell response of a sphere with dielectric function equations, whose complete development is found in textbooks (47). A power series expansion of the absorption and scattering coefficients is obtained, and their efficiencies are characterized by cross-sections, commonly given in cm2. , is the sum of the absorption and scattering The extinction cross section, cross sections: . In the limit of small particles compared to the , simple expressions are obtained wavelength, i.e., when the sphere radius (5, 47). for

where is the dielectric function of the material and is the dielectric constant of the medium. The quasi-static results (9) and (10) are valid for sub-wavelength spheres, and in the power series expansion (Mie theory) of the absorption and scattering coefficients they are set by considering only the first term. The quasi-static relationships have far reaching implications for analytical spectroscopic applications: The scattering scales with , and grows very fast with increasing particle size. The absorption cross-section varies as , and consequently absorption is more important than scattering for very small particles. The latter is illustrated in Figure 3 with Mie computations of absorption and scattering cross sections for Ag and Au spheres of . Absorption dominates for nanoparticles below 100 nm in size, and scattering overcomes absorption for larger NPs (43). 276 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 3. Absorption and scattering cross sections for Ag and Au spheres. Mie computations carry out with programs developed in reference (48).

Absorption-based detection methods are very sensitive, and large absorption cross sections permit development of new techniques, such as photothermal microscopy (49) for the detection of single absorbing nano-objects. Large absorption cross sections may be used directly to create local nanometric heating for biomedical applications (33). In the quasi-static limit, the theory of scattering and absorption of radiation by a small sphere predicts a resonant field enhancement due to a resonance in the g

factor (14, 40);

, if the Frölich condition

is satisfied, with the caveat that is a complex number, and the imaginary part plays an important role. Under these circumstances, the nanoparticle acts as an electric dipole, resonantly absorbing and scattering electromagnetic fields, placing it at the center of plasmon enhanced optical signals. Therefore, the optical , determines the region of the electromagnetic property of the material, spectrum where the effect would be observed, with distinctions induced by the environment, , the size and the shape of the nanoparticle (9). The final objective is a fundamental understanding that allows the optimization of these radiative (scattering) and nonradiative (absorption) properties for applications. Since the scattering scales with and grows very fast with increasing particle size, one should be expected to use larger nanoparticles for enhanced spectroscopy, as “the most intense SERS is really frequency-shifted elastic scattering by the metal. Under appropriate circumstances the field enhancement will scale as E4, where E is the local optical field”(Moskovits, see (51)). 277 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Field enhancement is an extraordinary phenomenon associated with metal nanoparticles excited by photons or electrons, at optical wavelengths, where local optical fields in metal nanostructures can achieve strengths that are orders of magnitude greater than that of the incident field (52). The spectral response of localized surface plasmon resonances both in the near-field and far-zone regimes is well understood in terms of classical electrodynamics (53, 54). Commonly, far-field quantities such as absorption, scattering, and extinction are measured to qualify the plasmonic response of a metallic nanoparticle; however, they do not provide information about the strength of the electromagnetic fields at the surface or at a distance from the particle surface. Therefore, near-field properties, such as the intensity and spatial distribution of the electromagnetic field enhancements, are also being measured and calculated (55). Although the exact relationship between the energy-loss map (ELM) for a given plasmon resonance and the optical near-field map is still an object of discussion, we have chosen to illustrate the near field with an energy-loss map of a plasmon resonance. Theory and experiments seem to indicate that ELM represents the electric field strength (56). The intensity of a plasmon, excited by electrons, has been mapped with high spatial resolution using EELS. This is illustrated for two gold spheres (35 nm and 25 nm) in Figure 4, adapted from reference (22). The EELS results are in full agreement with theory and optical measurements. The dependence of both near- and far-field measures on particle size has been comprehensively studied for spherical gold and silver nanoparticles, beyond the quasi-static approximation (44). It has been shown that for the peak wavelength of the primary resonance, the surface average of electric field intensity and scattering increase monotonically with particle size, while absorption does not. This size dependence of the primary resonance translates into a wavelength-dependent local field enhancement factor (EF) defined as a ratio of the normal component of the electric field on the surface of the Au nanosphere to the electric field of the incident electromagnetic wave at a point of observation. The profiles of the magnitude of the field EF as a function of incident wavelength for Au , have been reported by and Ag nanospheres, with Geshev, Klein, Witting, Dickmann and Hietschold (57), according to which even for spherical nanoparticles the field EF varies substantially with the excitation wavelength. For practical definitions of EF see also reference (58). Notably, the dipolar mode is the dominant excitation for sizes below 120 nm (59), while for larger diameters the multipolar excitations become important (47). Therefore, the most commonly used NPs for SERS have been of diameter below 100 nm, probably because for small sizes of nanospheres (20–50 nm) the electromagnetic enhancement effect can be successfully explained in terms of a simple electrostatic model. However, larger gold NPs (~100 nm) have been synthesized by various groups and their SERS activity thoroughly investigated (59–62). In fact, gold NPs as large as 120 nm are the most effective enhancers in shell–isolated nanoparticle enhanced Raman scattering (SHINERS) experiments (35). Similarly, large spherical silver NPs (~100 nm) have been tested for plasmon enhancement (63). In an early report on single molecule detection (SMD), enhanced vibrational Raman spectra from single hemoglobin molecules 278 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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attached to 100-nm-sized immobilized Ag particles were used (64). However, the extremely high field enhancement needed for SMD came from the dimer system, not from the single Ag sphere. Under appropriate illumination the spatial location between the two spheres becomes a “hotspot” for electromagnetic enhancement (41). Plasmon enhanced spectroscopic experimental results indicate that Ag or Au nanoparticle aggregation is a necessary condition for the hotspot effect to be observed. The most important part of this enhancement is due to an increase of the local electric field between Ag or Au nanoparticles (58), which is the next topic of discussion of plasmon properties.

Figure 4. A. STEM images of Au spheres. B. The corresponding EELS map of the intensity of the localized surface plasmon resonance at λ = 506 nm. Adapted with permission from reference (22). Copyright 2007 IOP Publishing.

Studies in the near-far-field (44, 53, 54) have revealed properties with important implications for analytical spectroscopy: a) The plasmon bandwidth (FWHM-full width at half maximum) increases with particle size, providing a 279 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

wider window for detection. b) The optimal wavelength for surface-enhanced spectroscopy, which depends on the near field, can be significantly red shifted compared to the commonly detected far-field extinction. c) The field strength decays exponentially in the direction perpendicular to the surface and reaches out further with increasing size of the metal core. d) The local field enhancement factor has strong wavelength dependence. In summary, for SERS and SEF, larger nanoparticles scatter strongly, dampening the LSPR and making it broader.

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Plasmon coupling “To develop the right substrate for efficient sensing of a specific adsorbate, it is therefore crucial to understand which microscopic properties determine the substrate’s plasmon resonances” (65). The Ag or Au spheres forming dimers (66) provide the simplest nanostructures leading to new resonances arising from the strong electromagnetic coupling between closely spaced particles, and these modes could be described using a plasmon hybridization model (67). A bonding interaction may be excited by incoming light polarized parallel to the interparticle axis giving rise to an attractive interaction, while the antibonding repulsive mode is excited when the electric field of the incoming light is polarized perpendicular to the interparticle axis. The modeling has continued to advance including two and three dimensional nanoparticle clusters with unique electrical, magnetic and Fano-like resonances (68). Coupled plasmon resonances are also predicted which cannot couple to plane wave incident radiation, because they do not have a net dipole, and hence, are called “dark” modes. By analogy, in the Mie series of a single nanoparticle, there is the dipole mode that couples most strongly to the incident light field, and quadrupoles, octupoles and so on, that are also localized resonances of the nanoparticle. The fabrication and characterization of metallic nanostructures exhibiting dark plasmon modes is now widely investigated (24, 68, 69). Dark modes may arise from the interaction of the bright modes of coupled nanoparticles, where the resulting collective plasmon mode has a net zero dipole moment. Dark modes can store electromagnetic energy more efficiently than bright modes, due to an inhibition of radiative losses. Correspondingly, dark plasmons have narrower spectral line widths that could lead to their use in sensors based on changes of refractive index. For practical analytical applications, when the coupled mode retains a dipole moment and can couple strongly to light, we have “bright” modes. In enhancing nanostructures, such as colloid clusters (70), the plasmonic response of closely-spaced nanoparticles produces a highly inhomogeneous local field that deviates significantly from that of the constituent nanoparticles; which is the result of strong near-field interactions, with field-intensity concentrated in hotspots. Given the high enhancement of optical signal that can be achieved with hotspots, there are extensive efforts towards engineering local field enhancements (71). The vast computational work on isolated nanoparticles (spheres and other shapes) showed EFs, which are not high enough for single-molecule detection using SERS. Very high EFs are predicted and experimentally observed only in highly localized regions, in the junctions between two particles, i.e. hotspots with EF that could be greater than ten orders of magnitude (15, 66). A table with the EF 280 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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range for non-hotspot and hotspot substrates for SERS can be found in reference (72). In any given plasmon enhancing substrate, hotspots are highly spatially localized regions exhibiting extreme field enhancements, and they are a sensitive function of the geometry and wavelength of excitation. Therefore, a substrate contains hotspots and many spatial locations with moderate enhancement factors, i.e., there exists a probability distribution of enhancement factors for a given substrate (58, 72). In practice, it is possible to define an average value of EF for a large area of the substrate (commonly the area illuminated by the microscope objective), and the site specific EF or hotspot. Hotspot computations have been carried out for several geometries. The EFs estimated are similar to those determined for the dimers. An excellent discussion and summary of results on hotspots can be found in a review by Schlucker (72). Strong EM fields may also be found at sharp edges and tips. Such hotspots can also provide single molecule sensitivity, which is the case of tip-enhanced Raman scattering (TERS) where the enhancement is achieved through the use of a sharpened metallic tip (73). So, it seems that in SERS, single molecule detection and hotspots go hand in hand. In fact, the technique developed for characterization of hotspots is based on SMD:“super-resolution imaging approach is inherently single molecule in nature, because it requires that only a single emitter be active at a given time, such that its position can be uniquely determined” (74). Experimentally, the approach requires that only a single species is active and detected within a diffraction limited spot at a given time. Spatial characterization of hotspots has been performed with super-resolution fluorescence imaging (75), and with SERS imaging at the single molecule limit (74). These techniques provide an indirect way of estimating the size of the hotspot (probably less than 15 nm (75)) based on molecular spectroscopic data at the molecule-plasmonic interface.

Surface Enhanced Raman Scattering (SERS) and Shell–Isolated Nanoparticle Enhanced Raman Scattering (SHINERS) Raman scattering is an established spectroscopic technique providing fingerprint information about molecular structure and functional groups in all classes of materials (76, 77). Despite enormous advances in technology (77), due to the low cross section in inelastic Raman scattering (as low as 10-30 cm2/molecule), it may be challenging to obtain good quality spectra in highly diluted systems. This limitation can be circumvented by plasmonic enhancement of the optical signal, as in surface-enhanced Raman scattering (SERS) or surface-enhanced resonant Raman scattering (SERRS) (51, 58). The effect is achieved placing the probe molecule close to an appropriate metallic nanostructure (mainly silver and gold). The plasmonic origin of SERS is accepted (13, 14, 78); however, when the molecule is directly adsorbed onto the metal nanoparticle, there are nuances (79) in the enhanced spectra discussed in several review articles (80, 81). The SERS phenomenon has been exploited in various analytical techniques, all of them based on nanostructures. In fact, nanostructure fabrication is a driving force in analytical applications of SERS (40, 82), and in practice, 281 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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each successful analytical application requires a finely tuned nanostructure or “SERS substrate” and optimal experimental conditions. Such techniques include Tip-Enhanced Raman Scattering (TERS) (73), electrochemical SERS, colloidal SERS (83), SHINERS (84), and many more. The introduction of shell-isolated nanoparticles (SHIN) in SHINERS by Li et al. (84) eliminates the issues related to metal-molecule interactions, particularly those due to chemical adsorption and formation of metal complexes giving rise to “first layer effects” (85). Therefore, SHINERS is a “clean” plasmonic enhancement and it is one of the main analytical techniques selected for this review, while we refer the reader to reviews on established SERS-based techniques. Furthermore, controlling the shell thickness permits to use SHINs in surface enhanced fluorescence (SHINEF) (86), which is a complementary plasmon enhanced technique. In both cases, SHINs nanoparticles may be spread as ‘smart dust’ over the probed surface, or work in the liquid phase. Literature in SHINERS has been expanding rapidly with applications to different systems, such as semiconductor materials (87), electrochemistry (88, 89), biological and food sciences (84, 90) and cancer detection (90). The key for this method is the synthesis of the core shell nanoparticles (SHINs), which may have variable core, shell, size and shape. In addition, one may design functionalized ultrathin shells capable of capturing target molecules, or fabricate two or three dimensional SHIN structures tailored for challenging applications (91). Figure 5 shows transmission electron microscope (TEM) images of three types of SHINs (nanospheres, nanorods and nanocubes).

Figure 5. TEM images recorded for SHINs of (a) spherical shape with 2 nm of silica shell, (b) nanorod with 4 nm of silica shell and (c) nanocube with 2 nm of silica shell. Adapted with permission from reference (92). Copyright 2012 Nature Publishing Group.

SHIN coatings have been designed (93, 94) to overcome the lack of compatibility of silica shells with high pH environments (87). For example, Qian, Liu, Yang and Liu (95) reported on tunable poly-(2-aminothiophenol) (PAT) shells on gold nanoparticles, which are claimed to display prominent advantages, including uniformity, chemical stability, being free of pinholes. These polymer shells were suitable for strong acid and alkaline environments with 282 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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pH ranging from 2.02 to 12.95. The goal of the work was to develop a platform for identification of trinitrotoluene (TNT) explosive, based on the formation of Meisenheimer complexes between TNT and amino groups coming from the shells. Figure 6 shows the three-step sequence of the procedure: (1) PAT assembly onto AuNP via electrostatic interactions, (2) formation of a Meisenheimer complex in the presence of TNT and (3) TNT detection via SHINERS. A clean SHINERS spectrum requires a “pinhole free” SHIN. However, a SHIN with pinholes could also be used, harnessing the high enhancement factor at the pinhole, and the chemical stability of the coated nanoparticle. This pinhole SHINERS-based approach has been implemented (96), and it may provide further opportunities for SERS applications.

Figure 6. Three-step sequence toward TNT identification: (1) PAT assemble onto AuNP via electrostatic interactions, (2) formation of a Meisenheimer complex in the presence of TNT and (3) TNT detection via SHINERS. Reproduced with permission from reference (95). Copyright 2012 The Royal Society of Chemistry.

SHINERS have also been used to characterize biological structures such as living cells. For instance, SHINs were incubated into Yeast cells and SHINERS spectra were taken directly from the cells, as shown in Figure 7a. The latter features spectra collected in different places of the same cell (I – III), the spectrum recorded from a substrate coated with SHIN (IV) and a normal Raman spectrum for yeast cells (V). A schematic representation of the SHINERS experiment on living yeast cells is depicted in Figure 7b. Taken together, these results demonstrate the use of SHINERS for in situ detection of cell components and as possible probes for the dynamics of living systems. 283 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 7. (a) SHINERS spectra (I – III) collected in different places of the same cell, spectrum recorded from a substrate coated with SHIN (IV) and a normal Raman spectrum for yeast cells (V). (b) Schematic representation of the SHINERS experiment on living yeast cells. Reproduced with permission from reference (84). Copyright 2010 Macmillan Publishers Limited.

In summary, in a typical SERS experiment the target molecule is brought to the metal surface in order to achieve the enhancement. The SERS-active substrate can be thermally evaporated thin films (97), freestanding 3D nanostructures (98), electrodeposited structures (99) or colloids with different geometries (100–102). SHINERS, similar to tip-enhanced Raman scattering (TERS), brings the SHIN (or the tip) to the target molecule to get SERS (103, 104). In both cases, one can control the distance between the target molecule and metal core (or metal tip) that acts as the Raman signal amplifier. In principle, TERS can be achieved from any surface with high spatial resolution (103). For instance, Boehme, Cialla, Richter, Roesch, Popp and Deckert (105) reported TERS on the nanoscale discrimination of protein-labeled supported lipid bilayer (SLB) structures. TERS information is based on the tip position and is characterized by lipid (orange circle) and/or protein marker modes. Therefore, TERS spectra could be attributed to different probed materials. The latter experiments are crucial for understanding lateral organization on the cell surface (106) and distribution of lipid domains (105). The total intensity of the Raman signal from the tip area is rather weak, so TERS is limited to molecules with large Raman cross-section. Besides, the 284 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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instrument is highly sophisticated and expensive, which may hamper practical applications. SHINERS is a simple technique based on plasmon enhanced electromagnetic field generated from the isolated SHINs or from nanoscale gaps (94) found among the SHIN aggregates to provide high-quality plasmon enhanced Raman spectra. One should distinguish two clearly different detection regimes in SERS spectroscopy: average SERS and Single Molecule regime of SERS detection (51). For the average SERS, it is possible to fabricate SERS substrates that will operate in a well-defined spectral region with an average enhancement factor (EF), typically 103-106, that may be used for analytical applications, including quantitative analysis (107). The EF provided by classical electrodynamics support the values in that range, and the computed magnitudes are smaller for the surface average of the nanostructure. On the other hand the experimental confirmation of the hotspot leads to Single Molecule Detection (SMD) regime of SERS (108).

Surface Enhanced Fluorescence (SEF) and Shell–Isolated Nanoparticle Enhanced Fluorescence (SHINEF) Fluorescence is based on the absorption of one photon by a fluorophore in the UV-visible range, followed by an emission of a second photon at lower energy. This highly efficient phenomenon has been widely used in optical devices, microscopy imaging, biology, medical research and diagnostics (109–111). Following light absorption, a fluorophore is usually excited to some vibrational level of a singlet excited state S1. Within a picosecond time internal conversion takes place and molecules in condensed phases relax to the lowest vibrational level of the singlet. Since fluorescence lifetimes are typically in the nanosecond range, internal conversion is usually complete prior to emission. Therefore, the fluorescence spectrum would be the mirror image of the S0 → S1 absorption, not necessarily the entire absorption spectrum. The two central properties of and the quantum yield (Q). the fluorophore are the fluorescence lifetime The quantum yield is defined in terms of the radiative decay rate , and the decay rate of non-radiative processes . Far from saturation is proportional to the quantum of the excited state, the fluorescence power yield and the absorption power . Using the absorption cross section , where is the irradiance. Statistically, the lifetime represents the life expectation of the excited molecule, i.e., the average time the molecule spends in the excited state; . Fluorescence emission is sensitive to external parameters and the local environment of the molecule. The concept was first presented in the Proceedings of the American Physical Society (1946) by Purcell (112), where the spontaneous emission was shown not to be an intrinsic property of the emitter, which could be modified by resonant coupling to the external electromagnetic environment (the Purcell 285 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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effect). Of particular interest here is the case when molecules are near metal surfaces. It requires understanding the behavior of a fluorophore (dipole) near a metal surface, as given in a detailed theoretical account of the dipole emission near interfaces (113). The latter study was prompted by experimental findings where for molecules located at “large distances from the metal surface the fluorescence lifetime oscillated as a function of the distance, while for small distances the lifetime went monotonically towards zero” (113). The changes in lifetime area due to modifications of both the radiative and non-radiative decay rates, and could result in strong quenching of fluorescence emission at the metal surface. In other words, nonradiative energy transfer to the metal surface may be an effective channel for the excited fluorophore, which can be produced by electronic coupling of the molecular orbitals with the extended band structure of the metallic electrons in the metal substrate. The latter leads to a connection between the frequency dependence of the energy transfer and the surface plasmon modes. Here it is appropriate to separate the surface plasmon resonance excited on flat metallic surfaces (usually using Otto and Kretschmann configurations[109]) and the corresponding analytical techniques (114), from the LSPR on nanometallic structures that supports collective electron excitations producing high local-field intensities (3, 115). Plasmon enhanced fluorescence (PEF), based on LSPR, was born under the tree of surface enhanced Raman scattering, and was termed surface enhanced fluorescence (SEF) (16, 17). In 2002 the physical phenomenon was renamed as metal enhanced fluorescence (MEF) (18). The strong local-fields near metallic nanostructures can induce changes in the light emission properties of emitters in close proximity. Assuming that the emitter quantum structure is preserved, the plasmonic structure can increase the optical absorption rate; it may alter the radiative and nonradiative decay rates and emission directionality. is proportional to , where p is the The fluorescence power dipole moment, and E is the enhanced optical electric field vector at the emitter position, leading to an increased intensity. The electromagnetic field surrounding the metallic nanostructures enhances the molecule absorption and, therefore, increases the quantum yield. However, the benefits of enhanced fluorescence are only observed for emitters located at a finite distance from the metal nanostructure (17). There are, thus, two competing effects: enhancement due to the local electromagnetic environment and nonradiative decay due to radiationless energy transfer that produces quenching. When excited molecules located near plasmonic nanostructures (nanoparticles or aggregates) can have efficient nearfield coupling to localized surface plasmons, large enhancements are obtained, especially if the molecular emission frequency matches the LSPR. Notably, once LSPR is excited, it can either decay nonradiatively or, most importantly, reradiate into free space. The latter is the analog of an optical antenna and is the main source of plasmon enhancement (4). In summary, the task for analytical applications is to design plasmonic nanostructures that can reradiate, increasing the optical signal, via non-radiative coupling to an excited molecule (placed at an optimum distance) with the localized surface plasmon of the metal (116). Yet, it is necessary to review the existing data 286 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

to appreciate the effect of contributing factors to the observed enhanced signal and help the experimental planning. To help the discussion, a detailed collection of reported PEF data is given in the Appendix.

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Distance Dependence A key parameter to get the most out of PEF is the thickness of the spacer layer between the fluorophore and the metal nanostructure to provide a critical distance that avoid quenching and maximize the enhancement. This metal-molecule distance dependence has been experimentally demonstrated in independent studies (117–123). Consequently, the design of a particular plasmonic substrate for SEF must include the optimization of the spacer layer, determining the nanostructure–molecule separation (124). SHINs originally developed for SHINERS (84) can be tuned for PEF, as an increase in shell thickness would provide the fluorophore with a continuous transition from fluorescence quenching (SHINERS) to fluorescence enhancement (86). This was accomplished by Guerrero and Aroca (86) in SHINEF for Langmuir–Blodgett monolayers, as depicted in Figure 8. SHINs constitute only one of the many avenues to preparation of plasmonic nanoparticles found in the literature, and enthusiastic research is ongoing in this direction (125).

Figure 8. Plasmon-enhanced fluorescence activated with Au nanoparticles coated with silica (SHINEF) over the analyzed surface (LB monolayer in this case). Reproduced with permission from reference (86). Copyright 2010 Wiley-VCH Verlag GmbH & Co.

287 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Hotspots and the Enhancement Factor (EF) PEF enhancement factors typically range from 2 to 100, which is modest compared to SERS, as indicated in the data provided in the Appendix. This is a consequence of PEF only benefiting from the enhanced local field, leading to a

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square dependence, , whereas SERS is based on the amplification of both incident and scattered field with an enhancement proportional to

, where E0 is the incident field and Eloc is the enhanced local field (14, 126, 127). The advent of SHINs allows one to tune the experimental conditions to simultaneously record SHINERS and SHINEF for a low quantum yield molecule, as illustrated in Figure 9. Experimental results for the same molecule confirm that PEF is proportional to |E|2 while SERS is given by |E|4.

Figure 9. Combined SHINERS and SHINEF spectra for an aqueous solution of crystal violet (CV) and comparison with normal Raman and fluorescence spectra. Reproduced with permission from reference (126). Copyright 2012 Wiley-VCH Verlag GmbH & Co.

The experiments depicted in Figure 9 were performed in solution, providing reproducible average values of the local field enhancement (126). Strongly confined fields between metallic nanostructures (hotspots), that can alter the light emission properties of nearby fluorophores, have been reported to yield an EF of 4.5 x106, the highest EF published so far for PEF (128). A large EF of 1340 has also been measured for single molecule fluorescence using gold bowties 288 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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(129). Similarly to SERS, reproducible average PEF enhancement factors can be attained for certain PEF substrates with quantitative analytical applications, while higher (but variable) EF can be achieved at hotspots. The contribution from hotspots to EF has also been shown in aggregation studies (130), including the use of SHINs (131). The near field response of a gold SHIN dimer embedded in water for different gap sizes was calculated. Figure 10 shows a plot of the near field intensity enhancement in the center of the gap in a dimer, formed from Au-SHINs of 50nm core diameter and 10nm SiO2 shell, illuminated at normal incidence and polarized along the dimer axis. As the gap is reduced, the interaction contributes to the enhancement of the near field intensity, in full agreement with observations. In addition, both the scattering and extinction cross sections increase and get spectrally red-shifted. This expected phenomenon is due to the coherent interaction of the plasmonic dipolar resonance excited in both SHINs.

Figure 10. SHIN dimer is illuminated at normal incidence and polarized along the dimer axis and near field intensity enhancement in the centre of the gap (point A). Reproduced with permission from reference (131). Copyright 2014 American Chemical Society.

