Fluorescence in Industry [1st ed.] 978-3-030-20032-9;978-3-030-20033-6

Table of contents :
Front Matter ....Pages i-x
Luminescence-Based Sensors for Bioprocess Applications (Idoia Urriza-Arsuaga, Guido Ielasi, Maximino Bedoya, Guillermo Orellana)....Pages 1-38
Fluorescence in Pharmaceutics and Cosmetics (Maurício da Silva Baptista, Erick Leite Bastos)....Pages 39-102
Instrumentation for Fluorescence Lifetime Measurement Using Photon Counting (David J. S. Birch, Graham Hungerford, David McLoskey, Kulwinder Sagoo, Philip Yip)....Pages 103-133
Applications of Submersible Fluorescence Sensors for Monitoring Hydrocarbons in Treated and Untreated Waters (Vadim B. Malkov, Jeremy J. Lowe)....Pages 135-171
Luminescence in Photovoltaics (José Almeida Silva, João Manuel Serra, António Manuel Vallêra, Killian Lobato)....Pages 173-211
Time-Gated Luminescence Acquisition for Biochemical Sensing: miRNA Detection (Emilio Garcia-Fernandez, Salvatore Pernagallo, Juan A. González-Vera, María J. Ruedas-Rama, Juan J. Díaz-Mochón, Angel Orte)....Pages 213-267
Thermally Activated Delayed Fluorescence Emitters for Light-Emitting Diodes and Sensing Applications (João Avó, Tiago Palmeira, Fernando B. Dias)....Pages 269-292
Explosives Detection: From Sensing to Response (Liliana Marques Martelo, Lino Forte Marques, Hugh Douglas Burrows, Mário Nuno Berberan-Santos)....Pages 293-320
New Trends in Fluorescent Reporters in Biology (L. Della Ciana)....Pages 321-339
Application of Fluorescence in Life Sciences for Basic Research and Medical Diagnostics (Gerhard Hawa)....Pages 341-363
Photoluminescent Glasses and Their Applications (César A. T. Laia, Andreia Ruivo)....Pages 365-388
Luminescence-Based Sensors for Aeronautical Applications (Bruno Pedras, Guillermo Orellana, Mário Nuno Berberan-Santos)....Pages 389-411
Back Matter ....Pages 413-419

Citation preview

Springer Series on Fluorescence  18 Series Editor: Martin Hof

Bruno Pedras Editor

Fluorescence in Industry

18 Springer Series on Fluorescence Methods and Applications Series Editor: Martin Hof

Springer Series on Fluorescence Series Editor: Martin Hof Recently Published and Forthcoming Volumes Fluorescence in Industry Volume Editor: Bruno Pedras Vol. 18, 2019 Perspectives on Fluorescence A Tribute to Gregorio Weber Volume Editor: David M. Jameson Vol. 17, 2016 Fluorescence Studies of Polymer Containing Systems Volume Editor: Karel rocházka Vol. 16, 2016 Advanced Photon Counting Volume Editors: Peter Kapusta, Michael Wahl and Rainer Erdmann Vol. 15, 2015

Advanced Fluorescence Reporters in Chemistry and Biology I Fundamentals and Molecular Design Volume Editor: A.P. Demchenko Vol. 8, 2010 Lanthanide Luminescence Photophysical, Analytical and Biological Aspects Volume Editors: P. Hänninen and H. Härmä Vol. 7, 2011 Standardization and Quality Assurance in Fluorescence Measurements II Bioanalytical and Biomedical Applications Volume Editor: Resch-Genger, U. Vol. 6, 2008

Far-Field Optical Nanoscopy Volume Editors: Philip Tinnefeld, Christian Eggeling and Stefan W. Hell Vol. 14, 2015

Standardization and Quality Assurance in Fluorescence Measurements I Techniques Volume Editor: U. Resch-Genger Vol. 5, 2008

Fluorescent Methods to Study Biological Membranes Volume Editors: Y. Mély and G. Duportail Vol. 13, 2013

Fluorescence of Supermolecules, Polymeres, and Nanosystems Volume Editor: M.N. Berberan-Santos Vol. 4, 2007

Fluorescent Proteins II Application of Fluorescent Protein Technology Volume Editor: G. Jung Vol. 12, 2012