For analytical applications, the main task may again be defined as the fabrication of a substrate, an optimized arrangement of suitable building blocks, leading to specific optical properties of the assembled nanostructure. There are various chemical methods to synthesize nanoparticles of different shapes and surface modifications, as well as approaches to assemble them and create predefined enhancing substrates (132). Therefore, surface coating of nanoparticles (mainly Ag and Au) with different sizes, shapes and materials, with different plasmon absorptions (126, 133), allows for many applications of PEF, where the core shape, the shell thickness and functionalization (134) can be tuned for specific tasks. 289 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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The Molecular Quantum Yield PEF has been measured for both high and low quantum yield molecules, as demonstrated in the data collected in the Appendix. However, some reports show a tendency to dismiss SEF or MEF for high quantum yield chromophores: “The enhancement effect is most significant for relatively weak and diluted absorbers and rather inefficient emitters that are placed in close proximity to the metal nanoparticles” (135). The experimental results show that high QY fluorophores can provide large fluorescence EFs. For instance, Gartia, Eichorst, Clegg and Liu (136) measured PEF for high quantum yield (HQY) fluorophores [R6G (Q0=0.9), and for fluorescein (Q0=0.95), together with three additional fluorophores of lower Q0. The unmodified Q0, the modified quantum yield Qmod with the estimated EFs are reproduced in Table 1, where the highest enhancement (EF=100) is found for the fluorophore with the highest Q0. From the data collected in the Appendix it can be concluded that there is no clear correlation between Q0 and EF. However, for a particular substrate such correlation has been reported (130).

Table 1. Unmodified and Modified Quantum Yields, in Addition to the Enhancement Factor for Two Fluorophores Q0

Qmod

EF

R6G

0.90

0.992

20.5

Fluorescein

0.95

0.991

100

Fluorophore

For a brief discussion of the role of the quantum yield, we can use the simplified approach to PEF enhancement presented by Bardhan, Grady and Halas (120), where the intensity ratio is given as the result of two contributions: . The local field enhancement has already been discussed. The second factor results from an increase in the radiative decay rate of the molecule leading to an enhancement of the quantum yield , with an EF equal to , which alone cannot account for the very large EF values measured experimentally. The approach neglects changes in the nonradiative decay that assumes a molecule-metal spacing greater than the critical distance. Within this approximation, a fluorophore with intrinsically high quantum yield will be mainly enhanced by . Therefore, tuning the plasmonic structure, particularly by increasing the hotspot distribution density in the PEF substrate, would further increase EF, as in aggregated nanostructures. The data collected in the Appendix support the assumption that the local field is the main source of enhancement. 290 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 11. Dual modal nanoprobe (DMNP) representation, TEM microscopy of the designed DMNP and outline of cancer marker detection using fluorescence-SERS dual DMNPs. Reproduced with permission from reference (141). Copyright 2012 The Royal Society of Chemistry.

Spectral Profile Modification An important consequence of the enhancement is the experimentally observed spectral profile modification [137] (SPM) of the fluorescence spectrum under PEF conditions. LSPR can affect not only the fluorescence intensity (enhancement or quenching) but also its spectral shape. The profile of the original fluorescence spectrum may be altered by the re-radiation of the plasmonic 291 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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nanostructure, as demonstrated by several groups (133, 137, 138), with the PEF spectrum being different from the original fluorescence spectrum. The role of nanostructure scattering (Mie scattering) is well established for both SERS and PEF (58). For example, Dragan, Mali and Geddes (139) summarize their findings stating: “the wavelength-dependent metal-enhanced fluorescence (MEF spectrum) correlates well with the plasmon specific scattering spectrum”. The latter is consistent with observation of large enhancements, particularly when the molecular emission frequency matches the LSPR. Similarly, maximum SERS enhancements are obtained when the excitation and Raman emission are within the plasmon wavelengths, as predicted by the plasmon enhancement theory (140). SPM is probably a common occurrence in SERS and PEF; however, since LSPR is very broad, the effect is more likely to be appreciated in fluorescence experiments. In summary, the fingerprints of the plasmon scattering may induce changes in PEF with respect to the original fluorescence spectral profile. We close with an example from fluorescence imaging that integrates in the plasmonic structure the basic ideas previously discussed. First, readily available fluorescent probes display relatively weak emission and are rapidly photobleached. This limitation was overcome by Lee, Chon, Yoon, Lee, Chang, Lim and Choo (141) with a highly sensitive optical imaging based on SERS and fluorescence combined in a dual modal nanoprobe (DMNP). The surface of Au nanoparticles (~40 nm diameter) was first coated with Raman reporter molecules (MGITC and Rubpy). Subsequent encapsulation with a silica shell was performed to prevent the release of the reporter molecules. The thickness of the silica shell was tuned to achieve the maximum fluorescence enhancement of the covalently attached fluorescent probes (fluorescent ITC-modified with FITC or RuITC). A final silica shell was added to minimize nanoparticle aggregation, avoid direct contact with the probed surface and to protect the fluorescent dye. Finally, the DMNP was attached to specific antibodies for targeting and imaging specific breast cancer markers in living cells, as shown by the outline in Figure 11. DMNP allows one to collect fluorescence as a fast track tool for cancer marker recognition and SERS as an accurate tool to determine the signature of specific molecular interaction. Moreover, the implementation of DMNP to early cancer diagnosis has shown to be straightforward.

Summary LSPR, which are resonantly driven oscillations in metal nanostructures, can be induced at specific optical frequencies producing a very strong charge displacement and local field concentrations. The characterization and assignment of these resonances is one of central tasks of plasmonics (3), and epitomize the starting point for analytical applications. The challenge for experimentalists is clear. The fabrication of nanostructures (nanoscale architectures) with increased complexity that provide the most useful optical properties for analytical techniques (142). The synthesis of nanoparticles and assembly of nanostructures with rationally designed dimensions can be realized using the “bottom-up” or the “top-down” approaches (125). 292 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Plasmonic nanostructures, under appropriate conditions, will lower the limit of detectable concentrations for materials, opening the field of ultrasensitive analysis using plasmon enhanced spectroscopy. When the target molecule is a fluorophore, plasmonic nanostructures are able to modify its radiative and non-radiative decay rates, changing both the fluorescence lifetime and quantum yield. In addition, an increased energy transfer may affect molecular photostability that can be used to increase the signal level when imaging fluorescent molecules. Finally, the introduction of shell-isolated nanoparticles has empowered the development and rapid growth of two analytical techniques: SHINERS and enhanced fluorescence (SHINEF). In fact, many new applications will flourish by taking advantage of the complementary nature of SERS-fluorescence techniques, a dual mode approach that takes advantage of both enhanced inelastic scattering and fluorescence.

Acknowledgments This work was supported by FAPESP, CNPq, Science Without Borders Program and nBioNet network (CAPES), Brazil.

Appendix. Reported Enhancement Factors in Plasmon-Enhanced Fluorescence Table A1 Molecule

Quantum yield

λexc

λemi

Average EF

Basic fuchsin

0.02

514.5

600

200

Indocyanine green (ICG)

0.012

785

850

2970

TPQDI: N,N0bis(2,6-diisopropylphenyl)1,6,11,16-tetra-[4(1,1,3,3-tetramethylbutyl)phenoxy]quaterrylene3,4:13,14-bis(dicarboximide)

0.025

780

820

Cy5 labeled oligonucleotide

Hot spot

Metal Ag (17)

4.5 x106

Au (128)

1340

Au (129)

635

15

Ag (143)

Atto 540Q-labeled DNA

1.6x10-3

532

740

Ag (130)

CY3-labeled DNA

0.08

532

37

Ag (130)

R6G-labeled DNA

0.17

532

17

Ag (130) Continued on next page.

293 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Table A1. (Continued)

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Molecule

Quantum yield

λexc

λemi

Average EF

Hot spot

Metal

Fluo-3, 1,2-bis(2-aminophenoxyethane) N,N,N_,N_tetraacetic acid

0.15

473

518

400

Ag (144)

AzoPTCD

~ 0.98

785

813

50

Ag (145)

Rhodamine B (ethanol)

0.49 (146)

540

581

260

Ag (147)

SiC nanocrystals

0.17 (148)

360

432

176

Ag (149)

A655-DNA

0.13

650

690

170

Ag (150)

470

640

189

Au (151)

627

7-12

Lanthanide ions; Pr+3 Rhodamine B (ethanol)

0.49 (146)

554

Rhodamine 6G (R6G)

0.95

633

Chromeo 642

0.17 (154)

642

676

6-(N-(7-nitrobenz2-oxa-1,3-diazol-4yl)amino)hexanoate (NBD) (156)

0.45

476

537

100

Ag (157)

Y3N@C80 Fullerene

0.02-0.05

633

710

~100

Au (158)

Fluorescein

0.95

440

100

Ag (136)

Octadecylrhodamine B (R18)

~ 0.5

514.5

575

94

Ag (126)

785

~800

241

IRDye 800CWlabeled streptavidin

164

125

Ag (152) Au (153)

136

2530

Ag (75, 155)

Ag (159)

AF790-SA

0.04

780

820

83

Au (160)

AF750-SA

0.12

740

800

10

Au (160)

AF488-SA

0.92

480

520

7.8

Au (160)

Indocyanine green (ICG)

0.012

785

850

50

Au (161)

Perylene

0.94

~500

50

Ag (162)

Tryptophan

0.12 (163)

270

339

40

Ag (164)

Terrylene

~1

532

~600

20

Au (165) Continued on next page.

294 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Table A1. (Continued)

Publication Date (Web): December 8, 2015 | doi: 10.1021/bk-2015-1215.ch014

Molecule

Quantum yield

λexc

λemi

Average EF

Hot spot

Metal

Fluorescein (FITC)

0.92

488

519

15

Ag (166)

Single walled carbon nanotubes (SWNTs)

0.01 – 0.03

658

1300

>10

Au (167)

Nile Blue

~1

637

661

10

Au, Ag (168)

IR800 conjugated with HSA

0.74

780

804

40

Au (169)

Crystal violet

5x10-5

514.5

740

59

Ag (131)

Eosin-Y

0.32

514.5

550

9

Au (131)

F8BT/MEH-PPV polymer

0.155

532

633

4

Au (170)

Rose Bengal

0.1

540

550

4

Au (171)

Eosin Y

0.32

540

550

2

Au (171)

CY3-labeled DNA

0.08

532

570

3

Ag (172)

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150. Gill, R.; Tian, L. J.; Somerville, W. R. C.; Le Ru, E. C.; van Amerongen, H.; Subramaniam, V. J. Phys. Chem. C 2012, 116 (31), 16687–16693. 151. Zhuo, S.; Shao, M.; Xu, H.; Chen, T.; Ma, D. D. D.; Lee, S.-T. J. Mater. Sci.: Mater. Electron. 2013, 24, 324–330. 152. Roth, A.; Shtoyko, T.; Taylor, B. K.; Pravitasari, A.; Gryczynski, Z.; Matveeva, E. G.; Gryczynski, I.; Chang, I. F. J. Chem. Educ. 2009, 86, 715–718. 153. Zhang, Z.; Yang, P.; Xu, H.; Zheng, H. J. Appl. Phys. 2013, 113, 033102/ 1–033102/5. 154. Wetzl, B.; Gruber, M.; Oswald, B.; Durkop, A.; Weidgans, B.; Probst, M.; Wolfbeis, O. S. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 2003, 793, 83–92. 155. Weber, M. L.; Willets, K. A. MRS Bull. 2012, 37, 745–751. 156. Rohacova, J.; Marin, M. L.; Martinez-Romero, A.; Diaz, L.; O’Connor, J. E.; Gomez-Lechon, M. J.; Donato, M. T.; Castell, J. V.; Miranda, M. A. ChemMedChem 2009, 4 (3), 466–472. 157. Sokolov, K.; Chumanov, G.; Cotton, T. M. Anal. Chem. 1998, 70, 3898–3905. 158. Bharadwaj, P.; Novotny, L. J. Phys. Chem. C 2010, 114 (16), 7444–7447. 159. Furtaw, M. D.; Anderson, J. P.; Middendorf, L. R.; Bashford, G. R. Plasmonics 2014, 9 (1), 27–34. 160. Xie, F.; Centeno, A.; Ryan, M. R.; Riley, D. J.; Alford, N. M. J. Mater. Chem. B 2013, 1 (4), 536–543. 161. Tam, F.; Goodrich, G. P.; Johnson, B. R.; Halas, N. J. Nano Lett. 2007, 7 (2), 496–501. 162. Zhang, Y. X.; Aslan, K.; Previte, M. J. R.; Geddes, C. D. Appl. Phys. Lett. 2007, 90 (5), 053107/1–053107/3. 163. Chen, R. F. J. Res. Natl. Bur. Stand., Sect. C 1972, A 76 (6), 593–606. 164. Caires, A. R. L.; Costa, L. R.; Fernandes, J. Cent. Eur. J. Chem. 2013, 11, 111–115. 165. Kuhn, S.; Hakanson, U.; Rogobete, L.; Sandoghdar, V. Phys. Rev. Lett. 2006, 97 (1), 017402/1–017402/4. 166. Zhang, R.; Wang, Z.; Song, C.; Yang, J.; Sadaf, A.; Cui, Y. J. Fluoresc. 2013, 23, 71–77. 167. Hong, G. S.; Tabakman, S. M.; Welsher, K.; Wang, H. L.; Wang, X. R.; Dai, H. J. J. Am. Chem. Soc. 2010, 132 (45), 15920–15923. 168. Bharadwaj, P.; Anger, P.; Novotny, L. Nanotechnology 2007, 18 (4), 044017/ 1–044017/5. 169. Bardhan, R.; Grady, N. K.; Cole, J. R.; Joshi, A.; Halas, N. J. ACS Nano 2009, 3 (3), 744–752. 170. Heydari, E.; Pastoriza-Santos, I.; Flehr, R.; Liz-Marzan, L. M.; Stumpe, J. J. Phys. Chem. C 2013, 117 (32), 16577–16583. 171. Kim, J.; Dantelle, G.; Revaux, A.; Berard, M.; Huignard, A.; Gacoin, T.; Boilot, J. P. Langmuir 2010, 26 (11), 8842–8849. 172. Wang, Y.; Li, Z.; Li, H.; Vuki, M.; Xu, D.; Chen, H.-Y. Biosens. Bioelectron. 2012, 32, 76–81.

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Chapter 15

Quantitative Comparative Techniques of Infrared Spectra of a Thin Film Publication Date (Web): December 8, 2015 | doi: 10.1021/bk-2015-1215.ch015

Takeshi Hasegawa* Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan *E-mail: [email protected].

Fundamentals of infrared (IR) spectroscopy for analysis of molecular adsorbates or a thin film on a flat surface are described in terms of surface spectroscopy. To fully understand IR spectra of a thin film deposited on a surface, theoretical backgrounds (such as both quantum mechanics and electrodynamics) of spectroscopy are necessary. Especially when an interface is taken into account, the electrodynamic approach is inevitable. In recent days, the ATR technique has spread over a wide range of research fields. In this situation, a very important matter must be known that the band position and relative band intensity of an ATR spectrum cannot be compared to those of a spectrum obtained by another technique such as the transmission and reflection-absorption (RA) spectrometries. Without an appropriate knowledge on this matter, incorrect discussion is generated, which makes the thin-film analysis highly confused. In this chapter, quantitative comparison of spectra is described by using some application studies.

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Infrared Spectroscopy for Surface Adsorbates Fourier transform infrared (FT-IR) spectrometry (1–3) is one of the most common spectroscopic tools spread over a wide variety of fields in chemistry. Unfortunately, the power of FT-IR is not fully recognized in many cases, even if much chemical information is necessary for chemical discussion. This situation is quite discouraging. FT-IR can provide more information than the primary chemical structure as well as molecular interaction via a quantitative analysis of the IR spectra. Absorption spectra provide molecular information through the band location (ordinate) and intensity (abscissa). Molecular vibration is measured by the interaction of a vibrating dipole moment with the oscillating electric field of IR ray (3, 4). This physical process is theorized in two manners of quantum mechanics and electrodynamics. The quantum-mechanical treatment yields a very important conclusion of “Fermi’s golden rule,” which is the starting point of discussing absorption spectroscopy, and it also provides an important concept of polarization analysis of “molecular orientation.” Oriented molecules are often found in a thin film, and FT-IR is quite suitable for analyzing the thin film due to the uniquely high sensitivity. To analyze a thin film, however, only the quantum-mechanics approach is insufficient, and another theoretical framework on electrodynamics is definitely necessary, since “an interface” comes up in the thin-film analysis (5). In this chapter, through some recent analytical examples, the spectroscopic concepts on the two approaches are described, so that IR spectra obtained by different techniques can directly be compared in a quantitative manner.

Fundamentals on Quantum Mechanics IR spectroscopy is one of the absorption spectroscopies that rely on the absolute principle of Fermi’s golden rule. This rule is a conclusion deduced from a physical model that a dipole along a chemical bond (or a chemical group) at a steady state is perturbed by oscillating electric field of light, which is described in an ordinary procedure using Schrödinger equation. The finally obtained golden rule is known as:

The left-hand side corresponds to peak area or absorbance, which is proportional to the squared transfer integral, , where the wave functions indexed by j and k respectively correspond to the initial and final . Dirac’s delta states of the transition induced by the perturbation operator, function means the conservative energy absorption between the two quantized energy levels. In the case of molecular vibrations observed by IR spectroscopy, 304 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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can be employed with a good approximation. Here, p(= er) and E are the dipole moment and the electric field applied to the moment, respectively. As a result, the transfer integral can be a dot product of the transition moment and the electric field (Eq. (1)).

Since the wavelength of the IR light is generally much longer than the length of the dipole, r, E can be treated as a constant value in the integral, and it can be put outside the integral. Fortunately, IR spectroscopy can be recognized to measure a vibrational transition from the ground state only. In this situation, the group theory tells us that the transition moment has the same direction as that of the normal mode. Therefore, Eq. (1) indicates that the absorption intensity becomes greater when the direction of the normal mode is parallel to the electric field; whereas no peak appears when they are orthogonal to each other. This is the fundamental of the orientation analysis using IR spectroscopy.

Fundamentals on Electrodynamics Eq. (1) provides a very simple principle for the orientation analysis of a molecule in vacuum. To quantitatively analyze the spectra of molecules adsorbed on a surface, however, only quantum mechanics is very insufficient, and electrodynamics is necessary to evaluate the electric field near the surface (4, 5). This is true of not only the orientation analysis, but all the analyses of the absorption spectra measured at an interface. The simplest example is presented by transmission measurements with the normal incidence of a thin film deposited on an IR transparent substrate. The absorbance, ATr, is defined as:

Here, Isample and IBG are single-beam spectra of the sample and background (BG) measurements, respectively. The BG spectrum can be interpreted as the apparatus function. On looking at this definition, one may consider that the chemical/physical information of the substrate is readily canceled to leave the information of the thin film only. Unfortunately, however, this expectation is denied when referring to Table 1.

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Table 1. Transmission Absorbance, ATr, of an LB Film on an IR Transparent Substrate Calculated at 2900 cm-1. n3 Is the Refractive Index of the Substrate (Phase 3). substrate

n3

ATr/10-3

ATr/ATr(air)

air

1.000

4.14

1.00

CaF2

1.415

3.43

0.83

KRS-5

2.380

2.46

0.59

ZnSe

2.455

2.40

0.58

Si

3.429

1.88

0.45

Ge

4.034

1.65

0.40

When an identical thin film is deposited on a different substrate, the absorbance (peak intensity) varies a lot. For example, the film on CaF2 exhibits a very strong peak more than twice that on Ge. This straightforwardly implies that the influence of the substrate still remains even after the division by the BG spectrum. To deduce the analytical expression of the transmission measurements, Isample and IBG are theorized by using the three- and two-phase optical models (Figure 1), respectively, and boundary conditions of the electric and magnetic fields are considered. As a result, the following equation (4) is obtained using a thin-film approximation

:

Here, d2 is the film thickness, and εx,2 is the surface-parallel component of the complex electric permittivity of the film. Here, no details is described for mj, but Eq. (2) apparently tells us that the absorbance is influenced by the optical parameter of the phase 3 (substrate). Another important point is that the transmission spectrum relies on Im(εx,2) that is called TO (transverse optic) energy-loss function.

Figure 1. A schematic of IR transmission measurements of a thin film on an IR transparent substrate. 306 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Note that the TO-driven spectra cannot be compared to a spectrum measured by the KBr pellet technique. The KBr-pellet spectra are free from the influence of an optical interface. As a result, the absorbance, AKBr, is physically represented as:

This is another expression of Beer’s law, and the KBr spectrum provides an “α-spectrum.” Here, d is the path length, and n″ is the imaginary part of the refractive index (n = n′ + in″)of the absorbing material (i.e., sample). Since the relationship of ε = n2 = (n′2 − n″2) + i2n′n″ holds, the function of Im(ε) = 2n′n″ is influenced not only by n″, but also by n′. In other words, IR bands in the transmission spectrum of a “thin film” are distorted by the dispersion curve of n′ with respect to the corresponding KBr spectrum. This explicitly shows an important character of “surface spectroscopy.”

Surface Selection Rules for Thin-Film Analysis Surface spectroscopy is a fundamental concept, which should be discriminated from normal spectroscopy on a bulk sample with no interface. Once the relationship between the surface and normal spectroscopy is understood, molecular adsorbates and a thin film can readily be discussed to reveal the molecular conformation, packing and orientation. In the previous section, the transmission spectra of a thin film depend on the TO energy-loss function of the surface-parallel component of the complex electric permittivity of the film. In short, this means that the surface parallel molecular vibrations appear in the transmission spectra. This rule is called “surface selection rule (SSR)” of transmission spectrometry. In other words, the surfaceperpendicular component is missed in the transmission spectra.

Figure 2. A schematic of IR RA measurements of a thin film on a metallic substrate using the p-polarized IR ray. To measure the surface-perpendicular molecular vibrations, the film should be put on a metallic surface, which is subjected to a grazing-angle reflection measurement. This grazing-angle technique on a metallic surface is called “reflection-absorption (RA)” spectrometry (Figure 2) (1, 4). The grazing angle is often set to near 80° from the surface normal. Note that reflection measurements on a “nonmetallic surface” are categorized into the “external reflection (ER)” 307 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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spectrometry (1, 4), which is strictly distinguished from the RA technique, since the analytical expressions are totally different from each other (4). The analytical expression of the RA technique is known as (4):

Here, the incident light comes through the air phase with the angle of incidence of θ1. The imaginary part of the inversed electric permittivity is called the LO energy-loss function, which determines the band shape of the RA spectrum. Although the TO and LO energy-loss functions look largely different from each other, the simulated spectra always yield very similar shapes, but they accompany some “band shift” especially for a strong absorption band. This claims an important point that the band position of an RA spectrum cannot directly be compared to a transmission spectrum, even if the analyte is a common compound. Of course, it cannot be compared to a KBr pellet spectrum, either. Eq. (4) indicates that the surface-normal (z) component of the molecular vibrations is selectively observed in the RA spectra, which is called the SSR of the RA spectrometry. In this manner, the SSRs of the transmission and RA spectrometries are complimentary with each other. In other words, the orientation of a transition moment is readily revealed by looking at the band appeared in the two spectra. Figure 3 presents IR RA and transmission spectra of a 7-monolayer LangmuirBlodgett (LB) film (6). Since the cadmium stearate is known to form a highly stable monolayer independent of the substrate, it is useful to compare the two techniques. We note that the anti-symmetric and symmetric CH2 stretching vibration (νa(CH2) and νs(CH2), respectively) bands appear in the transmission spectrum at 2916 and 2850 cm-1, respectively. These “band locations” respond to the “molecular conformation,” and the two values are typically found for the all-trans zigzag conformation of the alkyl chain (7). In other words, the chain should have a straight-line structure, which suggests that the molecules are highly packed. This is supported by the CH2 bending vibration (δ(CH2)) band, which is split into doublet at 1472 and 1462 cm-1. This splitting (factor-group splitting, or Davydov splitting (6, 8)) is known to be a good marker of the orthorhombic subcell packing of the alkyl chains. With the conformation and the crystallinity, the molecular orientation is expected to be nearly perpendicular to the surface. In fact, the νa(CH2) and νs(CH2) bands are both strong in the transmission spectrum; whereas they are largely suppressed in the RA spectrum. Judging from the SSRs of the two techniques, they consistently indicate that the transition moments are both nearly parallel to the substrate, which further indicates that the molecular axis is nearly perpendicular to the surface. This is supported by the band progression that is a coupled oscillation of the synchronous CH2 wagging (ωCH2) modes along the alkyl chain (9). The band progression is a proof of a highly ordered molecular configuration, and the appearance in the “RA spectrum only” indicates that the alkyl chain is nearly perpendicular to the surface. In this manner, the vibrational modes relating to the alkyl chain are all discussed in a 308 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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consistent manner, and a highly plausible model is reached. The discussion based on SSRs is a great benefit of using IR spectroscopy.

Figure 3. IR RA and transmission spectra of a 7-monolayer LB film of cadmium stearate deposited on a silver substrate (single side) and a ZnSe one (both sides), respectively. Adapted with permission from Reference (6). Copyright 1990 the American Chemical Society. In a similar manner, the terminal carboxylic group adds more information to the chemical structure. Since the group is fully anionized by the interaction with the cadmium cation, the C=O stretching vibration band disappears, and instead two bands of the anti-symmetric and symmetric COO- stretching vibration (νa(COO-) and νs(COO-), respectively) bands appear at 1543 and 1433 cm-1, respectively. The νs(COO-) band appears dominantly in the RA spectrum, while the νa(COO-) strongly appears in the transmission spectrum, which consistently indicates that the COO- group stands nearly perpendicular to the surface. In this manner, a chemical image of the molecule in the LB film has been obtained. To quantitatively discuss the orientation angle, a quantitative comparison of transmission and RA spectrometries is necessary, which requires the refractive indices of the film and the IR transparent substrate. This strategy is for obtaining the ratio of the surface-parallel and –perpendicular components of the electric permittivity, so that the molecular orientation would be calculated. In practice, the two components have different refractive index of the sample, which is close 309 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

to impossible to obtain without using the orientation angle of the molecule. Therefore, the quantitative analysis is possible only when the optical parameters are fortunately ready at hand, and the dispersion of the refractive index can be ignored.

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ATR Spectrometry Attenuated total reflection (ATR) spectrometry has rapidly spread in a recent decade, which is overwhelming the conventional KBr pellet technique. Note that, however, a KBr spectrum cannot be replaced by an ATR spectrum as shown later. ATR spectrometry is one of the internal reflection techniques: IR light traveling in a higher refractive-index matter reflects at an interface face to a lower refractive-index matter.

Figure 4. Optical scheme of the ATR spectrometry.

When the angle of incidence, , is greater than the critical angle, , the IR light is totally reflected at the interface (Figure 4), but only the electric field of the light is penetrated across the interface into the sample. Since the electric field oscillation is necessary to measure the absorption by the sample as presented in Eq. (1), this technique enables us to measure the sample near the interface only. The penetration depth of the electric field into the sample is about one tenth of the wavelength, which results in ca. 1 μm or less. An ATR spectrum has a component formulated as (4):

Here, and A is referred to literature (4). As found in the equation, ATR spectra depend on both TO and LO energy-loss functions. This is the reason an ATR spectrum has a distorted band shape and position in comparison to the corresponding KBr spectrum (Eq. (3)). Therefore, if we find a different band position in an ATR spectrum from the KBr spectrum, we should not conclude that the molecules are in a different chemical situation. As described in the next section, an appropriate conversion should be applied to the ATR spectrum to have a pure TO and LO function spectra for direct comparison to the transmission and RA spectra, respectively. This is particularly important for analyzing a strongly absorbing band, which is commonly found in perfluoroalkyl compounds. 310 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Application Study Using a Combined Technique of RA and ATR Spectrometries Here, an example study using IR RA spectrometry is described. A thin film of a perfluoroalkyl compound is employed as the analyte. A perfluoroalkyl (Rf) group is a fluorine-substituted alkyl group on all hydrogen atoms, and the character of an Rf group is often recognized on an extended line of the corresponding normal alkyl group. The material characters of an Rf compound is, however, totally different from those of a normal hydrocarbon. The uniquely high melting point (327°C for Teflon) (10) is, for example, a representative characteristic, which is not found in a normal hydrocarbon material. This Rf-specific property is attributed to the spontaneous molecular aggregation property of the Rf groups, which depends on the number of CF2 groups (Rf length) (11, 12). To discuss the molecular aggregation, IR spectroscopy works powerfully, since the aggregation influences the molecular conformation. Speaking of van der Waals force, one may consider London’s dispersive force (13). In fact, the dispersive force is the main factor accounting for the hydrophobic interaction between the normal hydrocarbons. Of Rf compounds, however, this concept is not true (11). The major factor of the van der Waals force between Rf groups is not the dispersive force, but the “orientation (or dipole-dipole interactive) force.” As a result of the spontaneous molecular aggregation due to the orientation force, the Rf groups are put together to generate a two-dimensional molecular aggregate, i.e., a monolayer film (12). In Figure 5, a myristic acid (MA) molecule having an Rf group at the terminal of the tail (MA-Rf(n); n is the number of CF2 groups) is illustrated. An Rf group is characterized by a twisted structure with a constant rate: the direction of the CF2 group is twisted by 180° over twelve C–C bonds (14, 15). In the case of an Rf group of n = 9, the twisted angle is thus 120° as in Figure 5.

Figure 5. A model compound, MA-Rf(n), having n CF2 groups. When a CF2 group is represented by a single dipole for simple visibility, the dipoles are easily found to be aligned linearly in the closest packing of the molecules, and a dipole array is generated over the hexagonal assembly (Figure 6). As a result, the MA-Rf(n) molecules spontaneously make a molecular aggregate with the hexagonal packing. This model is a result of the orientation force due to the “permanent dipoles,” which readily accounts for the high melting point of an Rf compound. In addition, the summation of the dipoles in various directions over 311 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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the molecular aggregate results in a low polarization density (11), under which a character of a low “bulk polarizability” appears, which further yields a low electric permittivity (11). This molecular aggregation model is named “stratified dipole-arrays (SDA)” model (12).

Figure 6. A top view of a molecular aggregate of the MA-Rfn=9 molecules in a hexagonal manner. To examine the SDA model, a series of Rf compounds, CF3(CF2)n(CH2)mCOOH denoted as MA-Rf(n), are prepared (n + m = 12). According to the SDA model in phase II (below 19°C), a short Rf chain would exhibit a dipole-interactive character; whereas an Rf chain of n = 7 or longer would exhibit a spontaneous molecular aggregation, in which the molecules would have a perpendicular stance to the water surface (Figure 7).

Figure 7. Schematic image of MA molecules with (a) n=3 and (b) n= 7 on the water surface. 312 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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The expectation on the SDA model was examined by using a Langmuir monolayer of each model compound on a water surface, which was transferred at a surface pressure of 15 mN m-1 onto a gold-evaporated glass surface to be an LB film. The surface pressure was chosen because the molecules with n = 7 or longer are expected to be aggregated “spontaneously” (12). IR RA spectra of the LB films are presented in Figure 8.

Figure 8. IR RA spectra of single monolayer LB films of MA-Rf(n) on gold. Adapted with permission from Reference (12). Copyright 2014 Wiley. As found in the figure, RA spectrometry is powerful to obtain high-quality IR spectra of a monolayer-level thin film on a metallic surface. The spectra are obtained by 2000 accumulations using a liquid nitrogen-cooled MCT detector (1). Here, it is of note that the spectral pattern largely depends on the Rf length. In general, normal alkyl compounds yields a nearly identical spectral shape except the methyl-related bands on a change of the chain length (16), and therefore the spectral change of the Rf compounds may look unusual (17). The most understandable band is the CF3 symmetric stretching (νs(CF3)) vibration band that appear at 1343 cm-1 for MA-Rf(9). Since only one CF3 group is available at the terminal end of the Rf group for all the compound, the orientation of the CF3 group equals to the orientation of the Rf group (12). In short, the band is useful for molecular orientation analysis of the Rf group. 313 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Here, let us remind the SSR of RA spectrometry: only the surfaceperpendicular component of a transition moment appears in the spectra. The IR RA spectra show that the νs(CF3) band strongly appears for n = 9 at 1343 cm-1 while it is suppressed for n = 3 at 1301 cm-1. This result straightforwardly implies that the molecules of n = 9 stand nearly perpendicularly to the surface while the molecules of n = 3 lie on the surface, which agrees with the expectation on the SDA model. On the other hand, the CF2 stretching vibration bands are difficult to be discussed. Since an Rf chain has a twisted structure, each CF2 group has different direction with a different tilt angle when the Rf group is tilted to the surface. This means that the bands cannot be employed for molecular orientation analysis. Instead, fortunately, the modes are still useful for discussing the molecular packing, since it is sensitive to the molecular conformation. For the analysis, the CF2 symmetric stretching vibration (νs(CF2)) band is used, since this band is separated from another band. Since the band location depends on the Rf length (17), IR spectra of bulk (un-oriented) solid samples are necessary. In recent days, for that purpose, the ATR technique is quite often conveniently employed.

Figure 9. IR ATR spectra of bulk compounds of MA-Rf(n) after converted to the LO energy-loss functions. Adapted with permission from Reference (12). Copyright 2014 Wiley. 314 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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IR ATR spectra of MA-Rf(n) are shown in Figure 9, in which the Rf-related bands appear. Here, we have to note that an ATR spectrum cannot directly be compared to an RA spectrum. An ATR spectrum involves a p-polarization component, which is a linear combination of both TO and LO energy-loss functions. Since an RA spectrum depends on the LO function only, the contribution of the TO function involved in the ATR spectrum must be removed for the mutual comparison especially for discussing the band location. To do that, a spectrum conversion available on a recent spectrometer is quite useful. For example, “Advanced ATR Correction” is the most useful function on a Thermo’s FT-IR, which generates an α-spectrum from an ATR spectrum (Eq. (3)), as if a film with a thickness of 2.303 (= ln10) μm is measured by the transmission technique having no influence by the air/film interfaces. The α-spectrum is defined as:

as found in Eq. (3).With this equation, the n″ spectrum is obtained, which can further be converted to be the real part of the refractive index, n′, by using the Kramers-Kronig relationship (18) with the limiting refractive index ( for an Rf compound, for example). With the complex refractive-index, the complex electric permittivity, ε, is readily obtained as:

which further yields the LO energy-loss function. Figure 9 is obtained in this manner. Now, the LO spectra of the bulk solid are ready to be compared to the RA spectra of the monolayers on gold. The νs(CF2) mode appears at the same position of 1153 cm-1 for both LO and RA spectra, which straightforwardly implies that the molecules in the monolayer have a highly condensed packing as found in the bulk solid. In this manner, the spontaneous molecular aggregation of the long Rf group with n = 9 has experimentally be proved. On the other hand, the same mode of the compound of n = 3 is located at 1138 cm-1 in the RA spectrum, which is higher than that of the LO spectrum at 1128 cm-1 by 10 cm-1. The higher wavenumber shift means that the molecular packing is loose in the monolayer, which agrees with the expectation on the SDA model. In this manner, an appropriate spectral conversion is necessary for precise comparison of the bulk and film samples.

IR External Reflection Spectrometry When a thin film deposited on a nonmetallic (dielectric) surface is measured by a reflection technique (Figure 10), the measurement is strictly discriminated from the RA technique, and it is called “external reflection (ER)” technique (1, 2, 4). 315 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 10. A schematic of IR ER measurements of a thin film on a nonmetallic substrate using the s- (gray arrow) and p-polarizations (solid arrow).

On the contrary to the RA measurements, the electric field of the s-polarization still remains on the surface, since the nonmetallic surface does not generate the mirror image beneath the interface (1, 2, 4). As a result, a totally different SSR is obtained for the ER spectrometry. To discuss the ER technique, absorbance and reflectivity should both be taken into account. The absorbance on a three-phase system is represented by the following equations.

Here, only the film phase (the 2nd layer) has a thickness while the rest phases have infinite thickness. The details of the coefficients, Cy and Cz, are referred to literature (4), but Eq. (6) apparently tells us that the ER spectra are of the TO and LO functions. One of the outstanding characteristics of ER spectrometry is that Cz can be both positive and negative. As a result, the p-polarized ER spectrum comprises both positive and negative bands. This character is intuitively understood by using Hansen’s approximation equations (2, 4, 19, 20). This approximation is based on the thin-film approximation and a non-absorbing substrate.

Here, can be calculated by Eqs.

. The ideally surface-parallel orientation (7) and (9); whereas the surface-perpendicular 316

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orientation is reflected in Eqs. (8) and (10). With these equations, band intensities of the s- and p-polarization measurements are readily calculated as presented in Figure 11.

Figure 11. Band intensity of a thin film on a GaAs substrate at 2850 cm-1. The thickness of the film is 22.5 nm. When a thin film on a single-side polished GaAs substrate (3-phase system) is subjected IR p-polarized ER spectrometry, for example, the surface-perpendicular component (Apz) of a transition moment yields a positive absorbance for a small angle of incidence, but it turns into negative when the angle is larger than Brewster’s angle ( (18)). On the other hand, the surface-parallel component yields negative band for the s-polarization, which is also indicated by Eq. (5).

Figure 12. Reflectance on the angle of incidence at a silicon surface for both polarizations. 317 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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This is the SSR of ER spectrometry, and both polarizations are useful. Figure 11 implies that a p-polarized spectrum is highly sensitive when the angle of incidence is near Brewster’s angle, but we have to pay attention to another issue, i.e., reflectance on the surface. The reflectance on a silicon surface for each polarization is presented in Figure 12. Since the p-polarization exhibits an extremely low reflectance near Brewster’s angle, the quality of the spectra using an angle of incidence near Brewster’s angle is degraded. As a result, we have to choose a well-balanced angle for obtaining high-quality ER spectra for the p-polarization. Figure 13 presents an IR ER spectrum of a self-assembled monolayer (SAM) of octadecylsilane (ODS) (21). The band positions of both νa(CH2) and νs(CH2) modes indicates a well-ordered packing of the alkyl chains having the all-trans zigzag conformation in the SAM. Since the angle of incidence is chosen as 60° that (Figure 11), the negative-absorbance bands indicate that the is less than transition moment of the two modes are both nearly parallel to the surface. This implies that the chain axis stands nearly perpendicular to the film surface. This discussion is supported by the rest two positive bands at 2966 and 2877 cm-1, which are assigned to the in-skeleton asymmetric CH3 stretching vibration (νs(CH3)IS) and the symmetric CH3 stretching vibration (νs(CH3)) bands, respectively (22).

Figure 13. IR ER spectrum of a SAM of ODS on a silicon substrate measured by using an angle of incidence of 60° (< θB). Adapted with permission from Reference (21). Copyright 2013 Japan Society for Analytical Chemistry.

IR p-polarized ER spectrometry has thus a benefit that molecular information of both surface-parallel and -perpendicular components is simultaneously obtained on a single spectrum. Although the signal-to-noise ratio can be inferior to the other techniques because of the low reflectance, the SSR of ER spectrometry is quite powerful. 318 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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IR MAIRS Spectrometry The ER technique has a limit that a transition moment with an oblique tilt angle gives no band, which is caused by overlapping the positive and negative bands. As an example, an IR p-polarized ER spectrum of a dip-coated film of linear polyethyleneimine (LPEI) on a silicon wafer is presented in Figure 14 (23). Since the angle of incidence (50°) is less than the Brewster angle (ca. 73°), the surfaceparallel and perpendicular components of a transition moment appear as negative and positive bands, respectively, due to the SSR. Of note is that there is no band in the circled area that is for the CH2 stretching vibration bands. Since LPEI has a number of CH2 groups, the bands must aappear in the wavenumber region. The disappearance is a result of an oblique tilting of the transition moment, but it is a quite ambiguous result.

Figure 14. IR p-polarized ER spectrum of a dip-coated film of LPEI on a single-side polished silicon wafer. Adapted with permission from Reference (23). Copyright 2008 American Chemical Society.

To overcome the limitation, the MAIRS (multiple-angle incidence resolution spectrometry (24–29)) technique is powerful. MAIRS is built on a unique concept that the surface-perpendicular component of a transition moment is measured by using a virtual light, in which the electric-field oscillation is parallel to the traveling direction of the IR light. IR MAIRS measurements are performed by using an IR transparent substrate with oblique-incidence transmission measurements as illustrated in Figure 15. A light intensity (single beam) spectrum is decomposed into two independent components, SIP and SOP. The single-beam spectrum of SOP corresponds to a virtual measurement using the virtual light, but it can be calculated by collecting single-beam spectra at multiple angles. 319 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 15. A schematic image of MAIRS: an oblique-incidence transmission measurements are decomposed into two normal-incidence transmission measurements denoted by SIP and SOP.

The collected single-beam spectra at various angles of incidence are stored in the matrix, S. These spectra involves a linear combination of the SIP and SOP spectra with weighting factors in R, which is presented by Eq. (11) involving a two-column matrix.

Regardless, other complicated components such as reflection of the substrate surface and multiple reflections in the substrate are not involved in the linear combination. This can readily be formulated by using a multivariate analysis formulated by Eq. (12).

This type of formulation is called classical least squares (CLS) regression, and the nonlinear response to R is automatically discarded into the undescribed matrix, U, by obtaining the least squares solution as Eq. (13).

As a result, two sets of SIP and SOP are obtained by the sample and background measurements, which yield two explicit absorbance spectra:

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Figure 16 presents IR MAIRS spectra of an LPEI dip-coated film on a doubleside polished germanium substrate (23). If the film is the same as that for Figure 14, the MAIRS-IP and –OP spectra would correspond to the negative and positive bands in the ER spectra, respectively. Although the CH2 stretching vibration bands disappear in the ER spectrum due to oblique orientations, they are apparently found in the MAIRS spectra: the νa(CH2) and νs(CH2) modes at 2908 and 2928 cm-1, respectively, in both IP and OP spectra with a nearly the same intensity. This is the reason, in the ER spectrum, the positive and negative bands are overlapped to make the bands disappeared.

Figure 16. IR MAIRS spectra of the LPEI film dip-coated on a germanium substrate. Adapted with permission from Reference (23). Copyright 2008 American Chemical Society.

Figure 17. A schematic of double helix of LPEI standing perpendicularly on a Ge substrate. Adapted with permission from Reference (23). Copyright 2008 American Chemical Society. 321 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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In addition, the surface-parallel orientated moment at 1247 cm-1 appears in the IP spectrum only; whereas the surface-perpendicular oriented moment at 1281 cm-1 appears in the OP spectrum only. Since these moments are known to parallel and perpendicular to the double-helix of LPEI, respectively (30), the MAIRS spectra straightforwardly implies that the double helices stand on the Ge substrate perpendicularly as illustrated in Figure 17 (23). Another notable band is the N–H stretching vibration (ν(N–H)) band found at 3222 and 3212 cm-1 in the IP and OP spectra, respectively, which exhibits a large shift of 10 cm-1. If this shift is caused by the TO-LO splitting (Berreman’s effect (31, 32)), the OP band should exhibit a higher wavenumber shift to the LO band. The opposite result to the TO-LO splitting is readily explained by the factorgroup splitting (33). When two closest N–H groups on the double helix vibrate symmetrically, the vibration mode has a lower vibration energy than that of the corresponding anti-symmetric vibration. Since the symmetric and anti-symmetric vibration are directed along and perpendicular to the helix, the shift in the MAIRS spectra implies again that the helix stands perpendicularly on the Ge surface. In this manner, IR MAIRS is quite useful for discussing molecular structure in a thin film. The MAIRS technique has a limitation, however, that only a high refractiveindex substrate can be employed for the measurements (26). Although a silicon substrate has a high refractive index of ca. 3.4 in the IR wavelength region, the oxidized surface has a low refractive index of ca. 1.4, which makes the MAIRS measurements largely degraded. To overcome this limitation, the s-polarization must be removed (26), and the following renovated weighting matrix, , is used.

This improved MAIRS technique is named pMAIRS, since only p-polarization is used. Unless otherwise stated nowadays, pMAIRS is strongly recommended to be used, since pMAIRS has a great advantage that the lowest limit of the analytical wavenumber region is down to 700 cm-1 (34, 35), while the original MAIRS is limited by 1100 cm-1. One of the most successful application studies using pMAIRS is the structural analysis of a spin-coated film of poly(3-alkylthiophene) (P3HT; Figure 18) on a silicon surface (35). P3HT is an extensively studied p-type organic semiconductor, which consists of polythiophene hanging a hexyl-chain tail on each thiophene ring. Thanks to the hexyl chain, it can easily be dissolved in various organic solvents, which can be subjected to the spin-coating to yield a thin film on a solid surface. After being spread on a solid surface, the molecular planarity along the polythiophene chain is improved to have a long π-π conjugation. In this situation, an exciton is created by irradiating visible light, which is suitable for a solar cell. Therefore, the “face-on” orientation of the compound has long been considered 322 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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to have a piled-up lamellar structure parallel to the film surface. In fact, this speculation was supported by an X-ray diffraction (XRD) analysis. Nevertheless, a spin-coated P3HT film having the face-on orientation often exhibits a very poor crystallinity, which yields an ignorable XRD peak. In this situation, the textbook image of the lamellar structure cannot be employed. To a thin film having poor crystallinity, IR pMAIRS works powerful.

Figure 18. Primary chemical structure of P3HT. pMAIRS has already been fully analyzed on electrodynamics to have mathematical expressions, which are also linear combinations of the TO and LO energy-loss functions (36). For the IP spectrum, for example, optimal parameters should be employed to make the weighting factor of LO function adequately small to leave the IP function only. The optimal parameters correspond to the optimized experimental condition. For example, for a quantitative analysis, the pMAIRS spectra require the condition: the angle of incidence is varied from 9° through 44° by 5° steps (35).

Figure 19. IR pMAIRS spectra of a spin-coated film of P3HT on a Si substrate. Adapted with permission from Reference (35). Copyright 2015 Royal Society of Chemistry. With the optimal experimental condition, IR pMAIRS spectra of a thin film of P3HT spin-coated on a silicon substrate are obtained as presented in Figure 19 (35). The left higher-wavenumber panel is of the hexyl chain. The locations of the νa(CH2) and νs(CH2) bands appear at 2927 and 2856 cm-1 are specific to the gauche 323 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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conformer, and the hexyl chain is found to have a largely disordered structure. The MAIRS shift (37) of the νs(CH2) band between the IP and OP spectra (2856 and 2857 cm-1) supports this discussion, too. In the lower-wavenumber panel, two useful bands are available: the anti-symmetric thiophene-ring (νa(C=C)) vibration at 1511 cm-1 and the C–H out-of-plane deformation vibration on the thiophene ring (γ(C–H)) at 826 cm-1 (34, 35). The νa(C=C) band appears stronger in IP than OP; whereas the γ(C–H) band exhibits the opposite relative intensity. Since the νa(C=C) has a transition moment along the long axis of the polythiophene and the γ(C–H) has a moment perpendicular to the thiophene ring, the pMAIRS spectra apparently indicate that the polymers are stacked in the face-on fashion. Since the film exhibited no XRD diffraction peak on a laboratory equipment (35), pMAIRS is powerful for the amorphous film. Another benefit of using pMAIRS is that the orientation angle, , is obtained quantitatively after the optimization of the measurement condition.

Here, IIP and IOP are band intensities of an identical vibrational mode appeared in the IP and OP spectra, respectively. In the same manner, the two key bands on the thiophene ring are analyzed for four kinds of similar polymer with a tail with a different length as found in Table 2.

Table 2. Molecular Orientation Angles Obtained by IR pMAIRS Spectra. (Adapted with permission from Reference (35). Copyright (2015) Royal Society of Chemistry.)

B, H, O and DD involved in a compound name stands for butyl, hexyl, octyl and dodecyl, respectively. IR pMAIRS reveal that the molecular orientation is greatly changed as a function of the tail length only. The spin-coating condition and the solvent are commonly fixed to all the samples. 324 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 20. Geographical relationship of the γ(C–H)) and νa(C=C) modes.

The ring-perpendicular orientation (γ(C–H)) is influenced by both short and long axis of the thiophene ring. If the short axis is fixed parallel to the surface (Figure 20), however, the angle of the γ(C–H) mode simply depends on the orientation of the νa(C=C) mode. In this case, as depicted in Figure 20, the summation of the orientation angles of the γ(C–H) and νa(C=C) modes should be equal to 90°. As found in Table 2, all the face-on type film yields the summation near 90° as expected, which paradoxically implies that the short axis of the polythiophene ribbon is kept unchanged parallel to the film surface; whereas the polymer chain exhibits a nearly random orientation (φC=C ≈ 55°). Since these “face-on” spin-coated films exhibit no XRD peak, the polythiophene film with the face-on orientation can be concluded to be not driven by the crystallinity, but by the planer interaction keeping the parallel orientation of the short axis (Figure 21).

Figure 21. A schematic of molecular piling of the face-on type polythiophene compounds. Adapted with permission from Reference (35). Copyright 2015 Royal Society of Chemistry. 325 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

This schematic image is largely different from that of the edge-on film of P3BT, in which the molecules are highly crystallized yielding an apparent XRD peak even on a laboratory-use X-ray equipment. In this manner, IR pMAIRS is quite useful to provide molecular orientation, which can be used for finer discussion of the film structure irrespective of the crystallinity of the film.

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Concluding Remarks IR spectroscopy has a great potential to reveal structural information in a thin film, and the sensitivity is also good enough to discuss a monolayer-level thin film. Since a vibrational spectrum provides plural number of absorption bands due to the 3N − 6 rule, a plausible chemical model can be constructed by discussing as many bands as possible. When we consider that a thin film always accompanies an optical interface, the SSR of each spectrometry works powerfully to help the discussion of the many bands. For quantitative discussion of the band locations, the mathematical expressions of the spectrometries are highly useful, and an appropriate spectral conversions considering the TO and LO energy-loss functions enable us to step into a deep insights of the thin films. Of another importance is that an IR analysis can be made on any sample irrespective of the degree of crystallinity, which suggests that IR spectroscopy should be the first choice for analyzing the thin film at hand. Since FT-IR has already spread over many chemical laboratories, the spectral data are expected to be analyzed more deeply to make the best use of the chemical information involved in the spectra.

References Griffiths, P. R.; de Haseth, J. A. Fourier Transform Infrared Spectrometry, 2nd ed.; Wiley: Hoboken, 2007. 2. Introduction to Experimental Infrared Spectroscopy; Tasumi, M., Sakamoto, A., Eds; Wiley: Chichester, 2015. 3. Nakamoto, K. Infrared and Raman Spectra of Inorganic and Coordination Compounds Part A; Wiley: Hoboken, 2009. 4. Tolstoy, V. P.; Chernyshova, I. V.; Skryshevsky, V. A. Handbook of Infrared Spectroscopy of Ultrathin Films; Wiley: Chichester, 2003. 5. Yeh, P. Optical Waves in Layered Media; Wiley: Hoboken, 1998. 6. Umemura, J.; Kamata, T.; Kawai, T.; Takenaka, T. J. Phys. Chem. 1990, 94, 62–67. 7. Snyder, R. G. J. Mol. Spectrosc. 1961, 7, 116–144. 8. Cameron, D. G.; Gudgin, E. F.; Mantsch, H. H. Biochemistry. 1981, 20, 4496–4500. 9. Yoshioka, Y.; Tashiro, K.; Ramesh, C. J. Polym. Sci., Part B 2003, 41, 1294–1307. 10. Starkweather, H. W., Jr. Macromolecules 1986, 19, 1131–1134. 11. Hasegawa, T. Chem. Phys. Lett. 2015, 626, 64–66. 1.

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12. Hasegawa, T.; Shimoaka, T.; Shioya, N.; Morita, K.; Sonoyama, M.; Takagi, T.; Kanamori, T. ChemPlusChem 2014, 79, 1421–1425. 13. London, F. Trans. Faraday Soc. 1937, 33, 8–26. 14. Bunn, C. W.; Howells, E. R. Nature 1954, 174, 549–551. 15. Clark, E. S. Polymer 1999, 40, 4659–4665. 16. Muro, M.; Itoh, Y.; Hasegawa, T. J. Phys. Chem. B 2010, 114, 11496–11501. 17. Hasegawa, T.; Shimoaka, T.; Shioya, N.; Morita, K.; Sonoyama, M.; Amii, H.; Takagi, T.; Kanamori, T. Chem. Lett. 2015, 44, 834–836. 18. Jackson, J. D. Classical Electrodynamics, 3rd ed.; Wiley: Hoboken, 1999. 19. Hansen, W. N. Symp. Faraday Soc. 1970, 4, 27. 20. Hasegawa, T.; Umemura, J.; Takenaka, T. J. Phys. Chem. 1993, 97, 9009–9012. 21. Norimoto, S.; Morimine, S.; Shimoaka, T.; Hasegawa, T. Anal. Sci. 2013, 29, 979–984. 22. Parikh, A. N.; Allara, D. L. J. Chem. Phys. 1992, 96, 927–945. 23. Kakuda, H.; Okada, T.; Hasegawa, T. J. Phys. Chem. B 2008, 112, 12940–12945. 24. Hasegawa, T. J. Phys. Chem. B 2002, 106, 4112–4115. 25. Hasegawa, T.; Matsumoto, L.; Kitamura, S.; Amino, S.; Katabe, S.; Nishijo, J. Anal. Chem. 2002, 74, 6049–6054. 26. Hasegawa, T. Anal. Chem. 2007, 79, 4385–4389. 27. Hasegawa, T.; Itoh, Y.; Kasuya, A. Anal. Sci. 2008, 24, 105–109. 28. Hasegawa, T. Anal. Bioanal. Chem. 2007, 388, 7–15. 29. Hasegawa, T. Appl. Spectrosc. Rev. 2008, 43, 181–201. 30. Hashida, T.; Tashiro, K.; Aoshima, S.; Inaki, Y. Macromolecules 2002, 35, 4330–4336. 31. Berreman, D. W. Phys. Rev. 1963, 130, 2193–2198. 32. Harbecke, B.; Heinz, B.; Grosse, P. Appl. Phys. A 1985, 38, 263–267. 33. Snyder, R. G. J. Mol. Spectrosc. 1961, 7, 116–144. 34. Shioya, N; Shimoaka, T.; Hasegawa, T. Chem. Lett. 2014, 43, 1198–1200. 35. Shioya, N; Shimoaka, T.; Eda, K.; Hasegawa, T. Phys. Chem. Chem. Phys. 2015, 17, 13472–13479. 36. Itoh, Y.; Kasuya, A.; Hasegawa, T. J. Phys. Chem. A 2009, 113, 7810–7817. 37. Hasegawa, T.; Iiduka, Y.; Kakuda, H.; Okada, T. Anal. Chem. 2006, 78, 6121–6125.

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Chapter 16

Protein Response to Chromophore Isomerization in Microbial Rhodopsins Revealed by Picosecond Time-Resolved Ultraviolet Resonance Raman Spectroscopy: A Review Misao Mizuno and Yasuhisa Mizutani* Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan *E-mail: [email protected].

Proteins function by changing their structures, and external stimuli facilitate the sequential change of specific structural sites. To understand the mechanism of protein functions, it is essential to clarify how external stimuli can bring about these site-specific structural changes. Time-resolved ultraviolet resonance Raman spectroscopy probes the structural dynamics of specific sites in protein structure by selectively enhancing the vibrational Raman bands assignable to aromatic amino acid side chains as well as polypeptide bonds. We have applied picosecond time-resolved ultraviolet resonance Raman spectroscopy to observation of protein response to chromophore isomerization in microbial rhodopsins.

Structural changes of proteins regulate their functions. Studies on protein dynamics is therefore important for elucidating mechanisms how protein functions. In photoreactive proteins, functionally-important structural changes of protein are initiated by light absorption to the chromophore. The local photoinduced structural change of the chromophore triggers a series of changes in the higher order structure, thereby facilitating function. Clarification of the

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propagation of structural changes induced by the local structural changes can help to understand the functional mechanism of protein. Microbial rhodopsins are typical photoreactive proteins. Figure 1a shows crystallographic structure of bacteriorhodopsin, which is the best studied microbial rhodopsin. Microbial rhodopsins consist of seven transmembrane α-helices. A retinal chromophore is covalently bound to the Lys residue through a protonated Schiff base linkage. All-trans configuration of the retinal chromophore is thermodynamically most stable. Absorption of a photon by the chromophore gives rise to isomerization from all-trans to 13-cis configuration as shown in Figure 1b. The photoisomerization of the chromophore takes place within subpicoseconds (1–4), which induces sequential changes in protein structure important for their functions.

Figure 1. (a) Crystallographic structure of bacteriorhodopsin (PDB ID = 1C3W). The red molecule represents the unphotolyzed retinal chromophore with the all-trans configuration. (b) Isomerization of the retinal chromophore from all-trans to 13-cis configurations. (see color insert)

Elucidation of the structural dynamics of the protein moiety associated with the chromophore isomerization is of great interest in understanding of protein mechanisms. To discuss protein response to chromophore isomerization, we have measured picosecond time-resolved ultraviolet resonance Raman (UVRR) spectra of four types of microbial rhodopsins, bacteriorhodopsin (BR) (5), sensory rhodopsin II (SRII) (6), sensory rhodopsin I (SRI) (7), and Anabaena sensory rhodopsin (ASR) (8). In this review, we summarize our recent studies on the protein response to chromophore isomerization of the microbial rhodopsins. 330 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Time-Resolved UVRR Spectroscopy UVRR spectroscopy is a versatile technique for studying protein structures because it enables us to observe of Raman bands of aromatic amino acid residues and polypeptide backbones with high selectivity (9–11). Several vibrational bands of aromatic residues can be utilized as structural markers of proteins; hence, time-resolved UVRR spectroscopy can provide site-specific information about protein dynamics. Although time-resolved UVRR spectroscopy has been successfully applied to protein dynamics in the nanosecond to second region (12, 13), application to protein dynamics in the picosecond region was limited (14). The most crucial factor was a lack of a light source that fulfills the requirements for small timing jitters, appropriate repetition rates, and the wavelength tunability of pulses applicable to time-resolved UVRR spectroscopy. Time-resolved UVRR measurements require pump and probe pulses. The wavelength of the pump pulse needs to fall within the electronic absorption band of the cofactor (retinal, flavins, heme, etc.) in proteins to photoexcite it. At the same time, the wavelength of the probe pulse has to be close to that of an electronic transition of the specific part of interest in the protein for resonance enhancement of Raman bands. Furthermore, the spectral width of the probe pulse has to be narrow enough to record well-resolved vibrational bands. To obtain the time-resolved UVRR spectra of a wide variety of proteins with high S/N ratios within a reasonable measuring time, it is necessary to generate independently tunable pump and probe pulses with a high repetition rate. We constructed an apparatus consisting of two widely tunable light sources for time-resolved UVRR spectroscopy using a 1-kHz picosecond Ti:sapphire laser/regenerative amplifier system (15).

Time-Resolved UVRR Apparatus and Measurements Figure 2 is schematic of the time-resolved UVRR measurement apparatus. A Ti:sapphire oscillator (Tsunami pumped by Millennia-Vs, Spectra-Physics) and amplifier (Spitfire pumped by Evolution-15, Spectra-Physics) system operating at 1 kHz provided 778-820 nm pulses, each with an energy of about 0.8 mJ, and duration of 2.5 ps in a nearly TEM00 mode under operation at 1 kHz. The whole laser system was covered with the plastic sheet, which was equipped with dust cleaners containing high-efficiency particulate air (HEPA) filters to keep the laser system free of dust. In the pump arm, a pump pulse of 530-600 nm was generated with a home-built optical parametric generator (OPG) and amplifier (OPA), which were pumped with the second harmonic of the output of the amplified laser. To generate a pump pulse with shorter wavelength (439-494 nm), stimulated Raman scattering in compressed methane or hydrogen gas was excited by the second harmonic of the laser output. Also, the second harmonic of the laser output (389410 nm) was directly applicable to a pump pulse. The tunability of the pump pulse is shown in the right panel of Figure 2c. In the probe arm, the second harmonic of the laser output was focused into a Raman shifter filled with methane or hydrogen gas to generate first Stokes stimulated scattering in 439-494 nm. For example, a UV probe pulse at 225 nm was generated with a BBO crystal as the second harmonic of the 450-nm output. In this way, a UV pulse was generated in the 331 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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wavelength range of 220-247 nm. Sum frequency generation between the second harmonic and the stimulated Raman scattering was generated to produce the UV probe pulse in 206-218 nm. The tunability of the probe pulse is also shown in the right panel of Figure 2b. Light components other than the probe pulse were eliminated spatially with a Pellin-Broca prism and spectrally with dichroic mirrors.

Figure 2. Picosecond time-resolved UVRR spectrometer. (a)Schematic optical setup of the spectrometer. (b) Optical configuration in the probe arm. (c) Optical configuration in the pump arm. L=lens, BS=beam splitter, ND=neutral density filter, HWP=half wave plate, SH=mechanical shutter, LBO=lithium triborate, BBO=β-barium borate, DM=dichroic mirror, PB=Pellin-Broca prism. (see color insert) 332 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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After the pump and probe beams were made coaxial using a dichroic mirror, they were focused with a spherical lens onto a flowing thin-film of the sample solution. Focused spot sizes were 150 μm (fwhm) for the probe beam, and 250 μm (fwhm) for the pump beam. At the sample point, energies of the probe and pump pulses were attenuated to 0.5 and 5 μJ, respectively, using Cr-coated quartz ND filters. The two beams were configured for 135° backscattering illumination and collection. The spectral features of photoproducts in the pump-probe spectra were also shown to be invariant to a two-fold change of the probe power. The pump power was selected so that no saturation effect or spectral changes occurred by a doubling of the pump power. Cross correlation trace of the pump and probe pulses measured by difference frequency generation with a thin BBO crystal indicated a width of 3.0-3.7 ps. Intensities of pump and probe pulses were monitored with photodiodes (S2387-1010R, Hamamatsu Photonics) and found to be stable within ±10%. Raman scattered light was collected by an F/2 quartz doublet achromat and focused by an F/4 quartz doublet achromat onto the entrance slit of a Czerny-Turner configured Littrow prism prefilter (16) coupled to a 50-cm single spectrograph (500M, SPEX). The spectrograph was equipped with a 1200 grooves/mm, 500 nm blazed grating operating in second order or a 2400 grooves/mm, 250 nm blazed grating operating in first order. Dispersed light in the spectrograph was detected with a liquid nitrogen-cooled CCD detector (SPEC-10:400B/LN, Roper Scientific) with Unichrome UV-enhancing coating. Raman shifts were calibrated with cyclohexane to an accuracy of ±4 cm-1. The time-resolved UVRR data acquisitions were carried out as follows. The sequence of the delay times in the time-resolved measurements were determined to be random in each scan. At each delay time, Raman signals were collected for three 20-second exposures with both the pump and probe beams present in the sample. This was followed by equivalent exposures for pump-only, probe-only, and dark measurements. This method enabled us to avoid the errors caused by a slow drift of laser power and to obtain quantitatively reproducible spectra from one day to the next, which is possible because of the excellent long-term stability of this laser system. The pump-only spectrum was directly subtracted from the pump-and-probe spectrum, yielding the “probe-with-photolysis” spectrum. The dark spectrum was directly subtracted from the probe-only spectrum, yielding the “probe-without-photolysis” spectrum, namely the spectrum of the photolyzed state. The probe-without-photolysis spectrum was subtracted from the probe-withphotolysis spectrum to yield the photoproduct spectrum. The scattering intensity for the change in the optical absorption of the sample at each time point was corrected by normalizing the data to the intensity changes of the OH stretching band of water (~3400 cm-1) of the sample solution. After normalizing the band intensities in all the spectra, the probe-without-photolysis spectrum was subtracted from the probe-with-photolysis spectrum to generate the difference spectra.

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Bacteriorhodopsin (BR) BR functions as a light-driven proton pump found in the purple membrane of halobacteria. So far, changes in the protein structure during a photocyclic reaction have been examined by time-resolved spectroscopy at room temperature (1, 2, 17–35) as well as cryo-spectroscopy (36–45) and crystallography (46, 47). In the photocycle, a series of intermediates, J, K, KL, L, M, N, and O are observed, each of which is characterized by a distinct absorption band. Structural information on the retinal chromophore in the photointermediate was studied based on time-resolved visible resonance Raman spectroscopy (48). The chromophore isomerizes around the C13=C14 bond from all-trans to 13-cis configuration upon the photoexcitation, to produce the J intermediate. The highly twisted chromophore with a 13-cis configuration is formed upon the J formation. In the K intermediate, the chromophore relaxes to a more planar 13-cis configuration. In the K-to-KL and KL-to L transition, the chromophore undergoes conformational changes although it keeps its configuration in 13-cis form. The Schiff base of the chromophore is deprotonated in the M intermediate and reprotonated in the N intermediate. The configuration of the chromophore returns to all-trans form in the O intermediate. For the time-resolved UVRR study, structural changes at Trp182 and Trp189 in helix F in the late intermediates, L, M, and N, were discussed based on microsecond time-resolved data (13). Nanosecond time-resolved experiments have been performed for M (49) and KL intermediates (50) by Mathies and co-workers. To study ultrafast protein response to the isomerization, we measured picosecond time-resolved UVRR spectra of BR (5). The UVRR spectrum of BR in the unphotolyzed state probed at 225 nm is shown in the top trace in Figure 3. This spectrum contains all the Raman bands of eight Trp and eleven Tyr residues in BR. Vibrational bands of Trp and Tyr side chains are noted as W and Y, respectively. The mode assignments made by Harada and Takeuchi (10) are shown in green and blue characters. The 1615-cm-1 band is attributed to the overlap of the W1 (Trp) and Y8a (Tyr) bands. The bands at 1555, 1357, 1013, and 763 cm-1 are assigned to the vibrational modes of Trp, W3, W7, W16, and W18, respectively. The other spectra in Figure 3 represent time-resolved UVRR difference spectra, obtained by subtracting the unphotolyzed BR spectrum from the spectrum measured at each delay time from -5 to 1000 ps. Upon photoexcitation, negative UVRR bands were clearly observed for Trp within the instrument response time. The negative bands indicate the depletion of the Raman intensity due to the change in protein structure during the photoreaction. The negative bands decayed from 10 to 50 ps, indicating that the band intensity of Trp recovers due to structural changes subsequent to the instantaneous change. In the region from 100 to 1000 ps, the difference spectra did not change. Figure 4 shows the temporal intensity changes of five remarkable bands observed in Figure 3. Data analysis revealed that the intensity of all the bands instantaneously decreases within the instrumental response time and recovers with a time constant, τrecovery, of approximately 30 ps. The retinal chromophore isomerizes from the all-trans to 13-cis form within ~0.5 ps upon the formation of the primary photoproduct of the J intermediate (1, 2). The initial UVRR 334 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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intensity depletion can be attributed to the protein response to the chromophore isomerization. The subsequent intensity recovery is likely to reflect the protein response to the structural change of the chromophore. It was revealed, for the first time, that the dynamics of the protein structure in the BR photocycle takes place with a time constant of 30 ps in this study (5).

Figure 3. Picosecond time-resolved UVRR spectra of BR. Probe and pump wavelengths are 225 and 565 nm, respectively. The top trace is the probe-only spectrum divided by a factor of 50, representing the UVRR spectrum of BR in the unphotolyzed state with an all-trans configuration. The spectrum of the buffer has been subtracted. The other spectra are time-resolved difference spectra generated by subtracting the probe-only spectrum from the pump-probe spectrum at each delay time. The accumulation time for obtaining each spectrum was 120 min. (Reproduced with permission from reference (5). Copyright 2009 American Chemical Society.) (see color insert) 335 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 4. Temporal intensity changes of the bands in the range from -10 to 100 ps. (a) 1615 cm-1, the overlap of W1 and Y8a; (b) 1555 cm-1, W3; (c) 1357 cm-1, W7; (d) 1013 cm-1, W16; (e) 763 cm-1, W18. Markers indicate the intensity changes measured at each delay time relative to the intensity in the probe only spectrum. Solid lines are the best-fit with a function of [A1 × exp(−t/τrecovery) + A2] convoluted with the instrument response function. The obtained parameter, τrecovery, for each trace is indicated in the figure. (Reproduced with permission from reference (5). Copyright 2009 American Chemical Society.) (see color insert) We also measured picosecond time-resolved UVRR spectra probed at 238 nm (Figure 5). The different spectral patterns were observed at 5 and 100 ps, implying that the different intermediates were detected. It was found that the intensity of the band at 1620 cm-1, attributable to the W1 (Trp) and Y8a (Tyr) bands, decreased within the instrument response time, and recovered with a time constant of 30 ps. The W18 band at 765 cm-1 exhibited an intensity loss within 30 ps (5). These intensity changes suggests that the 30-ps process detected under the 238-nm probe condition results from the protein response to the chromophore relaxation as observed in the spectra probed at 225 nm. The two time-resolved UVRR difference spectra shown in Figure 5 reflect the structure before and after the 30-ps process in the protein dynamics, respectively. We found that the temporal behaviors of the observed spectral changes in each Raman band of both Trp and Tyr were not uniform. The W16 and W18 bands were not observed in the 5-ps difference spectrum while negative bands emerged in the 100-ps spectrum due to the intensity decrease of these bands. On the contrary, the spectral pattern of the W3 band showed the sigmoidal form at 5 and 100 ps. If the observed spectral change arose from the structural change of single Trp residue, the temporal 336 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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behaviors of the spectral changes for these bands would be identical. Therefore, the non-uniform temporal behaviors of the Trp bands indicate that the spectral changes in Figure 5 are attributable to at least two residues. The same is true for the Tyr bands. The Y8a band showed the intensity bleach at 5 ps and recovered at 100 ps, whereas the Y7a band exhibited the sigmoidal form at both 5 and 100 ps. This implies that the observed spectral changes of the Tyr bands arise from at least two residues.

Figure 5. Picosecond time-resolved UVRR spectra of BR (probe laser, 238 nm; pump laser, 565 nm). The top trace is the probe-only spectrum divided by a factor of 50, representing the UVRR spectrum of BR in the light-adapted state. The spectrum of the buffer has been subtracted. The others are time-resolved UVRR difference spectra at (a) 5 ps and (b) 100 ps. The accumulation time for obtaining each spectrum was 295 minutes. (Reproduced with permission from reference (5). Copyright 2009 American Chemical Society.) (see color insert)

The present UVRR results provide structural information on the primary protein response associated with the photoreaction of the chromophore in BR. The observed process can be attributed to structural rearrangement of protein moiety in the vicinity of the retinal chromophore. Based on the spectral changes of the structural marker bands in the UVRR difference spectra, we discuss the primary protein response to the chromophore isomerization. In the picosecond region, photoexcited BR sequentially relaxes to the J, K, and KL intermediates. All the Trp bands probed at 225 nm (Figure 3) as well as the Y8a band in the 238-nm spectra (Figure 5) bleached within the instrumental response time. The initial intensity bleach is likely to arise from the J-intermediate formation which occurs within 0.5 ps (17, 18, 20). Because the J intermediate is supposed to convert to the K intermediate within 3 ps (17, 18), the negative bands in the UVRR difference spectrum at 5 ps are assignable to bands of the K intermediate. The subsequent K-KL transition is less well defined. Kaminaka and Mathies reported that the KL intermediate was observed in the 10-ns UVRR difference spectrum probed at 240 nm (50). The features of our UVRR difference 337 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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spectrum at 100 ps measured by using the 238-nm probe pulse (Figure 5b) are very similar to those of their 10-ns spectrum, indicating that the KL intermediate was observed in the 100-ps UVRR spectrum. Thus, the observed spectral changes suggest that the protein response with a time constant of 30 ps is associated with the formation of the KL intermediate from the K intermediate. In the time-resolved 225-nm UVRR difference spectra (Figure 3), the intensities of the Trp bands bleached in response to the photoreaction of retinal. The resonance enhancement of the Raman bands depends on the electronic transition to which the frequency of the probe pulse is resonant. The environmental changes around the Trp residue, such as changes in the hydrophobicity and the hydrogen-bond strength, give rise to the energy shift of the electronic transitions. Also the change in the dipole moment of retinal responding to the photoreaction would cause the changes in the transition dipole moment of Trp due to excitonic coupling (51). These changes affect the resonance enhancement and, thus, result in changes of the band intensities. The negative W7 band in the time-resolved difference spectra in Figure 3 showed an asymmetric form compared to the UVRR band in the unphotolyzed state. The peak of this negative band was located around 1360 cm-1, whereas the shoulder was detected at about 1340 cm-1. The W7 mode is known to show a doublet, of which intensity ratio (I1360 / I1340) is a marker of hydrophobicity around Trp. When the Trp residue is located in a hydrophobic environment, the intensity ratio becomes larger (52). Since the spectral width of our laser was as wide as 20 cm-1, the Trp doublet could not be resolved. The enhancement of the shoulder band at 1340 cm-1 relative to the band at 1360 cm-1 in the difference spectra may indicate that the intensity ratio I1360 / I1340 in the early photointermediate is smaller compared to that in the unphotolyzed state. Thus, it is likely that the hydrophobicity of a Trp residue is reduced in the early picoseconds. Under the 238-nm probe condition (Figure 5), the W3 mode exhibited a sigmoidal form arising from the frequency shift in the difference spectra at both 5 and 100 ps. The W3 frequency is correlated with the torsion angle, χ2,1, which is defined as the dihedral angle of the C2-C3-Cβ-Cα linkage of the indole side chain (53, 54). The observed downshift implies a decrease in the torsion angle χ2,1 of the indole ring in the K and KL intermediates. Negative bands appeared at the positions of the W16 and W18 bands at 100 ps in Figure 5b. These negative bands indicate that the intensities of the W16 and W18 bands in the KL intermediate are smaller compared to that in the unphotolyzed state. The Raman intensities of the W16 and W18 modes are enhanced in resonance with the Ba and Bb states (55, 56). The absorption band of the Bb transition is blue-shifted when the hydrophobicity around the Trp residue decreases (57, 58). Because the probe wavelength, 238 nm, is located at the red side of the maximum of the Trp Raman excitation profile, which is located at 224 nm (59), the blue-shift of the absorption band results in decrease of the resonance enhancement. Therefore, the intensity loss of the W16 and W18 modes is associated with the reduction of the hydrophobicity in the KL state. For Tyr bands observed in Figure 5, two apparent marker bands were detected. A derivative-like feature was observed in both the 5- and 100-ps UVRR difference spectra at the position of the Y7a mode. This is caused by the lower frequency shift 338 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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of the Y7a band in the K and KL intermediates. The Y7a frequency is an indicator of the degree of the proton-donating state in the hydrogen bond on the phenolic OH group (60). The lower frequency shift suggests that the Tyr has more proton-donor character in the K and KL states. A large negative band of the Y8a mode instantaneously appeared upon photoexcitation, decaying within 30 ps. This indicated that the Y8a band intensity bleached in the K intermediate and subsequently recovered in the KL intermediate. The Raman intensity of the Y8a mode is resonantly enhanced by the Franck-Condon A-term mechanism via the La absorption (peak wavelength 222 nm). The maximum of its Raman excitation profile is around 225 nm (61). Thus, under the present probe condition, the intensity of the Y8a band decreases when the Raman excitation profile exhibits blue shift. It has been reported that the La absorption band systematically blue-shifts when the Tyr residue is in a more protic environment (57). The present results show an increase of the hydrogen-bond strength in the J and K intermediates and the subsequent decrease in the KL intermediate. In picosecond time-resolved UVRR spectra of BR at room temperature, we observed spectral changes of both the Trp and Tyr bands, which reflect the primary protein response to the photoreaction of the retinal chromophore. The time constant of the primary process in the protein moiety was determined to be 30 ps, for the first time. The time constant of 30 ps suggests that this change is associated with the transition from the K intermediate to the KL intermediate, which has been less defined.

Sensory Rhodopsin II (SRII) SRII serves as a negative phototaxis receptor found in halobacteria. It forms the signaling complex with its cognate transducer protein, HtrII, in the cell membrane. SRII is activated by a blue light around 500 nm and regulates the kinase phosphorylation. The structure of SRII and its complex with HtrII have been studied by X-ray crystallography (62, 63) and various other spectroscopic methods (64–66). Structural differences between the unphotolyzed state and the cryogenically trapped photointermediates have been deduced from FTIR studies (67–70) and X-ray diffraction on protein crystals (71). The primary chromophore reaction dynamics of SRII are quite similar to those of BR (3, 72). For instance, the formation time constants of the J and K states are approximately 0.5 and 3 ps, respectively (3). However, little is known about the primary protein dynamics in SRII. We investigated the primary protein response to retinal isomerization of SRII in the early intermediates on the basis of picosecond time-resolved UVRR spectra (6). The time-resolved UVRR spectra clarified the structural changes that occur around the Trp and Tyr residues located in the vicinity of the retinal chromophore. Figure 6a shows picosecond time-resolved UVRR spectra of SRII probed at 225 nm. Similarly to the BR spectra, the negative bands were clearly observed at the positions of the Trp bands after 450-nm pump pulse irradiation. Based on the temporal intensity changes in Trp bands as shown in Figure 6b, it was found that 339 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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the Trp band intensities instantaneously bleached within the instrumental response time and recovered with a time constant of ~30 ps. The initial UVRR intensity depletion can be attributed to the protein’s response to chromophore isomerization associated with the formation of the J intermediate. The subsequent intensity recovery reflects protein response to the relaxation of the chromophore in SRII in the transition from the K to the “post-K” (the KL state analogous to the case of BR) with a time constant of 30 ps, which is comparable to the time constant reported for the BR protein response.

Figure 6. (a) Picosecond time-resolved UVRR spectra of SRII (probe laser, 225 nm; pump laser, 450nm). The top trace is the probe-only spectrum divided by a factor of 40. The other spectra are time-resolved difference spectra. The accumulation time for obtaining each spectrum was 80 min. (b) Temporal intensity change of the W3 band. Markers indicate the intensity changes measured at each delay time relative to the intensity in the probe only spectrum. Solid line is the best-fit with a function of [A1 × δ(t) + A2 × exp(−t/τrecovery) + A3] convoluted with the instrument response function. (Adapted with permission from reference (6). Copyright 2011 American Chemical Society.) (see color insert) 340 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 7. (a) Picosecond time-resolved UVRR difference spectra of WT-SRII (red) and the Y174F mutant (blue) in the 1400−1800 cm-1 region. Probe and pump wavelengths were 238 nm and 450 nm, respectively. The accumulation times for obtaining WT and Y174F mutant spectra were 79 and 76 min, respectively. (b) Details of the crystallographic structure of the SRII−HtrII complex in the unphotolyzed state (PDB ID=1H2S) in the vicinity of the retinal chromophore, Tyr174 and Thr204. Black dashed lines indicate hydrogen bonds. The helices of HtrII are in contact with the transmembrane helices F and G of SRII, in which Tyr174 and Thr204 are involved, respectively. SRII Tyr199 is hydrogen-bonded to HtrII Asn74. The HtrII helix involving Asn74 is displayed by a yellow-colored helix. (Reproduced with permission from reference (6). Copyright 2011 American Chemical Society.) (see color insert)

Another important result on picosecond protein response of SRII is that we succeeded to detecting structural and/or environmental changes of Tyr174, of which functional importance was pointed out previously (68, 73), taking the advantage that Raman band intensities of Tyr residues in proteins are greatly enhanced in UV excitation. Figure 7a shows a comparison of the picosecond time-resolved UVRR spectra of WT SRII and the Y174F mutant probed at 238 341 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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nm. Red and blue traces show the spectra of WT SRII and the W174F mutant, respectively. The spectral features of all the Trp bands observed in the difference spectra of the Y174F mutant were consistently close to those of WT SRII (data not shown), indicating that the mutation does not significantly perturb the Trp residues. However, Figure 7a shows that the negative bands of the Y8a mode in the Tyr174 spectra were much weaker than the corresponding bands in the WT spectra. This is strong evidence that Tyr174 is responsible for the intensity change of the Y8a band and that the structure and/or environment of Tyr174 changes in SRII following chromophore photoisomerization. Figure 7b shows the X-ray crystallographic structure of the SRII-HtrII complex around Tyr174 and Thr204, which are located close to the retinal chromophore and form a hydrogen bond with each other (74). One of the HtrII helices is displayed by a yellow-colored helix. The all-trans-retinal molecule binds to Lys205 via a protonated Schiff base linkage (magenta-colored side chain). The HtrII helices are in contact with the SRII transmembrane helices F and G (62), in which Tyr174 and Thr204, respectively, are located. Assay measurements of SRII mutants showed that Tyr174 and Thr204 are key residues in the SRII signal transduction pathway (73). Sudo et al. found that phototaxis function was lost in Thr204 or Tyr174 mutants and claimed that these residues are functionally important (68). They also demonstrated the presence of steric constraint between the C14H group and Thr204 (75). Furthermore, the extent of the steric constraint correlated with the physiological phototaxis response (68). They therefore proposed the model that the light signal is transmitted to HtrII from the energized interhelical hydrogen bond between Thr204 and Tyr174. The energized hydrogen bond is located in both the retinal chromophore pocket and in helices F and G that form the membrane-embedded interaction surface, and is transmitted to the signal-bearing second transmembrane helix (TM2) of HtrII (76). Thus, while the roles of Thr204 in structural changes and in negative phototaxis are clearly understood, the role of Tyr174 in these processes is not. Our data clearly show that the structure and/or environment of Tyr174 does change in accordance with chromophore photoisomerization. If the steric constraint between the C14H group and Thr204 is present, its effect would be exerted on Tyr174 simultaneously with photoisomerization. In fact, the contribution of Tyr174 to the Y8a band intensity was observed immediately upon photoisomerization. Thus, the present data strongly support the model described above.

Sensory Rhodopsin I (SRI) SRI was discovered in the halobacterium, regulating both negative and positive phototaxis. Kitajima-Ihara and co-workers characterized a new SRI-like protein from the eubacterium, Salinibacter ruber, and named this photosensor protein SrSRI (77). SrSRI showed remarkable stability compared to that of halobacterium, Halobacterium salinarum, SRI (HsSRI), which has been less studied due to its thermal instability. In SrSRI, it is suggested that the Cl− ion 342 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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binding site is located around the β-ionone ring of the retinal chromophore (78). Photochemical properties and structural changes of SrSRI are very similar to those of HsSRI (77). To analyze the protein response in the SRI, we employed picosecond time-resolved UVRR spectroscopy (7).

Figure 8. (a) Picosecond time-resolved UVRR spectra of SRI (probe laser, 225 nm; pump laser, 549nm). Red and blue traces are the spectra in the presence of 1 M NaCl (with Cl−) and 333 mM Na2SO4 (without Cl−), respectively. The top trace is the probe-only spectrum divided by a factor of 40. The other spectra are time-resolved difference spectra. The accumulation times were 100 and 70 min for obtaining the spectra with and without Cl−, respectively. (b) Temporal intensity change of the W3 band. Filled and open circles indicate the intensity changes measured at each delay time relative to the intensity in the probe only spectrum with and without Cl−ion, respectively. Solid lines are the best-fit with a function of [A1 × exp(−t/τrecovery) + A2] convoluted with the instrument response function. (Adapted with permission from reference (7). Copyright 2014 American Chemical Society.) (see color insert)

Figure 8a shows picosecond time-resolved UVRR spectra of SRI with and without Cl− ion probed at 225 nm. After photoexcitation, negative bands due to Trp residue were clearly observed within instrumental response. The negative bands represent depletion of the Raman intensity resulting from the change in protein structure upon photoisomerization. The negative bands decayed from 10 to 343 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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50 ps. This indicates that the Trp band intensity recovers due to further structural changes following the intensity depletion associated with photoisomerization. After 100 ps, the difference spectra did not change. The temporal intensity change of W3 band is shown in Figure 8b. The band intensity instantaneously bleached with in the instrumental response and recovered within 100 ps. The retinal chromophore isomerizes from the all-trans to 13-cis configuration in 0.64 ps (7). Thus, initial intensity depletion is attributed to the protein response to the chromophore isomerization. The time constants of band recovery of SRI with and without Cl− ion were estimated to be 17 ± 3 ps and 12± 2 ps, respectively. Both were similar to each other, indicating the similar structural changes of the Trp residue(s) following the K formation in the chromophore structure in both conditions.

Anabaena Sensory Rhodopsin (ASR) ASR is found in a freshwater cyanobacterium and exhibits a unique photoreaction different from other microbial rhodopsins. In the ground state, ASR has two stable configurations of retinal, all-trans, 15-anti (ASRAT) and 13-cis, 15-syn (ASR13C) (79), which exhibit photoinduced interconversion (80). Thus, the photoreaction of ASR is not cyclic but photochromic. The retinal chromophore is predominantly of the all-trans configuration in the dark-adapted state (DA-ASR) and contains a large fraction of the 13-cis configuration in the light-adapted state (LA-ASR) (80–82). In the photochromic reaction of ASR, ASRAT is photoconverted to the primary photointermediate, K-ASRAT, with the 13-cis, 15-anti configuration, and ASR13C is converted to the K-ASR13C intermediate with the all-trans, 15-syn configuration. Taking an advantage of the photochromic character of ASR, we can compare the protein responses upon the all-trans→13-cis and 13-cis→all-trans isomerization. Spectroscopic studies indicated that the photoreaction of both ASRAT and ASR13C includes several distinct intermediates (4, 80, 82–87). FTIR spectroscopy revealed that the distortion of the chromophore in K-ASRAT is localized in the Schiff base region, while that in K-ASR13C is distributed widely along the polyene chain. In addition, although the hydrogen-bond strength between the Schiff base and the water molecule in ASRAT is similar to that in ASR13C, the hydrogen bond is broken in K-ASRAT but not in K-ASR13C (83, 84). Femtosecond absorption spectroscopy demonstrated that the gross appearance of transient absorption of DA-ASR and LA-ASR is similar whereas the time constants of both intersystem crossing and buildup of K-ASR13C are much faster than those of K-ASRAT (4). We obtained the picosecond time-resolved UVRR spectra of DA- and LA-ASR to compare the primary protein dynamics beginning with ASRAT and ASR13C (8). Figure 9 shows picosecond time-resolved UVRR spectra of ASR probed at 225 nm. The initial states of photoreaction for green and pink traces in Figure 9 are the DA and LA states, respectively. The top traces are the probe-only spectra of DA-and LA-ASR, representing the UVRR spectra of each initial state. No difference was observed between UVRR bands for DA- and LA-ASR, suggesting that no significant difference exists in the structures of the Trp and Tyr residues 344 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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in DA- and LA-ASR. Figure 9 also shows time-resolved difference spectra. The difference spectra were obtained by subtracting the probe-only spectrum of the initial state from the pump-probe spectrum measured at each delay time from -10 to 1000 ps. In the time-resolved difference spectra, negative bands were clearly observed for Trp vibrational modes. The negative bands represent the intensity bleach of the Trp Raman bands due to the change in protein structure upon photoexcitation of the retinal chromophore. It should be noted that the frequency of the negative W3 band in the 10-ps difference spectrum of LA-ASR was higher than that of DA-ASR, suggesting that the Trp residue in LA-ASR giving rise to spectral changes upon retinal isomerization had a higher frequency component of the inhomogeneously broadened UVRR bands of the W3 mode than that of DA-ASR.

Figure 9. Picosecond time-resolved UVRR spectra of ASR (probe laser, 225 nm; pump laser, 549nm). Green and pink traces are the spectra of DA- and LA-ASR, respectively. The top trace is the probe-only spectrum divided by a factor of 40. The other spectra are time-resolved difference spectra. The accumulation times for obtaining the spectra of DA- and LA-ASR were 48 and 100 min, respectively. (Adapted with permission from reference (8). Copyright 2013 Elsevier B.V.) (see color insert)

The negative bands of Trp appeared within the instrument response. The negative bands decayed from 10 to 50 ps. Based on the temporal intensity changes of the Trp bands (Figure 10), the time constant of the intensity recovery was calculated to be 34 ± 7 ps for DA-ASR and 31 ± 4 ps for LA-ASR. Accordingly, the time constant of the change in protein structure during the photoisomerization of LA-ASR was almost the same as that of DA-ASR. In the unphotolyzed state of both DA- and LA-ASR, no difference was observed between the UVRR spectra. Under the present experimental condition, HPLC analyses revealed that LA-ASR contains 62.5% ASR13C, while DA-ASR is composed of 98.6% ASRAT. The UVRR spectrum of DA-ASR reflects the protein 345 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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structure of ASRAT. Although the UVRR spectrum of LA-ASR is a mixture of the spectral contributions of ASR13C and ASRAT, the spectral contribution of ASR13C can be examined by looking at the differences in the LA- and DA-ASR spectra.

Figure 10. Temporal intensity change of W3 band of (a) DA-ASR and (b) LA-ASR. Markers indicate the intensity changes measured at each delay time relative to the intensity in the probe only spectrum. Solid line is the best-fit with a function of [A1 × exp(−t/τrecovery) + A2] convoluted with the instrument response function. In panel (b), the black solid and gray dashed curves represent the temporal change of ASR13C and ASRAT, which was calculated based on the isomer composition in the unphotolyzed state of LA-ASR. (Adapted with permission from reference (8). Copyright 2013 Elsevier B.V.) (see color insert)

In the primary intermediate states appearing in the picosecond temporal frame, the intensity change of the Trp bands was observed both in the DAand LA-ASR spectra (Figure 9). Because DA-ASR solely consists of ASRAT, the observed temporal intensity changes of Trp bands in the time-resolved spectra starting in DA-ASR (Figure 10a) is originated from the photoreaction of ASRAT. Therefore, for the intermediate starting in ASRAT, the intensity of Trp bands bleached within the instrumental response time and recovered with a time constant of 30 ps. On the other hand, it was not straightforward to determine the spectral contribution of ASR13C in the time-resolved spectra starting in LA-ASR (Figure 10b), because the absorption coefficients at the pump pulse wavelength (80) as well as the quantum yields of photoisomerization (88) are different in ASRAT and ASR13C. The black solid and gray dashed curves Figure 10b shows estimated spectral contributions of ASRAT and ASR13C in the temporal intensity changes in the time-resolved UVRR spectra of LA-ASR, based on the isomer composition. Wand and co-workers investigated the ultrafast relaxation 346 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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process in photoreactions of the chromophore both in ASRAT and ASR13C by time-resolved absorption spectroscopy (4). They succeeded in isolating the spectral contributions of ASRAT and ASR13C in the LA-ASR spectra. Both in the photoreaction of ASRAT and ASR13C, the photoisomerization of retinal occurs within sub-picoseconds. Accordingly, the initial intensity bleach observed in the present time-resolved UVRR spectra is attributable to the protein structural change due to retinal photoisomerization occurring within a picosecond. The intensity of the Trp bands recovered with a time constant of 30 ps both for ASRAT and ASR13C. The photoisomerized ground-state intermediate is formed within sub-picoseconds (4). It does not convert back to the original state because the barrier height for the torsion around the C13=C14 bond is too high to pass over it in thermal reaction. The observed intensity recovery can be explained not by a recovery of the original structure, but by a transition from the K intermediate to the subsequent intermediate showing different band intensities. So far, the 30-ps process has not been reported in the photoreaction of ASR. This process is attributed to further rearrangements of the protein moiety around retinal. The rate of the structural rearrangement in K-ASRAT is similar to that in K-ASR13C. The robustness of the relaxation rate implies that the mode of structural change of retinal does not significantly affect the protein response of surrounding residues.

Comparison of Primary Protein Responses in Microbial Rhodopsins We carried out real-time observation of the primary protein response to the chromophore isomerization in microbial rhodopsins based on the spectral changes in Trp bands. During the picosecond region, it is expected that residues in the vicinity of retinal change their structures. Figure 11 shows the crystallographic structure in the vicinity of the chromophore of BR. Three Trp residues are located in the retinal biding pocket. Trp86 and Trp182 sandwich the polyene chain of retinal. Trp189 are positioned near the β-ionone ring. Many amino acid residues are conserved among microbial rhodopsins. The three Trp residues are also conserved for BR, SRII, SRI, and ASR. Owing to the selective Raman enhancement, we can utilize the Raman bands of Trp as good probes to discuss the structural change around the chromophore in microbial rhodopsins. The temporal changes of the Trp band intensities among the four microbial rhodopsins described above were compared. It was commonly found that the intensities of Trp band in these rhodopsins bleached upon photoisomerization of retinal and recovered with tens of picoseconds. The intensity recovery of the Trp band was attributed to the rearrangement of the protein moiety following the structural relaxation of the chromophore. The comparison shows that the rates of rearrangements of the protein moiety are insensitive to the functions, ion binding, and direction of the isomerization among these rhodopsins, suggesting that the primary structural response of the protein moiety to the chromophore isomerization is very similar in the microbial rhodopsins. 347 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 11. Crystallographic structure in the vicinity of the retinal chromophore of BR (PDB ID=1C3W). Three Trp residues (Trp86, Trp182 and Trp 189 in BR) are conserved at the same positions among BR, SRII, SRI, and ASR. (see color insert)

The similar primary structural response of the protein moiety to the chromophore isomerization observed for the four microbial rhodopsins. Two pathways can be proposed for the propagation of structural changes from the chromophore to the protein moiety. One is a pathway through hydrogen bonding network including the protonated Schiff base. The orientation of the hydrogen bond of the protonated Schiff base changes upon the photoisomerization and can quickly perturb the protein moiety. The other is through van der Waals contacts between the retinal and the surrounding amino acid residues. In the time-resolved visible resonance Raman spectra, an intense Raman band due to the hydrogen-out-of-plane (HOOP) wagging mode of the retinal was observed in the J intermediate of BR (1). The HOOP band gains its intensity when the polyene chain is distorted (89). In fact, no intense HOOP band is observed for polyenes in solution because the polyene quickly adopts the stable planar form following the photoisomerization in solution. The distorted polyene chain with measurable lifetime in the proteins would be due to highly packed structure around the retinal. The propagation of structural changes through the van der Waals contacts is possible in such highly packed environment around the chromophore. It is interesting to compare the primary protein response of the microbial rhodopsins with that of visual pigment rhodopsin. Time-resolved UVRR spectra of visual pigment rhodopsin have been reported by Kim and co-workers. Visual pigment rhodopsin undergoes the 11-cis to all-trans isomerization (14). They reported that the intensity of negative Trp band gains up to 20 ps using probe wavelength of 233 nm. This temporal behavior is similar to that of BR probed at 238-nm excitation. The similarity of the protein response to the chromophore isomerization suggests that the protein response is insensitive to the retinal configuration and that a time constant of tens of picoseconds is common as the protein response to the retinal isomerization. 348 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Concluding Remarks

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It is essential to clarify how external stimuli can bring about site-specific structural changes to understand the mechanism of protein functions. For that purpose, measurement technique with high time resolution and site-selectivity is necessary. Time-resolved UVRR spectroscopy is able to probe the structural dynamics of specific sites in protein structure by selectively enhancing the vibrational Raman bands assignable to aromatic amino acid side chains as well as polypeptide bonds with picosecond time resolution. We here demonstrated high potential ability of time-resolved UVRR spectroscopy in observation of primary protein response to chromophore isomerization in microbial rhodopsins.

Acknowledgments We are grateful to Professors Hideki Kandori (Nagoya Institute of Technology) and Yuki Sudo (Okayama University) for kindly supplying the protein samples and stimulating discussions. We thank Mr. Seisuke Inada (Osaka University) who carried out time-resolved UVRR measurements of ASR. We also thank Dr. Mikihiro Shibata and Mr. Junya Yamada (Nagoya Institute of Technology) for hard work for preparation of a large amount of BR samples. The works presented in this review were supported by a Grant-in-Aid for Scientific Research in the Priority Area “Molecular Science for Supra Functional Systems” (No. 19056013) to Y.M. from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a Grant-in-Aid for Scientific Research on Innovative Areas “Soft Molecular Systems” (No. 25104006) to Y.M. from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a Grant-in-Aid for Scientific Research (B) (No. 20350007) to Y.M. from the Japan Society for the Promotion of Science, and a Grant-in-Aid for Young Scientists (B) (No. 23750015) to M.M. from the Japan Society for the Promotion of Science.

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Chapter 17

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Biophysical Methods for the Studies of Protein-Lipid/Surfactant Interactions Shuo Sun,1,3 Caleb I. Neufeld,2 Ramil F. Latypov,3 Bernardo Perez-Ramirez,2,3 and Qiaobing Xu*,1,2 1Department

of Chemical and Biological Engineering, Tufts University, 4 Colby Street, Medford, Massachusetts 02155, United States 2Department of Biomedical Engineering, Tufts University, 4 Colby Street, Medford, Massachusetts 02155, United States 3BioFormulations Department, Global BioTherapeutics, Sanofi, 1 The Mountain Road, Framingham, Massachusetts 01701 United States *E-mail: [email protected].

Comprehensive characterization of protein-lipid/surfactant interaction is crucial for both biopharmaceutical formulation development and drug delivery research. Revealing the mechanism of this interaction will facilitate novel ideas for drug delivery system design, improve protein stability, and optimize protein formulation. Protein/lipid interaction must be analyzed by many different techniques, using various perspectives, in order to approach the full view of conformational, stoichiometric and calorimetric changes accompanying different interaction stages. Among these methods of analysis, biophysical technology is well-suited to monitor the atomic and molecular changes resulting from two-component interactions. The purpose of this chapter is to present some of the most popular biophysical methods employed for such investigations. The basic principles, advantages and limitations of each technique, their roles, applications and limitations, and a few case studies of each are discussed.

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Introduction Surfactant molecules are organic compounds that are amphiphilic: they contain hydrophobic groups, usually an alkyl tail, and hydrophilic head groups, typically a polar moiety. At a sufficiently high aqueous concentrations (the critical micelle concentration or CMC), surfactant molecules associate via their hydrophobic chains to form micelles, composed of a hydrophobic inner phase and a hydrophilic water-exposed exterior (1). Surfactants have a wide range of applications from agriculture and the food industry, to cosmetics and the pharmaceutical industry (2, 3). In pharmaceutics, surfactants can reduce surface tension and thus a formulation’s free energy, reducing protein-protein and protein surface interaction (4, 5). They are commonly used in protein formulation to prevent physical instability during purification, filtration, transportation, freeze-drying, spray drying, storage, and delivery (6–12). Interaction of surfactants with biomolecules depends on the charge on surfactant head groups, hydrophobic tail lengths, and the nature of the biomolecule interacted with (13). Additionally, proteins interact differently with the monomeric and the micellar forms of surfactants (1). In order to evaluate their properties, roles and mechanisms in protein formulation, protein–surfactant interactions must be studied by different biophysical techniques, providing full view of the structural, stoichiometric and calorimetric changes accompanying different interaction stages. Nonionic surfactants bind weakly to proteins. They are widely used as stabilizers in protein formulations to prevent protein aggregation due to transportation or sorption. Polysorbate 20 (PS20) and Polysorbate 80 (PS80) are the two most frequently used non-ionic surfactants in protein formulations (7). PS20 and PS80 are used to stabilize monoclonal antibodies including Rituxan®, ReoPro®, and Humira® (14). However, due to the potential formation of peroxides in PS20 and PS80, they can oxidize proteins they are formulated with, negatively impacting their stability (15, 16). Moreover, recent studies showed that polysorbate raw material can give rise to free fatty acid (FFA) particles which are granular in shape and several microns in size (17, 18). The degree of particle formation tested under recommended storage conditions was found to be dependent on polysorbate type and concentration. Because therapeutic protein formulations must exhibit sufficient stability over a long period of time to be commercially appealing, polysorbate’s propensity to form particles is an undesirable formulation characteristic. New, FFA-free surfactants are intensely needed for use in place of polysorbates. However, only a few surfactants are currently FDA-approved as excipients for inclusion in parenteral medications (19). One example is Poloxamer 188, (known as Pluronic F68) which is found in commercial formulations, many of which are delivered intravenously or subcutaneously, at concentrations up to 0.6% w/v. Another promising surfactant is Triton X-100, which is used mainly in topical formulations such as gels and ointments. A notable exception is Fluarix® from GSK, an FDA-approved injectable product which contains Triton X-100 at 0.0085% w/v (19). Ionic surfactants are usually not used to stabilize proteins because they can bind to both polar and nonpolar groups in proteins, causing denaturation (20, 21). 356 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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For example, sodium dodecyl sulfate (SDS) has been extensively used to denature proteins for subsequent electrophoresis (from whence the term SDS-PAGE). However, these surfactants have been reported to posess a protein-stabilizing effect as well. SDS has been found to play two opposite roles in the folding and stability of proteins: At low surfactant/protein molar ratios, it acts as a structure-stabilizing additive, but at increased amounts, it behaves as a destabilizer (20, 22, 23). The intravenous protein drug Proleukin™/Aldesleukin (Novartis) is an example of this interaction. At 0.18 mg per 1.2 mL (between 95-250 µg SDS per mg of interleukin-2), SDS acts to mitigate ionic and hydrophobic interactions, which would otherwise lead to the formation of non-covalent microaggregates (24). Cationic surfactants, such as cetyltrimethylammonium bromide (CTAB), bezalkonlum chloride (BZK) and cetrimide, have been widely applied as penetration enhancers in transdermal drug delivery. Despite their potential to damage human skin, they cause a larger level of transdermal flux than anionic surfactants (25–28). Cationic surfactants act on the keratin fibrils of cornified cells, resulting in a disrupted cell/ lipid matrix. Additionally, they interact with skin proteins via polar interactions and hydrophobic binding. These interactions result in pendant ionic head groups and subsequently swelling of the stratum corneum (26). Lipids play a key role in biological, pharmaceutical and medical research (29, 30). Drugs and biologically active molecules present in the extracellular medium must either cross the membrane to act inside the cell, or bind to membrane receptors or the lipid matrix of the membrane, (31). Liposomes were first described in 1965 (32). They are lipid bilayer nanoparticles or colloidal carriers, usually 50-500nm in diameter, which form spontaneously when certain lipids are hydrated in aqueous media (32, 33). Due to the “designable” nature of lipids, liposomes as drug delivery systems have undergone significant improvement. Liposomes are one of the few systems that have successfully translated to the clinic and the market (34). For example, DaunoXome® (daunorubicin citrate liposome injection), launched in 1996 in the US, is a prescription drug indicated as a first line cytotoxic therapy for advanced HIV-associated Kaposi’s sarcoma, and is delivered in a liposomal formulation (35). Protein delivery is also carried out in liposomal formulations: For example, PEGylated liposomal delivery of streptokinase dramatically improved its pharamacokinetics in a rat model (36). The activity of many biomolecules and drugs depend upon binding to biological membranes or translocation to the inner lipid leaflet (31). Furthermore, surfactants used as a protein excipient must remain stable over a substantial period of time to effectively be marketed. Understanding of the mechanisms involved in protein and lipid/surfactant interaction provides the basis for rational strategies to optimize these results. Thus, in the following section, we will discuss the principles of some key biophysical characterization methods essential to mechanistic characterization of protein/lipid interactions, presenting a few interesting examples of each technique, as well as their limitations. This review will serve the protein-focused investigator in choosing appropriate analytical techniques for each interaction he wishes to characterize. 357 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Methods

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Circular Dichroism Spectroscopy (CD) Plane-polarized light can be viewed as vectors made up of 2 circularlypolarized components of equal magnitude, one rotating counter-clockwise (left handed, L) and the other clockwise (right handed, R). When a light source passes through a sample, due to the differences in absorbance of left and right circularly-polarized light by the chromophores, elliptical polarization can be determined. The numerical relationship between the difference in absorbance of the L and R circularly-polarized components (ΔA = AL − AR) and ellipticity (θ, in degrees), is θ= 32.98 ΔA. In proteins, chromophores of interest include the peptide bond, aromatic amino acids, and disulfide bonds. Detailed information on their absorption range is described in Table 1.

Table 1. Summary of Absorbent Information of CD Spectroscopy Absorbent

Wavelength measured

Structure of protein

Comments

Peptide bond

Below 240 nm

Secondary structure

α-helix, β-sheet and turns can be measured in the far UV region

Aromatic amino acid

Between 260-320 nm

Tertiary structure

Absorbent aromatic amino acids: Phenylalanine, tyrosine, tryptophan

Disulfide bonds

Around 260 nm

Tertiary structure

Weak broad absorption bands around 260 nm

Peptide bonds are measured mainly by far-UV CD, with a wavelength between 240-180 nm. Different secondary structures (α-helix, β-sheet, random coil etc.) of proteins have their own particular CD absorbance signals due to their unique structural properties. Figure 1 shows the CD fingerprints of different secondary protein structure components. α-Helical structure displays dual minima at 222 nm and 208 nm, while β-sheet topology possesses a minimum around 216 nm, providing a secondary probe for protein conformations. By applying an empirical database-based algorithm to deconvolute protein spectra, estimation of the identified secondary structure compositions can be calculated. SELCON, VARSLC, CDSSRR and CONTIN have been widely used in various applications (37–40). 358 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 1. Far UV CD spectra associated with various types of secondary structures. Solid line, α-helix; long dashed line, anti-parallel β-sheet; dotted line, type I β-turn; cross dashed line, extended 31-helix or poly (Pro) II helix; short dashed line, irregular structure. Reproduced with permission from reference (41). Copyright 2005 Elsevier.

Recombinant human growth hormone (rhGH) contains only α-helix and random coil structures. Thus, the 222-nm signal in the CD spectra is a good indication of changes in α-helix content. Thus, CD may be used to asses the stability of the protein upon interaction with surfactants. When rhGH was spray dried without any excipients, the α-helix content was decreased in comparison to that of bulk rhGH. By formulating rhGH with PS20(0.05%, w/w) for spray drying process, however, α-helix content remained close to that of the control. Thus, the interaction of PS20 can effectively protect the protein against aggregation and 359 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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maintain the conformational stability of rhGH by excluding protein molecules from exposure to the air–liquid interface at the surface of droplets (42). The interaction between human serum albumin (HSA)/bovine serum albumin (BSA) and various lipids has been evaluated by CD spectrum to examine the effects of lipid complexation on protein conformational stability (40, 43). It has been reported that there were no major changes in α-helix content for cholesterol (Chol) and dioleoylphosphatidylethanolamine (DOPE)–protein complexes; however, a major secondary structural transition from α-helix to β-sheet was noted for 1,2-dioleoyl-3-(trimethylammonium)propane (DOTAP) and (dioctadecyldimethyl)ammonium bromide (DDAB)-protein complexes. These observations indicate that the interaction between DDAB/DOTAP and protein resulted in partial protein unfolding, but cholesterol and DOPE stabilized protein conformation, confirming the complex nature of protein/lipid interaction. CD spectroscopy has been widely used to analyze structural changes in biomolecules, in qualitative and even semi-quantitative fashions. Applications of CD are not limited to conformational assessments of protein, thermodynamics and kinetics of folding-unfolding of macromolecules; protein-lipid/surfactant interaction studies also benefit from CD. However, due to the low sensitivity to structural changes, relatively higher sample concentrations are required. In some cases, this may lead to precipitation of complexes, resulting in a paradoxical reduction of signal intensity. Nevertheless, circular dichroism remains an essential method of biophysical protein analysis. Ultraviolet–Visible Absorption Spectroscopy (UV-vis) In spite of the vigorous development of more sophisticated techniques, UVVis spectroscopy remains an indispensable tool for obtaining an initial insight into the interaction between protein and surfactants or lipids. UV-Vis is based on the absorption of energy by the studied molecule upon interaction with specific light sources (e.g. xenon flashing lights and deuterium lamps). The energy of the light promotes electrons from the ground state to an excited state, leading to a decrease in transmitted light. Figure 2 shows the various kinds of electronic excitation that may occur in organic molecules. Of the six transitions outlined, only the two lowest energy ones (n→π* and π→π*) are achieved by the energies available in the UV-Vis spectrum, and are thus the transitions observed in protein absorption spectra. (44). Spectra are obtained by measuring the absorption of light as a function of its wavelength. Molecules with electrons in delocalized aromatic systems often absorb light in the near-UV (150–400 nm) or the visible (400–800 nm) region. One example of UV-vis spectroscopy in protein structural characterization can be observed in the interaction between SDS and acid-denatured cytochrome c (45, 46). At low SDS concentration (1.0 would imply enthalpic governed binding, and slope 13 mN·m-1, the orthorhombic lattice structure with NNN-tilted alkyl chains is found in the whole pressure range. The surface-pressure−temperature phase diagram of 1-monostearoyl-racglycerol monolayers demonstrates the temperature effect on the phase behavior in more detail (Figure 5). It is seen that already at 20 °C, the intermediate oblique phase disappears in the racemic monolayer and a direct NN → NNN transition of the orthorhombic lattices takes place.

Figure 5. Surface pressure−temperature phase diagram of the racemic 1-stearoyl-rac-glycerol monolayer. Reproduced with permission from reference (36). Copyright 2015 American Chemical Society.

The GIXD results obtained over large pressure interval at 5, 10, 15 and 20 °C indicate impressively that also in the racemic 1-monostearoyl-rac-glycerol monolayer symmetry breaking takes place at low temperatures in a small pressure region. In this low-temperature region, an oblique (Obl) intermediate phase is observable in the racemic monolayer between orthorhombic chain lattices with NN and NNN tilt directions. The extension of this oblique phase increases with decreasing temperature. The NN→NNN phase transition splits into two successive NN→Obl and Obl→NNN transitions. The appearance of an oblique phase between the two rectangular phases in monolayers of racemic surfactants is a new and surprising finding, not yet described by a theoretical phase diagram of monolayers (26). 385 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

The phase diagram of the 1-monostearoyl-rac-glycerol monolayer was constructed on the basis of reliable two-dimensional lattice structures. So far, it is the first phase diagram of a monoacylglycerol monolayer published in the literature. It is much simpler than the theoretical phase diagram satisfying the symmetry requirements of the Landau theory and the thermodynamic and structural data on fatty acid monolayers (26) often discussed in the literature and reviews. The presented results of 1-monostearoyl-rac-glycerol monolayer (36) can help to avoid the impression that the fatty acid phase diagram can be a standard for other homologous surfactant series.

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Stearylamine-Glycerol: Chiral Discrimination The functionality of these molecules depends crucially on their chiral structure. Chiral objects are abundant in nature and are operative in many biological systems at both microscopic as well as macroscopic levels, so that there is a continued interest in studying the effect of chirality on the monolayer morphology (37). In the most cases, only one enantiomer is biologically active, and the biological units lose their functionality when their chiral structures are altered. A further challenge is to understand the well-known problem of homochiral evolution in nature. A chiral molecule has at least one asymmetric carbon atom connected to four different chemical groups so that permutation of any pair of these groups leads to the mirror image of the original molecule. Amphiphilic monolayers with chiral centers represent unique systems to study chirality-dependent interactions under defined conditions (38). Generally, two categories of enantiomer pair mixtures are distinguished: (1) homochiral discrimination with favored interaction between the same enantiomers (ED-D or EL-L > ED-L), (2) heterochiral interaction with favored interaction between the different enantiomers (ED-D or EL-L < ED-L), where for strong interactions racemic compounds can be formed. The combination of surface pressure studies with imaging techniques and GIXD measurements is optimal for the experimental characterization of chiral discrimination in amphiphilic monolayers. The shape and inner texture provide valuable information about the long-range ordering of the condensed phase domains. A variety of chiral discrimination in mesoscopic scale has been evident. This concerns not only general differences in shape and inner texture of the condensed phase domains between the enantiomeric forms and the racemic mixtures but also between the two enantiomeric forms. Typical examples for handedness of the two enantiomeric forms in opposite directions have been demonstrated by the mirror-image relation of the respective condensed phase domains. In the case of very small energetic differences, textural and structural differences between the different chiral forms are relatively small or cannot be observed at extreme experimental conditions. In the following, chiral discrimination effects of stearylamine-glycerol monolayers are discussed by comparison of mesoscopic domain shapes and GIXD results (39, 40). 386 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Obviously, at most, small energetic effects occur at the complete mixing of the two enantiomers. Although the π-A isotherms of the enantiomeric forms and the racemic mixtures agree largely to each other, the filigree domain patterns show remarkable differences, obviously driven by the chirality. This is demonstrated by fluorescence microcopy of nonequilibrium domains, shown in Figure 6. The domains of the R(+) enantiomer are curved clockwise, those of the S(-) enantiomer counterclockwise, whereas those of the racemic mixtures are without a specific sense of direction.

Figure 6. Chiral discrimination of 1-stearylamine-glycerol monolayers: (left) Fluorescence microscopic images of nonequilibrium domains with the enantiomeric forms (above) and racemic mixture (below); (right) contour plots of the S(-) enantiomeric monolayer at 20 °C; above at 1 mN/m, middle at 10 mN/m, below at 35 mN/m. Adapted with permission from reference (39) . Copyright 2015 American Chemical Society.

The lattice structures of the 1-stearylamine monoglycerol monolayers are also affected by the chirality of the molecules. The enantiomeric monolayers have an oblique lattice (Figure 6, right, middle), where at compression the tilt direction changes continuously from angles nearly toward NN direction (Figure 6, right, above) to angles nearly toward NNN direction (Figure 6, right, below). In opposite, the condensed phases of the racemic mixtures give rise to rectangular-centered lattices (Figure 7). Here, a phase transition occurs that is accompanied by a change in the tilt direction from NN at 1 mN·m-1 to NNN at 5 mN·m-1. Both enaniomeric monolayers and their racemic mixtures have highly tilted molecules at low surface pressures. In agreement with the filigree domain patterns, the low position correlation allows the conclusion of low ordering of the alkyl chains in the filigree domain structure. 387 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 7. BAM image of a racemic 1-stearylamine-rac-glycerol domain in equilibrium (LEFT) and contour plots of racemic 1-stearylamine glycerol monolayer at 20 °C (RIGHT) above at 1 mN/m, below at 10 mN/m. Adapted with permission from reference (39). Copyright 2015 American Chemical Society. Adapted with permission from reference (40). Copyright 2015 American Chemical Society.

Similar chiral discrimination effects display the monoglycerol ether monolayers. In the enantiomeric monolayers domain morphology, chiralitydependent spirals (41, 42) curved in the same sense grow from initially compact 1-O-hexadecylglycerol domains at the end of the plateau region of the π-A isotherm., whereas the racemic monolayers develop spirals curved in the two directions of both enatiomeric forms. Domain morphology and lattice structure textures of the racemic 1-stearylamine-rac-glycerol monolayer in equilibrium are presented in Figure 7 bottom (42). After compression stop within the main phase transition region at T ≥ 30°C, the fractal-like nonequilibrium domain patterns initially formed, are slowly transformed to circular, faceted, or cardioid domains subdivided in six or seven segments at equilibrium. Figure 7, left shows a 6-fold segmented domain. It is seen that the contrast of the condensed phase patterns is comparably low. The racemic 1-stearylamine-rac-glycerol monolayers form a rectangular centered lattice characterized by changing the tilt direction of the alkyl chains from NN to NNN already at low surface pressures (see the contour plots of Figure 7, right). Effect of Unsaturation in Fatty Acids Unsaturated fatty acids abundant in biological systems are important constituents of biological membranes (43, 44). The variation of the unsaturated chains in type, number, position, and geometric configuration of unsaturated carbon-carbon bonds is obviously related to the broad diversity in the biological properties of lipids. The effect of unsaturated fatty acids on the membrane 388 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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characteristics concerns essentially the improvement of fluidity and the control of the phase transition behavior. Correspondingly, unsaturated fatty acids found versatile practical applications in the fields of food, pharmaceutical, and cosmetic industries. For a better understanding of the role of unsaturated fatty acids in biological membrane systems, insoluble monolayers have been used as indispensable model systems very early. Already in the pioneering works of Langmuir (45) and Adam (46) the effect of unsaturation in fatty acids on the spreading behavior of the monolayers has been studied. Following studies of unsaturated fatty acids directed attention to the effect of cis-trans isomerization, position, and number of the double bonds on the surface pressure properties of fatty acid monolayers (47–53). Despite the permanent interest and studies of unsaturated fatty acid monolayers, extensive examinations of their structure and morphology are seldom. Only Kato et al. (54) described some morphological structures of several cis unsaturated fatty acids monolayers. In the following, some results on the effect of unsaturation in fatty acids on the main characteristics of monolayers obtained in ref. (55), are reviewed. Unsaturated cis and trans fatty acid monolayers with the same hydrocarbon chain length having the double bond at the same position (ω9, Δ13) should be compared. The effect of cis and trans bonds on the conformation of the hydrocarbon chain can be seen in Figure 8, left, exemplifying the chemical structure of the studied amphiphiles. The effect of the hydrocarbon chain length was investigated with additional studies of the homologous cis-nervonic acid (ω9, Δ15) and trans-elaidic acid (ω9, Δ9). The results of the effect of unsaturation of fatty acids on the main characteristics of their monolayers are based on the combination of π-A isotherms, BAM, and GIXD measurements.

Figure 8. Effect of unsaturation in fatty acids on the main characteristics of Langmuir monolayers. (left) Chemical structure of selected cis- and trans-unsaturated fatty acids; (right) contour plots of the corrected diffraction intensities as a function of the in-plane (Qxy) and out-of-plane (Qz) components of the scattering vectors of the unsaturated fatty acid monolayers measured at 5 °C: (a) brassidic acid, 10 mN/m, (b) erucic acid, 15 mN/m, (c) nervonic acid, 10 mN/m, and (d) elaidic acid, 15 mN/m. Adapted with permission from reference (55). Copyright 2015 American Chemical Society. 389 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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The thermodynamic analysis, performed on the basis of the π-A isotherms, has shown that the presence of double bonds in the hydrocarbon chain affects the thermodynamic characteristics of the monolayer in a similar way as shortening the alkyl chain length. The effect of unsaturation is much smaller for the trans isomers than for the cis isomers indicating the stronger polar character of the cis double bond. The mesoscopic condensed phase domains formed in the fluid/condensed coexistence region reveals considerable differences between the cis and the trans conformations at the same molecular structure of the unsaturated fatty acid. At the beginning of the undisturbed growth process of the nonequilibrium erucic acid and nervonic acid domains, six main axes grow from the center (Figure 9 b, c, left). The crystalline nature of these nonequilibrium domains prevents a fast transformation to equilibrium shapes. On the other hand, the transition of well-shaped dendritic nonequilibrium domains of the trans-brassidic acid to the equilibrium domains occurs already at a slow compression rate. The round equilibrium domains are subdivided by straight lines into seven segments of different reflectivity meeting in the center. At the main axes the chain tilt azimuth jumps by a defined angle (Figure 9 a, right).

Figure 9. BAM images of (a) equilibrium trans-brassidic acid domains, (b) nonequilibrium cis-erucic acid domain, (c) nonequilibrium cis-nervonic acid domain. Reproduced with permission from reference (55). Copyright 2015 American Chemical Society. Despite the similarity of the brassidic acid domains to those of 1monoglycerides (36), their morphological characteristics, such as the tendency to form irregular shapes, the smaller differences in the reflectivity of the segments, and the less sharp domain and segment boundaries, indicate a more soft condensed phase and smaller line tension. Completely different to the behavior of saturated fatty acid monolayers, for all cis and trans unsaturated fatty acids studied, only an orthorhombic lattice with NN tilted alkyl chains is found, and no indication for a phase transition between different condensed phases could be observed, independent of different surface pressures and temperatures (see the contour plots of Figure 8, right). The conformity of the cis and trans unsaturated fatty acid monolayers to only one lattice type having an orthorhombic unit cell with NN tilted alkyl chains correlates obviously with the obstructive effect of the double bond in the alkyl chain on the molecular ordering. 390 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Amide Amphiphiles Based on Derivatized Ethanolamine The role of the amide group as integral part of a number of important membrane phospholipids and other amphiphiles is of outstanding interest in biological systems (56–58). According to their far-reaching significance in biological systems, the main characteristics of the monolayers of selected amide amphiphiles based on derivatized ethanolamine should be demonstrated. Ethanolamine is the central building block (59) in numerous membrane phospholipids and also structural component of N-acylethanolamines (NAEs), N-,O-diacylethanolamines (DAEs) and N–acylphosphatidylethanolamines (NAPEs) Therefore, the amphiphilic derivatives of ethanolamine found special interest not only because of their occurrence in a wide variety of animals, plants, and microbes (3,5) but also due to their interesting biological, pharmaceutical, and medicinal properties (60–63). In addition, ethanolamine is also the structural component of N-acylethanolamines, N-,O-diacylethanolamines (4) and N–acylphosphatidylethanolamines, which are present in a wide variety of organisms. In the following, we are focussing on the characterization of selected amide amphiphile’s monolayers of different chemical structures, in particular of mimetic monolayer systems based on derivatized ethanolamine. Acylethanolamines (NAEs) Because of numerous biological and medicinal properties of interest, NAEs have been of permanent interest. Obviously, their production in a variety of organisms increases dramatically as a response to stress, such as injury in animals and dehydration in plants (58, 64). NAEs reveal anti-inflammatory, antibacterial, and antiviral properties, which may have considerable therapeutic potential (58). In the following, the tailored amide amphiphile N-myristoylethanolamine (tetradecanoic acid- (2-hydroxyethyl) amide, TDAHA) is selected to obtain information on its specific interfacial characteristics. It is important to note that it was necessary to synthesize TDAHA of high purity to avoid artifacts in the characteristic features (65). The combination of π-A isotherms, the morphology of the condensed phase domains, the lattice structure of the condensed phase, and the role of hydrogen bond systems (-NH···OC-) was most advantageous to characterize the monolayers (65). The results are presented in Figures 10 and 11. The characteristics of the π-A isotherms measured between 3 and 20 °C indicate the main first-order phase transition from the fluid phase to the condensed phase and are similar to those of usual amphiphiles (Figure 10). However, at temperatures of 27°). It is seen that the oblique lattice structure found in the monolayers is independent of the process of monolayer formation. The results of the two-dimensional lattice structures obtained by GIXD indicate that real crystalline structures are formed in the condensed phase of the adsorbed monolayers. The condensed phase formed at the adsorption of DHBAA in the mesoscopic scale shows an interesting phenomenon. Two different types of topographical textures of the condensed phase are formed above and below 10 °C (Figure 15, left). The ordering of the domain textures was determined using BAM by rotating the analyzer in the reflected laser beam and comparison at the parallel and crossed polarizers. The main growth directions of the condensed phase textures were correlated with the two-dimensional lattice structure (see Figure 15, right) (72). As can be seen, for both domain types, the main or preferred growth directions of the dendritic domains are related with the low indexed lattice directions although both texture types have the same lattice structure. For the lower temperature region lattice (T ≤ 10°C), the preferred growth directions correspond to the [01] and directions so that the bisector of the main domain direction is parallel to the 397 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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lattice direction. For T ≥ 10°C, the growth directions correspond to the [01] and [10] lattice directions for intersection angles between the growth directions of 120° and are parallel to the and [10] lattice directions for intersection angles between the growth directions of 150° (77).

Figure 15. Correlation of mesoscopic textures (left) with 2D lattice structures (right) of DHBAA for lower (T=5 °C, top) and higher (T=15 °C, bottom) temperatures. The positions of the molecules are represented by filled circles. The thick grey lines in the crystal structure illustrate the growth directions in the domains. The arrows symbolize the azimuthal tilt direction of the molecules. A scheme of the unit cell is inserted. Reproduced with permission from reference (77). Copyright 2015 American Physical Society.

Phase Transition at Coadsorption of Dodecanol/SDS SDS represents the most widely used model surfactant in colloid and interface research. However, caused by the synthesis, n-dodecanol is the most frequent contaminant of SDS and hardest to remove. Therefore, the effect of n-dodecanol, even in trace amounts, on the coadsorption of SDS/dodecanol mixtures from aqueous solutions has been studied over decades (78–83). The efforts to provide a more comprehensive characterization of the coadsorption of SDS/dodecanol mixtures were motivated by the evidence that a first-order phase transition can occur in adsorbed monolayers (Gibbs monolayers) of amphiphiles dissolved in aqueous solution. (76, 84–86). 398 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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It was possible to clarify the reasons for the dominant effect of dodecanol traces on the surface properties of SDS by coupling dynamic surface tension π(t), BAM and GIXD results (86). Comparative studies of the pure components SDS and dodecanol as well as their mixtures indicated that the adsorption kinetics of the two single components and the properties of their adsorbed monolayer are fundamentally different. The adsorbed monolayers of SDS do not show any phase transition, even at bulk concentrations above the critical micelle concentration and low temperatures, so that a condensed phase cannot be formed. The adsorbed dodecanol monolayers show a first-order phase transition and the subsequent growth of condensed phase domains at very different experimental conditions. Also the adsorbed monolayers of the dodecanol/SDS solutions, having dodecanol only in trace amount, undergo a first-order phase transition with subsequent formation of condensed phase domains. In adsorption equilibrium, the surface is largely covered by the condensed phase. Figure 16, top shows the π(t) coadsorption of an aqueous 12 µM dodecanol/3 mM SDS solution and the corresponding development of the condensed phase at the indicated times.

Figure 16. π(t) Coadsorption of dodecanol/SDS from an aqueous 12 µM dodecanol/3 mM SDS solution and the corresponding BAM images at the indicated times. All images have the same scale of 325 µm × 325 µm. Reproduced with permission from reference (86). Copyright 2015 American Chemical Society. 399 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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The BAM studies demonstrate not only large similarity in the domain shape of the adsorbed monolayers of dodecanol/SDS mixtures and pure dodecanol (Figure 17, top) but also the increasing coverage of the surface with the condensed phase domains over a large range of the experimental conditions. The morphological features of the condensed phase formed during the coadsorption of SDS/dodecanol mixtures are dominated by the fundamental features of dodecanol, although present only as minor components in the aqueous solution. Reaching the adsorption equilibrium, the surface is largely covered by the condensed phase.

Figure 17. (top) Similarity of the BAM images obtained after the break point of the π(t) adsorption curves at 10 °C (a) for pure 12 µM dodecanol solution, (b) for a mixed 10 µM dodecanol/3 mM SDS solution. (bottom) Comparison of the contour plots of the corrected X-ray intensities vs the in-plane component Qxy and the out-of-plane component Qz of the scattering vector Q of pure dodecanol and dodecanol/SDS mixtures on water. (Left) Dodecanol spread from a 1mM chloroform solution and compressed to 3 mN/m, 2.5 °C. (Middle) Adsorbed from a 15 µM aqueous dodecanol solution, equilibrium surface pressure π = 26 mN/m, 4 °C. (Right) Coadsorption from a 15µM dodecanol/3mM SDS solution, 49 mN/m, 5 °C. Adapted with permission from reference (86). Copyright 2015 American Chemical Society. Additional information on the structure of the condensed phase can be provided by GIXD measurements. Figure 17, bottom shows contour plots of the corrected intensities as a function of the in-plane scattering vector component 400 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Qxy and the out-of-plane scattering vector component Qz for single-component dodecanol and dodecanol/SDS mixtures on water: (left) spread dodecanol, (middle) adsorbed dodecanol, (right) coadsorption from a 15 µM dodecanol/3mM SDS solution. Experimental details are discussed in ref. (86). In all cases, only one diffraction peak is observable, indicating a hexagonal packing of nontilted dodecanol molecules. The condensed monolayer phase formed by the dodecanol/SDS mixtures has exactly the same lattice structure as that observed in pure dodecanol layers. A small shift of the Qxy values at the same surface pressure indicates, however, a small expansion of the lattice structure, obviously caused by incorporation of a small proportion of SDS into the dodecanol lattice. The equilibrium surface pressures of the mixed dodecanol/SDS solutions correspond approximately to the sum of those observed for the single components, so that the presence of a certain amount of SDS in the adsorbed layer can be concluded from this result. Oleanolic Acid as Omnipresent Triterpenoid Oleanolic acid (OLA) is a ubiquitous triterpenoid in plant kingdom, medical herbs, and it is an integral part of the human diet. Triterpenoids represent a promising and expanding basis for biologically active natural compounds. However, their capability for unique and potentially usable biological effects is currently only partially utilized. Model monolayer studies have been used to obtain information on the role of various triterpenoids in more complex biological systems (87–90). From the biological standpoint, oleane triterpenoids belong to the most important triterpenoid structures. In particular, the omnipresence of oleanolic acid in epicuticular waxes suggests its important role for the cuticle performance. However, the chemical structure of oleanolic acid with a multicyclic planar structure deviates from the typical structure of amphiphiles consisting of a polar head group and alkyl chain(s). Despite its untypical amphiphilic character, oleanolic acid is exclusively located in the cuticula. First studies of model monolayers represent a promising possibility for obtaining insights into the specific behavior of oleanolic acid (91, 92). GIXD and specular X-ray reflectivity (SR) were used to characterize the interfacial ordering of pure oleanolic acid monolayers. The π–A isotherm of the oleanolic acid spread on pure water at 25°C (see Figure 18, left) confirms unambiguously that insoluble monolayers can be formed by oleanolic acid in the 0≤π≤8 mN·m-1 range. GIXD gives information about the two-dimensional symmetry of condensed monolayers on the Å-scale. Figure 18 , right shows the GIXD results obtained at 8 mN·m-1 and 25 °C. It is seen the contour plot as a function of the in-plane and out-of-plane scattering vector components Qxy and Qz (Figure 18, right at bottom left), the corrected X-ray intensities as a function of the in-plane scattering vector component Qxy (Bragg peak, Figure 18, right at top). Only one Bragg peak located at a Qxy-value of 1.017 Å-1 can be observed, just as at all accessible lateral pressures. According to the Bragg peak position a hexagonal packing of the oleanolic acid molecules in the monolayers (LS phase) with a cross-section area of 44.1 Ų·molecule-1 is calculated (92) in good agreement with the estimated value of the cross-section 401 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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area of 44.2 Ų·molecule-1using oleanolic acid molecular data and assuming free rotation around the long molecular axis with a diameter of 7.5 Å of the cylindrical molecule.

Figure 18. (left) Chemical structure and π–A isotherm of oleanolic acid monolayer spread on water at 25 °C. (right) GIXD data at 8 mNm-1 and 25°C. The diffracted intensity is plotted as contour lines of equal intensity (bottom left). Integration of the measured intensity over a Qz range from 0 to 0.5 Å−1 yields a single Bragg peak (top), whereas integration over a Qxy range from 0.9 Å−1 to 1.14 Å−1 yields the Bragg rod (bottom right). Reproduced with permission from reference (92). Copyright 2015 Wiley.

Figure 19. Schematic illustration of the upright orientation of oleanolic acid molecules in a monolayer on water at 10 mN m−1. Reproduced with permission from reference (92). Copyright 2015 Wiley. The thickness of the diffracting oleanolic acid layer can be estimated from the full-width at half maximum (FWHM) of the Bragg rod (Figure 18, right at bottom right) using Lz~0.9(2π)/FWHM(Qz). The thickness Lz of the oleanolic acid monolayer amounts to 14.9 Å resulting from the average value of the FWHM(Qz) of 0.38 Å-1. 402 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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The finite size Lxy of the 2D crystallites of the monolayer can be estimated by Lxy~0.9(2π)/FWHM(Qxy) and FWHM (Qxy) corresponds to the correlation length ξ = 2/FWHM (Qxy). The measured FWHM corrected by the instrumental resolution results in Lxy ≈ 260 Å (ξ ≈ 93 Å) for the LS phase of the oleanolic acid monolayer which is considerably smaller compared with those of typical amphiphilic monolayers. The thickness of oleanolic acid monolayers was additionally determined using specular X-ray reflectivity (XR) data. The thickness of (15.2 ± 0.4) Å obtained with the XR data is in good agreement with that determined by GIXD. The side view packing of oleanolic acid molecules in a monolayer at the air/water interface calculated using Cerius2 (Version 4.6. Accelrys) is schematically presented in Figure 19. The perpendicular orientation of the oleanolic acid molecules in a rotator phase (LS) in the whole lateral pressure region is consistent with the GIXD and XR results. The COOH group is directed towards the aqueous subphase because of its higher polarity and more options to form hydrogen bonds, and, consequently, the less polar OH group must be exposed to the hydrophobic air. The strong lateral hydrophobic interactions between the tightly packed molecules compensate obviously the energetically unfavorable direction of the OH groups. Double-Chain Phospholipids Biological membranes are enclosing and separating units for various ingredients and organelles of a cell (93). Additionally, biological membranes are responsible for cell-cell-interactions and signal transduction (94). For outstanding performance, a biological membrane has to maintain a fluid-like character and at the same time to form highly organized domains (lipid rafts) (95). This flexibility of the membrane is based on physical-chemical properties based on the amphiphilic character of its main components - glycerophospholipids. Glycerophospholipids form a large variety of organized structures. At a given temperature, the structure of a polymorphic form depends not only on the chemical structure of the hydrophilic and hydrophobic parts of the molecules but also on important variables such as ionic strength and pH. Under physiological conditions, most of the membrane lipids form lamellar gel or liquid-crystalline phases, but also non-lamellar phases such as hexagonal or cubic phases have been observed. Many studies are focused on the main transition from a gel to a liquid-crystalline phase. Such transitions can be induced by changes in temperature (thermotropic behavior), hydration (lyotropic behavior), pressure, as well as by changing the ionic strength or pH of the aqueous phase. The functional role of different structures is still far from being completely understood, especially in complex out-of-equilibrium systems like biological membranes. Chemical modifications of synthetic phospholipids allow the study of structure-properties relationships and the better understanding of lipid polymorphism and its biological significance. Another interesting approach is the use of phospholipid monolayers as convenient models of biomembranes, as they represent half a membrane. Studies of Langmuir monolayers have encountered an increase in activity, 403 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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although they are not directly relevant for applications, but because of their high definition, they are excellent models for biophysical studies. One major difference between monolayers and bilayers is the thermodynamical stability of the phases. Condensed phases of monolayers are mostly meta-stable, whereas bilayers in multilamellar systems represent thermodynamically stable states. In many cases, the structures of condensed monolayers can be directly compared with those of gel phases. However, the unit cell dimensions depend on the number of juxtaposed layers (monolayers, single hydrated bilayers, and multilayers) as shown in the recent works of Ziblat et al. (96). As a consequence, using one or the other physical model to describe real biological membranes can lead to different conclusions about structures and the influence of other biological molecules as peptides or proteins on the lipid structures. The advantage of Langmuir monolayers is that the investigated systems can be easily manipulated by simple compression using one or two movable barriers (97–99). In this way, the lipid density increases and phase transitions between disordered (LE – liquid-expanded) and ordered (LC – liquid-condensed) or between different condensed phases can be triggered. Using homologous series of phospholipids allows the determination of generic phase diagrams (structures formed at different temperatures and surface pressures) of amphiphilic monolayers (100). Keeping the head group structure unchanged, the increase in chain length shifts the transition pressure from LE to LC to lower values. This is similar to Traube’s rule stating that for every extra CH2 group in a surfactant molecule the surface activity approximately triples. Shorter chain length molecules have less surface activity than higher chain length molecules. This translates to monolayers of insoluble amphiphilic molecules in the following way - shorter chain length phospholipids form mainly LE phases and exhibit high transition pressures whereas higher chain length molecules form mainly LC phases. GIXD experiments using phospholipid monolayers had a great importance for understanding the nature of phase transitions during compression. The nearly horizontal part of the surface pressure/area isotherm could be clearly assigned to a first-order fluid/gel phase transition. Changes in slope, indicating changed compressibility of the layer, have been ascribed to a second-order phase transition from tilted to nontilted LC phases (101) or from LC to a solid phase (102). X-ray data demonstrate that positional correlations in double-chain phospholipid monolayers extend only over tens or hundreds of angstroms (21) indicating that the condensed phases are not perfect crystalline phases. Phospholipids with ethanolamine (PE) head groups exhibit Langmuir isotherms with molecular areas determined by the hydrophobic chains. Thus, the area requirement of the PE head group is quite small and does not disturb the chain packing. In contrast, the bulkier and more hydrated head group of phosphatidylcholine (PC) lipids determines the larger minimal area per molecule (~45 Å2). The LC phases formed by DMPE (1,2-dimyristoyl-phosphatidylcholine) were defined as mesophases with diffraction peaks much broader than the instrumental resolution. At low surface pressures, an oblique lattice has been determined transforming upon compression into a hexagonal lattice of non-tilted chains with a cross-sectional area per tail around 20 Å2. Increasing the chain length from DMPE (C14) to DPPE 404 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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(C16) and DSPE (C18) leads to increased van der Waals attractions between the hydrophobic chians and decreases the cross-sectional areas from 20.4 Å2, to 19.9 Å2 and to 19.5 Å2, respectively. In case of PC lipids, the chains are always tilted to optimize their van der Waals interactions, even at very high surface pressures. The most studied phospholipid, DPPC, exhibits a structural order in monolayers that is defined by a tilted tail arrangement. The large tilt angle (~ 30°) at 30 mN·m-1 agrees very well with the tilt angle in bilayers. A systematic study demonstrates clearly the influence of the methylation degree of the head group on the monolayer properties (103). The smallest head group, PE, extends by 1–2 Å further into the subphase, while the head group aligns progressively closer to the lipid/water interface as the methylation degree increases. This leads to an increased area requirement and therefore to a higher tilt angle of the chains. In bulk, four different phases have been found in fully hydrated 1,2-DPPC being annealed for several days at 4 °C (104, 105). A subgel phase Lc characterized by the packing of entire molecules forms only after several days at low temperature and transforms at 18.4 °C into an usual gel phase Lβ’. The head group is only weakly hydrated in the subgel phase. The gel phase of glycerophospholipids exhibits the typical orthorhombic packing of acyl chains which are strongly tilted to the bilayer normal. The head group is much stronger hydrated. The gel phase transforms into a so-called ripple phase Pβ’ at Tp = 35.1 °C which melts at Tm = 41.1 °C into a lamellar Lα phase with molten acyl chains (trans-gauche conformational change). The existence region ΔT (ΔT = Tm – Tp) of the ripple phase decreases with increasing chain length. In monolayers, only LE and LC phases with strongly tilted molecules have been found. A subgel phase has never be found. The head groups of 1,2-DPPC are aligned almost parallel to the bilayer horizon and the glycerol backbone perpendicular to it (106). In contrast, 1,3-DPPC shows a backbone orientation that runs parallel to the membrane horizon. The main transition temperature Tm (37 °C) is 4 °C lower than that of 1,2-DPPC (41.5 °C). Three different lamellar phases have been found in aqueous dispersions of 1,3-DPPC: (i) below 18 °C, a hydrated "crystalline" bilayer phase, Lc, with a bilayer periodicity d = 58 Å; (ii) between 30 °C and 35 °C, a more hydrated but interdigitated gel phase, with hexagonal chain-packing and d = 47 Å and (iii) above 37 °C, a highly hydrated liquid-crystalline Lα phase with d = 65 Å. The hydrocarbon chain interdigitation is deduced from the small bilayer periodicity, and an in-plane area per molecule of ~80 Å2. Zumbuehl et al. have recently reported on 1,3-phospholipids containing amides replacing the common ester linkers (107, 108). Large extruded unilamellae are mechanosensitive: a dye loaded into the aqueous lumen of the vesicles would not be released if the liposomes were left standing untouched. As soon as the vesicles were mechanically stimulated, the vesicle cargo was released. On the other hand, giant unilamellar vesicles made from 1,3-diamidophospholipids showed various geometries, faceted vesicles in particular. In monolayers, GIXD revealed a homogeneous structure for the longer-chain compounds. Pressure-area isotherms of monolayers of the compounds (1) – (4) have been measured at the air/water interface at 293 K (Figure 20). As expected from the low main phase transition temperature (Tm) values, the compounds 405 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Lad-PC-Lad (1,3-dilauramidopropan-2-phosphocholine) (1) and Mad-PC-Mad (1,3-dimyristamidopropan-2-phosphocholine) (2) with the shorter chains form only liquid-expanded monolayers. The coumpounds with the longer chains, Pad-PC-Pad (1,3-dipalmitamidopropan-2-phosphocholine) (3) and Sad-PC-Sad (1,3-distearamidopropan-2-phosphocholine) (4), exhibit a plateau region characterizing the first-order phase transition from the fluid (liquid-expanded (LE)) to the condensed (liquid-condensed (LC)) phase. The transition pressure increases with decreasing chain length or equivalent with increasing temperature. The temperature dependence of the transition pressure πt can be described by a linear function (Figure 21A), from which the experimental data points deviate only in the vicinity of T0 (the lowest temperature of the existence of the liquid-expanded phase). Below T0, the transition into the condensed phase starts directly from the gas-analogous state. The temperature dependence of the entropy change ΔS = ΔH/T for the phase transition is presented in Figure 21B. The absolute ΔS and ΔH values increase as the temperature decreases, indicating that the ordering of the condensed phase increases as the temperature decreases. Three Bragg peaks (the two ones at higher Qz values are strongly overlapping and the third one is slightly above the horizon) have been observed at all pressures at 293 K showing that the structure of the condensed Sad-PC-Sad monolayer is oblique with strongly tilted chains (Figure 22). The chain cross-sectional area is between 19.9 and 20.0 Å2 as found for long-chain PCs. The tilt angle t with respect to the surface normal decreases only marginally with increasing pressure. The existence of strong head group interactions due to the formation of a hydrogen bond network was demonstrated by IRRAS. The observed monolayer rigidity is correlated to marginal changes of the chain tilt upon compression. The strong hydrogen-bonding interactions between the head groups could also be responsible for the observed facetted shape in 3D aggregates. A clear difference between bilayer and monolayer stabilization was found by analyzing the main phase transition temperature Tm of the bilayer and the critical temperature Tc (the temperature above which the monolayer cannot be compressed into the condensed state) of the monolayer (109). Tm is significantly higher than the extrapolated Tc. An explanation for this effect could be a chain interdigitation in bulk which cannot be realized in monolayers. For further particulars we refer the reader to detailed reviews describing the structural changes occurring in phospholipid monolayers during compression (110, 111). Another important topic, especially for biophysical studies, is the effect of chirality on the packing properties in model membranes. GIXD measurements have been performed using the enatiomeric L-α-dipalmitoylphosphatidylethanolamine (L-DPPE) and its corresponding racemic mixture (DL-DPPE) (112). No diffraction pattern from the head groups have been measured, most probably due to static or dynamic disorder (113, 114). However, the GIXD study revealed a different structural arrangement between the optically pure L-DPPE monolayers and the racemic ones. The structural differences are more pronounced at lower surfaces pressures. The chiral resolved compound exhibited an oblique unit cell while the racemic mixture formed a pure rectangular unit cell (with NN tilted chains). At higher surface pressures, the chirality did 406 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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not have an effect on the monolayer structure demonstrating that this a weak force compared with other forces determining the monolayer struture. The head groups of the pure enantiomer are interlinked by hydrogen bonds formed between NH3+ and PO4- groups of neighboring head groups, while for the racemate a two-dimensional network of hydrogen bonds between the head groups was proposed.

Figure 20. π-A isotherms of Lad-PC-Lad (1, black line) and Mad-PC-Mad (2, green line), Pad-PC-Pad (3, blue line) and Sad-PC-Sad (4, red line) monolayers on water measured at 293 K. Reproduced with permission from reference (109). Copyright 2015 American Chemical Society.

Figure 21. A) Temperature dependence of the main phase transition pressure πt of Pad-PC-Pad (●) and Sad-PC-Sad (▴) monolayers spread on water. B) Temperature dependence of the entropy change at the main phase transition of Pad-PC-Pad (●) and Sad-PC-Sad (▴) monolayers spread on water. Reproduced with permission from reference (109). Copyright 2015 American Chemical Society. 407 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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Figure 22. Grazing incidence X-ray diffraction data for Sad-PC-Sad spread on water at 293 K and compressed to 10 (A), 20 (B) and 35 (C) mN·m-1. The diffracted intensity, corrected for polarization, effective area, and Lorentz factor, is plotted as contour lines of equal intensity versus the in-plane component Qxy and the out-of-plane component Qz of the scattering vector. Reproduced with permission from reference (109). Copyright 2015 American Chemical Society.

Complex Monolayers Bulky and strongly hydrated head groups lead to monolayer structures with strongly tilted acyl chains (e.g., 1,2-DPPC has a tilt of ~30° at a lateral pressure of 30 mN·m-1). The corresponding gel phase in multilamellar dispersions is a lamellar Lβ’ phase with tilted chains. The chain tilt is the same in monolayers and bilayers at a comparable lateral pressure (~30 mN·m-1). The reason for the chain tilt is the area mismatch between the head group and two ordered acyl chains. The reduction of the effective head group area leads to a decrease of the tilt angle. This has been obtained by changing the hydration shell around the head group or by changing the head group conformation. The change of PC head group orientation to a more vertical arrangement leads to the reduction of the area requirement mismatch. Such a behavior was found, e.g., for the binding of an enzyme (phospholipase A2) to a D-DPPC monolayer. The protein binding enforces a dehydration and reorientation of the PC head group (115, 116). This process is highly cooperative. Another possibility to reduce the tilt angle is the insertion of alkanes into the hydrophobic part of the monolayer (117). In this case, the effective head group area remains unchanged, but the alkanes can be incorporated into the ordered lipid arrays leading to increased van der Waals interaction between non-tilted chains. A third possibility is a partial dehydration of the head group as observed for a subphase containing alcohols (118). To keep the membrane in a fluid state, nature uses unsaturated fatty acids, for instance oleic and linoleic acid and also fatty acids with additional double bonds (119). The unsaturated fatty acids are mainly attached at the sn-2 position of the glycerol backbone of glycerophospholipids. Such phospholipids exhibit only disordered LE phases due to the disordering effect of double bonds on the packing properties (120, 121). Another effect of double bonds in phospholipids is the reaction with oxygen resulting in a degradation of the fatty acid (protection 408 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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against oxidative stress). The unsaturated fatty acids can be easily deacylated by phospholipase A2 (122). Membranes adjust their composition according to the environmental conditions. For example, bacteria alter their membrane lipid composition, and therefore the membrane fluidity, in response to changes in growth temperature (123). Another mechanism to ensure membrane fluidity was found in the lipids of eubacteria and archaebacteria. Mainly iso- and anteiso-methyl branched fatty acids have been found. The position of the methyl branching has a dramatic effect on the Tm. Another example for methyl branches in a fatty acid chain are ceramides (124).

TRXF (Total Reflection X-ray Fluorescence) We have shown that X-ray scattering is a powerful tool to study the structure of planar soft interfaces at molecular length scales. While GIXD yields information about the in-plane structure of an interface, X-ray reflectometry reveals density profiles perpendicular to the interface. However, it is difficult to elucidate molecular conformations and elemental distributions from such density profiles. In contrast, X-ray fluorescence techniques are suited to determine element-specific density profiles across an interface at sub-nm resolution. Standing waves (SW) have been widely used for this purpose. Close to a Bragg condition the SW exhibits periodic nodes and antinodes (minima and maxima) which are located in defined spatial relations to the atoms of the crystal lattice and therefore give rise to enhanced or reduced fluorescence from various elemental species (125). As a result, the position of elements in the crystal lattice is encoded in dependence of the fluorescence intensity and can be reconstructed. SW fluorescence was also used to determine the precise positions of adsorbate atoms at crystal surfaces (126, 127) For details, the reader is referred to a review by Zegenhagen (128). The first SW fluorescence study at a liquid/gas interface was performed by Bloch et al. (129) and described the distribution of Mn-labeled polymers near the interface between air and liquid DMSO. The liquid phase is probed with the exponentially decaying evanescent tail of the SW under total reflection at the liquid/gas interface. Bloch et al. (130) determined also the excess of Mn2+ ions at a negatively charged stearic acid monolayer. Daillant et al. (131) used the same approach and determined excesses of counter- and co-ions at a charged lipid monolayer as a function of the lateral density of amphiphiles and found a constant counter-ion to lipid ratio along the isotherm. In the last years, TRXF (total reflection X-ray fluorescence) has been developed by Shapovalov et al. as a really simple and quantitative analytical method for the characterization of monolayers at liquid/air interfaces (132). The monochromatic synchrotron X-ray beam hits the liquid surface at a grazing angle αi ~ 0.8·αc (αc is the critical angle of total reflection) giving rise to the high surface sensitivity (~8 nm). The absorbed X-ray energy depends on the type of the element to be detected since the gap between the energy of the incoming X-ray beam and the edge energy should not be too large, otherwise the coupling between 409 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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the X-ray and electron is inefficient. Due to instrumental limitations and low X-ray fluorescence yields, light elements cannot be detected. The determination of the concentration of elements (e.g., ions) at the interface from experimental X-ray fluorescence data requires information about both the intensity distribution of the exciting X-ray and the element distribution near the interface (23). In a first study, the competitive adsorption excess of various monovalent and divalent cations to negatively charged behenylsulfate monolayers was determined and pronounced ion specificity was observed and interpreted in terms of different hydrated ion sizes (132). Small monovalent cations were found to be even able to compete with large divalent cations under certain conditions. TRXF was also employed to determine the protonation state of new cationic lipids in monolayers at the air/water interface by quantification of the counter ion excess (Br-) as a function of pH and lipid lateral density (133) and to quantify the amount of adsorbed DNA (134). The monolayers of cationic lipids were formed on subphases with a constant concentration (2 mM) of Branions and the X-ray fluorescence intensity of Br, which is proportional to the amount of Br- anions coupled to the lipid head groups, was measured in dependence on the bulk phase pH. Selected X-ray fluorescence spectra of CIII (N-{2-[bis(2-aminoethyl)amino]ethyl}-2,N¢-dihexadecyl-propandiamide) are presented in Figure 23. The Br Kα and Br Kβ fluorescence peaks are clearly present. Their intensity decreases with increasing pH and is close to 0 at pH 11.

Figure 23. Selected X-ray fluorescence spectra of the CIII Langmuir monolayer at 40 Å2·molecule-1 on Br- containing subphases at pH 3 (▪), pH 6 (□), pH 8 (□), and pH 11 (○). Reproduced with permission from reference (133). Copyright 2015 American Chemical Society. Plotting the integral intensity of fluorescence peaks versus pH one can obtain a titration curve for each investigated lipid (Figure 24). Except for compound IV (2-amino-3-hexadecyl-oxy-2-hexadecyloxymethyl-propan-1-ol), the slope of these curves levels off within the limits of the error bars below pH 4. Above 410 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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pH 9, the intensity decreases to zero indicating that the head groups are fully deprotonated. In order to get quantitative information about the protonation rate and/or the number of protonated groups in the head group of the cationic lipids, the X-ray fluorescence spectrum of a DODAB monolayer on a Br- containing aqueous subphase was measured as a reference (133).

Figure 24. Integral X-ray fluorescence intensity from Br- coupled to the Langmuir monolayers of DODAB at 50 Å2·molecule-1 (▪), CI (2-tetradecylhexadecanoic acid-{2-[(2-aminoethyl)amino]ethyl}amide ),at 64 Å2·molecule-1 (○), CII (2-tetradecylhexadecanoic acid-2-[bis(2-aminoethyl)amino]ethylamide),at 60 Å2·molecule-1 (●), CIII N-{2-[bis(2-aminoethyl)amino]ethyl}2,N¢-dihexadecyl-propandiamide),at 40 Å2·molecule-1 (Δ), and CIV (2-amino-3-hexadecyloxy-2-hexadecyloxymethyl-propan-1-ol) at 43 Å2·molecule-1 (▾) in dependence on the subphase pH. Reproduced with permission from reference (133). Copyright 2015 American Chemical Society.

In order to use the TRXF technique for the quantification of DNA binding to positively charged monolayers, DNA from salmon testes was labeled by covalently bound bromine (134). The bromination degree was quite low but sufficient to be detected. The additionally present bromide ions are not competitive with Br-DNA for the binding at the positively charged monolayer. An important result of these studies was the finding that higher salt concentrations (representing physiological conditions) lead to an increased amount of adsorbed DNA. This has been explained by the decrease of the effective charge of the DNA molecules with decreasing Debye screening length. More recently, excesses and approximate distributions of monovalent and divalent cations at comparatively realistic, negatively charged models of bacteria surfaces were determined in the absence and presence of cationic antimicrobial peptides (135). 411 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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New NPs (nanoparticles) are constantly synthesized for biomedical applications. As an example, Fe3O4 NPs capped with catechol-terminated random copolymer brushes of 2-(2-methoxyethoxy) ethyl methacrylate (MEO2MA) and oligo(ethylene glycol) methacrylate (OEGMA) (MEO2MAx-co-OEGMA100-x), promising magnetic resonance imaging contrast enhancers and cell manipulation agents, have been investigated at the air/water interface as pure NP monolayers and as mixed NPs–DPPC (136) model membrane systems. The physical-chemical properties of those stimuli-responsive copolymer-capped Fe3O4 NPs were dictated by the molar ratio of the two copolymers (137). TRXF was used to detect the presence of these NPs at the interface in pure (138, 139) and mixed systems The NPs can be dispersed both in water and chloroform, therefore, NP layers at the interface can be prepared either by adsorption of the NPs from the aqueous bulk solution (Gibbs films) or by spreading from a chloroform solution (Langmuir films). A maximal surface pressure of approximately 23 mN·m-1 can be reached after 20 h of adsorption. By compression a Langmuir film, the surface pressure increases continuously up to approximately 25 mN·m-1. This pressure has been called the critical pressure (πc) of the NP film. During further compression, a plateau region appears at which the surface pressure increases only slightly. No hysteresis of the compression/expansion isotherms is observed when the interfacial film is compressed only to surface pressures below the critical pressure of the Langmuir layer. The authors have shown that all NPs from the bulk solution adsorb at the interface and that the particles spread are trapped at the interface and do not exchange with the subphase.

Figure 25. A) AFM image of a NP Langmuir layer transferred onto mica support by the LS technique and the height profile measured along the line (detail image 1x1 µm2); B) The X-ray fluorescence spectrum in the region of the Fe Kα and Fe Kβ peaks of the NP interfacial layer at 1.1 mN·m-1 (blue line) and 5.4 mN·m-1 (black line). Reproduced with permission from reference (139). Copyright 2015 American Chemical Society. The redistribution of the NPs into the subphase occurs only above the critical pressure indicating that the NPs are in a metastable state, being trapped at the air/water interface. The squeezed-out NPs adsorb again at the free interface behind the barrier of the Langmuir trough. The presence of the NPs behind the barrier was confirmed by AFM measurements of samples prepared by the Langmuir-Schaefer (LS) technique. Additionally, the enrichment of the squeezed-out iron oxide NPs 412 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

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behind the barrier was determined by measuring in situ TRXF spectra. An increase of the intensity of the characteristic Fe Kα and Fe Kβ X-ray fluorescence peaks upon compression of the adsorption layer was recorded (Figure 25) (138, 139). When dispersed in aqueous solution, the NPs are able to penetrate into a DPPC monolayer and to occupy a certain part of the interface due to their own surface activity. The mixed layers are largely phase separated, but the NPs are able to change the DPPC (1,2-dipalmitoyl-phosphatidylcholine) monolayer structure in a highly cooperative way by inducing a tighter in-plane packing. IRRAS verified that the effective size of the phospholipid polar head group is reduced due to partial dehydration and reorientation. Above a critical pressure, the phase separated NPs are squeezed-out but the ones interacting with the DPPC molecules stay in the layer up to much higher lateral pressures (136). TRXF elucidated also the binding of Cu2+ and Zn2+ ions to amyloidogenic model peptides adsorbed at the air/buffer interface (140, 141). Comparing single metal ions experiments with competition experiments using both metal ions in the subphase, the preference for Cu2+ binding was clearly observed. The result is based on the higher binding affinity of Cu2+ for the histidine binding sites compared to Zn2+ and to the additional binding sites of Cu2+ probably present on the peptide backbone. Additionally, the binding of Zn2+ ions is strongly dependent on the possibility of chelate formation. Therefore, the presence of Zn2+ ions in the subphase accelerated the transformation to a β-sheet conformation when chelate formation was not possible in the α-helical state.

Concluding Remarks and Outlook This review has attempted to present an overview of possibilities to characterize Langmuir or Gibbs monolayers at the liquid/air interface by using synchrotron based X-ray methods as GIXD and TRXF. We present to the readers some interesting examples of chemically very different molecules to get a flavor of the recent progress. The importance of synchrotron based X-ray scattering methods is highlighted. The polymorphism in monolayers is rich and new and more highly ordered structures have been found in the last 20 years. By synthesizing new molecules with promising application potential the competing interactions between the different parts of amphiphilic molecules can be tuned and new monolayer structures will be observed. Especially strong hydrogen-bond networks can induce never before observed highly ordered ‘subgel’ phases in Langmuir monolayers. Proteins, peptides, polymers, and nanoparticles are meanwhile hot topics in monolayer research with a high potential for medical applications. X-ray methods can elucidate how monolayer structures of amphiphilic molecules can be modified by the interaction with such biological molecules and therefore contribute to answer important questions as: How do antimicrobial peptides interact with bacterial membranes? What are the triggers to force peptides to change their secondary structure to β-sheets that aggregate to amyloid fibrils? 413 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

How do new nanoparticles designed for medical applications influence the model membrane structure? For this, it is important to design beamlines at synchrotron sources for the special needs. The limitation of beamtime is one of the main problems since there are only a limited number of suitable beamlines worldwide. For the future, we can imagine the coupling of different surface sensitive techniques which is an important task to study more complex systems which are the better models of biological membranes.

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Subject Index

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A α-helix facilitates proteins, 89 discussion, 105 introduction, 90 α-helix, illustration, 90f Langmuir monolayer, formation, 92 β-sheet, parallel and antiparallel, 91f results aequorin, circular dichroism spectra, 99f aequorin, surface potential-area isotherms, 94f aequorin, surface properties and orientation, 93 aequorin Langmuir monolayer, IRRAS, 97 aequorin Langmuir monolayer, IRRAS spectra, 100f aequorin Langmuir monolayer, pH effect, 96 compression-decompression cycles, 95f Langmuir monolayer,stability curves, 103f secondary structures of aequorin, fraction, 99t surface pressure of 10 mN/m, incident angles, 104f surface pressure-area isotherms, 102f surface pressure-area isotherms, effect of pH, 98f α-syn, sequence, 101f Analytical spectroscopy, plasmonics introduction, 269 web of science, retrieved results, 271f localized surface plasmon resonances, properties Ag and Au spheres, absorption and scattering cross sections, 277f Au spheres, STEM images, 279f localized surface plasmon resonances,spectral response, 278 LSPR, origin, 276 nanoparticles, plasmons, 275 plasmon coupling, 280 shell–isolated nanoparticle enhanced raman scattering (SHINERS), 281 SHINERS spectra (I – III), 284f SHINs, TEM images, 282f

TNT identification, three-step sequence, 283f surface enhanced fluorescence (SEF) and shell–isolated nanoparticle enhanced fluorescence (SHINEF), 285 analytical applications, task, 286 Au nanoparticles, plasmon-enhanced fluorescence, 287f combined SHINERS and SHINEF spectra, 288f distance dependence, 287 dual modal nanoprobe (DMNP) representation, 291f hotspots and the enhancement factor (EF), 288 molecular quantum yield, 290 SHIN dimer, 289f spectral profile modification, 291 unmodified and modified quantum yields, 290t surface plasmon resonances bulk plasmon, 272 dispersion of the transverse bulk plasmon, diagram, 274f transverse mode, propagation equation, 273

C Chromatin biology, single-molecule fluorescence microscopy methods labelling strategies and dyes, 130 tag-meditated labeling proteins, schematic representation, 132f TALEN-mediated genomic modification unit, schematic representation, 131f summary, 134 superresolution microscopy, visualizing chromatin structure, 133 Chromophore isomerization, protein response, 329 anabaena sensory rhodopsin (ASR), 344 ASR, picosecond time-resolved UVRR spectra, 345f W3 band, temporal intensity change, 346f bacteriorhodopsin (BR), 334 bands, temporal intensity changes, 336f

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BR, picosecond time-resolved UVRR spectra, 335f BR, UVRR spectra, 337f time-resolved 225-nm UVRR difference spectra, 338 bacteriorhodopsin, crystallographic structure, 330f primary protein responses in microbial rhodopsins, comparison, 347 vicinity of the retinal chromophore, crystallographic structure, 348f sensory rhodopsin I (SRI), 342 SRI, picosecond time-resolved UVRR spectra, 343f sensory rhodopsin II (SRII), 339 SRII, picosecond time-resolved UVRR spectra, 340f WT-SRII, picosecond time-resolved UVRR difference spectra, 341f time-resolved UVRR apparatus and measurements, 331 picosecond time-resolved UVRR spectrometer, 332f time-resolved UVRR data acquisitions, 333 time-resolved UVRR spectroscopy, 331

D Diverse near-infrared resonant gold nanostructures biomedical applications, 215 biological tissues, optical windows, 216f drug delivery and light-controlled drug release, 219 photoacoustic imaging (PAI), 217 photodynamic therapy (PDT), 218 photothermal therapy (PTT), 218 scattering- and luminescence-based optical imaging, 216 surface-enhanced spectroscopy-based imaging, 217 diverse NIR-resonant gold nanostructures Au nanocages, SEM image, 228f Au nanoechinus, 227 Au nanoparticle ensembles, 233 Au nanorods, 219 Au nanoshells, 232 Au nanostars, 224 Au nanostars, SEM images, 225f AuNCs, phototherapy agent, 229

AuNCs as intrinsic inorganic photosensitizers, schematic illustration, 231f AuNP-DOX conjugates, schematic illustration, 235f biodegradable Au vesicles, self-assembly, 238f formation of SiO2@AuNSs, TEM images, 232f nanorods, transmission electron microscopy (TEM) images, 220f NIR laser-induced targeted thermo-chemotherapy, 223f NIR light-encoded logic gate, schematic representation, 230f NIR-responsive delivery platform, DNA assembly, 222f organic photosensitizer-mediated PDT, 221 PEGylated mixed-charge Au nanostars, schematic illustration, 226f pH-induced aggregation of individual gold nanoparticles, TEM images, 234f plasmonic vesicles, 236 synthesis of SERS-active amphiphilic AuNRs, schematic illustration, 237f introduction, 356 localized surface plasmons, excitation and decay, 215f Dynamic light scattering, 157 introduction, 158 food and environmental toxins, list, 162t NanoDLSay, schematic illustration, 159f proteins, viruses, and virus antigens, list, 164t single and double-stranded DNAs and microRNAs, list, 163t small chemicals and biomolecules, list, 161t toxic metal ions, list, 160t literature survey adenosine, 169 DNAs and RNAs, 172 food and environmental toxins, 170 lead (Pb2+), 166 melamine, 171 mercury (Hg2+), 168 protein biomarkers, 174 small chemicals and biomolecules, 168 toxic metal ions, 165 viruses and virus antigens, 175

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F Fluorinated fatty alcohols and dipalmitoylphosphatidylcholine (DPPC), 1 experimental section F4H11OH, F6H9OH and F6H11OH, chemical structures, 4s materials, 3 methods, 4 results and discussion π-A and ΔV–A isotherms, 7f atomic force microscopy (AFM), 20 binary DPPC/F4H11OH monolayers, fluorescent micrographs, 14f binary DPPC/F4H11OH monolayers, typical AFM topographic images, 18f binary DPPC/F6H9OH monolayers, AFM topographic images, 19f binary DPPC/F6H9OH monolayers, fluorescent micrographs, 15f binary DPPC/F6H11OH monolayers, AFM topographic images, 21f binary DPPC/F6H11OH monolayers, fluorescent micrographs, 16f DPPC, two-component monolayers, 8 excess Gibbs free energy changes, 11f fluorescence microscopy (FM), 13 pure systems, Langmuir monolayer, 6 ratio of ordered domain area, surface-pressure dependence, 17f thermal properties, 5 two-component DPPC/F4H11OH, π–A isotherms, 9f variation of the transition pressure, two-dimensional phase diagrams, 12f

G Graphene oxide and biomolecules, interactions adsorption study, 46 GO, fluorescence quenching of Trp, 48f tested amino acids, concentrations, 47t Trp or Tyr by GO, fluorescence quenching, 47 fluorescence quenching of GO, protein detection, 51 GO, antibacterial activity, 59

POPC/ POEPC (3:1) lipid membrane, schematic representation of the formation, 60f GO, orientation, 57 GO, structure and characterization, 44 proposed chemical structure, 45f GO and amino acids, interaction, 45 graphene oxide, tapping mode AFM image, 46f GO and lipid bilayers, interactions, 58 possible orientations of GO, schematic diagrams, 59f GO and lipids, interaction, 57 graphene oxide and oligonucleotides, interactions, 55 DNA, fluorophore, 56f ternary mixture of LYZ/OVA/HSA, electrophoresis, 56f lysozyme, adsorption and desorption, 53 concentration of GO, lysozyme, 54f lysozyme, fluorescence spectra and UV-vis absorption spectra, 54f lysozyme, selective adsorption, 55 peptides or proteins, intrinsic fluorescence, 50 different proteins aqueous solution, Stern-Volmer plot, 51f peptides or proteins and GO, interaction, 49 surface of GO, protein adsorption, 52 Trp or Tyr and GO, electrostatic interactions, 49 Trp or Tyr and GO, hydrophobic interaction study, 48 Trp, Sterm–Volmer plot, 49f

L Langmuir monolayers, role, 65 biomembrane model system, Langmuir monolayer, 66 colloidal biomembrane model systems, comparison, 67t lipids and membrane proteins, monolayer, 67f biomembrane organization, 68 biomineralization studies, 80 Langmuir and Langmuir-Blodgett films, adsorbed enzymes, 71 exclusion surface pressure, determination, 73f functionalized biomimetic membrane, 74

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Publication Date (Web): December 8, 2015 | doi: 10.1021/bk-2015-1215.ix002

LMs and/or LB films, enzymes incorporated, 75t mixed enzyme-lipid LB film, scheme, 75f Langmuir films, bioactive compounds, 76 Langmuir monolayers, peptides and proteins, 68 antimicrobial peptides (AMPs), 69 hydrophobic proteins, 70 LM and LB films, incorporation of nanoparticles, 77 nanoparticles in LM, scheme for the incorporation, 78f palmitic acid (PA), presence, 78

M Metal oxide nanoparticulate cytotoxicity, 137 discussion case I, 147 Case II, 150 Hut-78 cancer cells,cell viability, 152f TiO2 induced apoptosis and necrosis, 148f TiO2 inhibited cell proliferation, 147f ZnO-I and ZnO-II NP, FTIR spectra, 149f ZnO-I and ZnO-II NP, zeta potentials, 151f experimental procedures chemicals and materials, 139 cytotoxicity assay, 141 microscopy and brunauer-emmettteller (BET), 139 NP solid surface, surface analysis, 140 statistical analysis, 141 X-ray photoelectron spectroscopy (XPS), 139 results, 141 A549 cells, transition metal NPs induces cell death, 143f available particle surface binding sites, 146t measured specific surface areas, 142t NP toxicity, correlating physicochemical properties, 143 O 1s orbitals, XPS, 144f PZC initial pH versus final pH plots, 144f surface binding sites, relative number, 145

4th period metal oxide NPs, morphology and size distribution, 142f Microfluidics, bioanalysis, 245 airborne pathogen analysis, challenges, 246 all-glass impinge (AGI) sampler, 247 Anderson sampler, 247 chemical tracers, 250 cultivation methods, 248 filter sampler, 248 genomic DNA, amplification, 251 laser-induced breakdown spectroscopy, 253 light detection and ranging (LIDAR), 253 microarray technology, 252 microscopy techniques, 249 nucleic acid sampling and extraction from bioaerosols, 250 on-line auto fluorescence methods, 252 restriction fragment length polymorphism techniques (RFLP), 251 sequencing methods, 252 static sampler, 248 airborne pathogen analysis, microfluidics, 254 future perspective, 258

O Organic-inorganic supramolecular gels, 199 experimental section, 201 results and discussion, 201 base-pair modified Anderson type POMs, schematic illustration, 206f cationic surfactant, chemical composition, 203f gadolinium-incorporated POM complexes, 207 Gd-POM complex for magnetic resonance imaging contrast agent, 208f gelation of TBA-Py-MnMo6 grating pyridyl groups, schematic drawing, 205f Mn-12-C18/C18EO10 aggregates, formation process, 209f MRI CAs, manganese cluster single-molecular magnets, 208

430 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Publication Date (Web): December 8, 2015 | doi: 10.1021/bk-2015-1215.ix002

organically grafted POM complexes, responsive gels, 204 POM-cored complexes, supramolecular gels, 202 Oxidized nanocarbons, hydrophilization, 25 bottom-up methods, nanocarbons synthesized, 34 CQDs, fluorescence spectra, 36f graphene, precisely synthesized model compounds, 34f semiconductive SWCNT, 34 nanocarbons, oxidation, 26 graphene oxide, dispersions, 31f graphene oxide, TEM images and size distributions, 30f graphene oxide, zeta potential, 30f multi-walled carbon nanotubes, TEM image, 28f nano-GO, size, 29 water-soluble fullerene derivatives, chemical modifications, 26 oxidized nanocarbons, chemical modification, 31 graphene oxide, structural model, 32f nano-sized GO, TEM images, 33f

P Polymeric nano/microstructures results and discussion, 182 100 nm nanowire device, I-V curve, 188f 80 nm wide PPy, AFM image, 188f conducting polymer nanostructures, fabrication process, 184f fine structures of the mold, SEM images, 191f high density PPy, AFM image, 192f light diffraction, CCD camera image, 193f polyaniline, electrode shaped structures, 189f polyaniline nanostructure, AFM images, 195f polymer nanostructures, fabrication, 187f polymer nanowire arrays, fabrication process, 190f PPy conducting polymer nanowire arrays, gold pads, 194f PPy nanowire based nanosensor, sensing behavior, 186f

research method, schematic illustration, 183f single PPy nanowire, AFM image, 185f Protein and peptide selectivity, 109 materials and methods binding parameters, determination, 113 materials, 111 RP2 and tLRAT, cloning and expression, 112 RP2 and tLRAT, purification, 112 results and discussion binding parameters, measurement, 113 binding parameters of RP2, determination, 120f Cter-R9AP, monolayer binding parameters, 122 determination of a MIP value, typical example, 114f fast-throughput method, monolayer methodology, 115 histograms, 119f microtroughs, commercially available apparatus, 115f MIP and synergy of Cter-R9AP peptide, histograms, 123f phospholipid monolayers, physical state, 118 phospholipids, presence, 117 polyunsaturated pho0pholipid oxidation, 119 RP2, MIP and synergy, 116f selectivity of RP2, assessment, 116 tLRAT monolayer binding, 121 values, histograms, 122f Protein-lipid/surfactant interactions, studies, 355 introduction, 356 biological, pharmaceutical and medical research, role of lipids, 357 methods absorbent information of CD spectroscopy, summary, 358t circular dichroism spectroscopy (CD), 358 differential scanning calorimetry (DSC), 367 fluorescence signals, changes, 363 fluorescence spectroscopy, 361 fourier transform infrared (FTIR) and raman spectroscopy, 368 isothermal titration calorimetry, 364 ITC enthalpogram, changes, 366f

431 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

mixed amphiphilic acid amide monolayers, 394f main phase transition pressure π, temperature dependence, 407f mesoscopic textures, correlation, 398f monoacylglycerols, 382 1-monostearoyl-rac-glycerol, chemical structure, 383f 1-monostearoyl-rac-glycerol monolayers, GIXD data, 384f oleanolic acid monolayer, chemical structure, 402f omnipresent triterpenoid, oleanolic acid, 401 phospholipid monolayers, 406 racemic 1-stearoyl-rac-glycerol monolayer, surface pressure-temperature phase diagram, 385f racemic 1-stearylamine-rac-glycerol domain, BAM image, 388f Sad-PC-Sad, grazing incidence X-ray diffraction data, 408f stearylamine-glycerol, 386 1-stearylamine-glycerol monolayers, chiral discrimination, 387f tetradecanoic acid-(2hydroxyethyl)amide (N-myristoyl ethanolamine), typical domain shapes, 392f upright orientation of oleanolic acid molecules, schematic illustration, 402f TRXF (total reflection x-ray fluorescence), 409 Br-, integral x-ray fluorescence intensity, 411f CIII Langmuir monolayer, selected x-ray fluorescence spectra, 410f NP Langmuir layer, AFM image, 412f

Publication Date (Web): December 8, 2015 | doi: 10.1021/bk-2015-1215.ix002

Jablonski energy level diagram, illustration, 362f light scattering, 370 molecular orbital diagrams, illustration, 361f secondary structures, UV CD spectra, 359f small-angle scattering (SAS), 371 three interactions, demonstration, 365f ultraviolet–visible absorption spectroscopy (UV-vis), 360 vibrational spectroscopies, 369

S Synchrotron-based X-ray methods, 377 concluding remarks, 413 grazing incidence X-ray diffraction (GIXD), brief description, 379 diffractometer, experimental setup, 380f rodlike linear amphiphilic molecules, side view, 381f monolayers by GIXD, structural studies acylethanolamines (NAEs), 391 adsorbed amide amphiphile’s monolayers, phase transition, 395 amide amphiphiles, miscibility behavior, 396f amide amphiphiles, two-dimensional miscibility behavior, 395 BAM images, similarity, 400f coadsorption of dodecanol/SDS, phase transition, 398 common ester linkers, 405 complex monolayers, 408 derivatized ethanolamine, amide amphiphiles, 391 dodecanol/SDS, coadsorption, 399f double-chain phospholipids, 403 equilibrium trans-brassidic acid domains, BAM images, 390f fatty acids, effect of unsaturation, 388 fatty acids, effect of unsaturation on Langmuir monolayers, 389f Gibbs monolayer of DHBAA, contour plots, 397f GIXD contour plots, 393f GIXD experiments, 404 Lad-PC-Lad, π-A isotherms, 407f long-chain n,o-diacylated ethanolamine, dominance, 393 long-chain n,o-diacylated ethanolamine, dominance in

T Thin film, infrared spectra, 303 ATR spectrometry, 310 ATR spectrometry, optical scheme, 310f concluding remarks, 326 electrodynamics, fundamentals, 305 IR transmission measurements, schematic, 306f transmission absorbance, 306t IR external reflection spectrometry, 315

432 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

Publication Date (Web): December 8, 2015 | doi: 10.1021/bk-2015-1215.ix002

spin-coated film of P3HT,IR pMAIRS spectra, 323f quantum mechanics, fundamentals, 304 RA and ATR spectrometries, application study, 311 bulk compounds of MA-Rf(n), IR ATR spectra, 314f MA molecules, schematic image, 312f MA-Rf(n), model compound, 311f MA-Rfn=9 molecules, top view of a molecular aggregate, 312f single monolayer LB films, IR RA spectra, 313f surface adsorbates, infrared spectroscopy, 304 thin-film analysis, surface selection rules, 307 IR RA measurements, schematic, 307f 7-monolayer LB film, IR RA and transmission spectra, 309f RA technique, analytical expression, 307

GaAs substrate, band intensity of a thin film, 317f IR ER measurements of a thin film, schematic, 316f SAM of ODS, IR ER spectrum, 318f silicon surface, reflectance on the angle of incidence, 317f IR MAIRS spectrometry, 319 γ(C–H)) and νa(C=C) modes, geographical relationship, 325f dip-coated film of LPEI, IR p-polarized ER spectrum, 319f double helix of LPEI, schematic, 321f IR pMAIRS spectra, molecular orientation angles, 324t LPEI film dip-coated on a germanium substrate, IR MAIRS spectra, 321f MAIRS:, schematic image, 320f molecular piling of the face-on type polythiophene compounds, schematic, 325f P3HT, primary chemical structure, 323f

433 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.