Fluorescence Spectroscopy in Biology Volume Editor: M. Hof Vol. 3, 2004

Fluorescent Proteins I From Understanding to Design Volume Editor: G. Jung Vol. 11, 2012 Advanced Fluorescence Reporters in Chemistry and Biology III Applications in Sensing and Imaging Volume Editor: A.P. Demchenko Vol. 10, 2011

Fluorescence Spectroscopy, Imaging and Probes Volume Editor: R. Kraayenhof Vol. 2, 2002 New Trends in Fluorescence Spectroscopy Volume Editor: B. Valeur Vol. 1, 2001

Advanced Fluorescence Reporters in Chemistry and Biology II Molecular Constructions, Polymers and Nanoparticles Volume Editor: A.P. Demchenko Vol. 9, 2010

More information about this series at http://www.springer.com/series/4243

Fluorescence in Industry

Volume Editor: Bruno Pedras

With contributions by J. Avó
E. L. Bastos
M. Bedoya
M. N. Berberan-Santos
D. J. S. Birch
H. D. Burrows
L. Della Ciana
M. da Silva Baptista
F. B. Dias
J. J. Díaz-Mochón
E. Garcia-Fernandez
J. A. González-Vera
G. Hawa
G. Hungerford
G. Ielasi
C. A. T. Laia
K. Lobato
J. J. Lowe
V. B. Malkov
L. F. Marques
L. M. Martelo
D. McLoskey
G. Orellana
A. Orte
T. Palmeira
B. Pedras
S. Pernagallo
M. J. Ruedas-Rama
A. Ruivo
K. Sagoo
J. M. Serra
J. A. Silva
I. Urriza-Arsuaga
A. M. Vallêra
P. Yip

Volume Editor Bruno Pedras CQFM-IN and iBB-Institute for Bioengineering and Biosciences Instituto Superior Técnico Universidade de Lisboa Lisboa, Portugal

ISSN 1617-1306 ISSN 1865-1313 (electronic) Springer Series on Fluorescence ISBN 978-3-030-20032-9 ISBN 978-3-030-20033-6 (eBook) https://doi.org/10.1007/978-3-030-20033-6 © Springer Nature Switzerland AG 2019 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG. The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Series Editor Martin Hof Academy of Sciences of the Czech Republic J. Heyrovsky Institute of Physical Chemistry Department of Biophysical Chemistry Dolejskova 3 16223 Prague 8 Czech Republic [email protected]

Aims and Scope Fluorescence spectroscopy, fluorescence imaging and fluorescent probes are indispensible tools in numerous fields of modern medicine and science, including molecular biology, biophysics, biochemistry, clinical diagnosis and analytical and environmental chemistry. Applications stretch from spectroscopy and sensor technology to microscopy and imaging, to single molecule detection, to the development of novel fluorescent probes, and to proteomics and genomics. The Springer Series on Fluorescence aims at publishing state-of-the-art articles that can serve as invaluable tools for both practitioners and researchers being active in this highly interdisciplinary field. The carefully edited collection of papers in each volume will give continuous inspiration for new research and will point to exciting new trends.

Preface

Every now and then, scientists feel that their research topic may lead to an important practical application. This vision, not always clear, is frequently not pursued, owing to lack of adequate partners or insufficient funding, but is mostly impaired by unawareness of the real potential of the project. In the field of fluorescence (or broadly speaking, luminescence, since phosphorescence can also be involved), which has experienced a swift growth in the last decades, this mindset is particularly relevant given the numerous areas of industrial and commercial application of its methods and techniques. This volume is aimed at filling a gap in the literature devoted to fluorescence, by providing several examples of its industrial applications, described by both industrybased researchers and academics who hold strong collaborations with industrial partners. After Professor Martin Hof kindly invited me for this challenging task, my first guideline for choosing topics and authors was to reach a broad audience, having in mind what the reader can expect to learn from such a specific book. Hence, the purpose of this volume is to serve a wide variety of readers coming from both university laboratories (no matter how small or economically constrained) and companies with substantial funding for R&D activities. The former might have incomplete acquaintance with industrial applications and processes, in spite of their large experience in fundamental research; likewise, the latter may want to expand or update their knowledge on fluorescence/luminescence science. In both cases, this volume is intended to serve as a reference work and to stimulate an interplay of expertise. The edition of the present volume implied a major effort from all the parts involved and has passed by many stages until it reached the present form. I am obliged to all the authors for their outstanding contributions, which reflect a fragment of the state-of-the-art industrial work on fluorescence. Likewise, I would like to express my deep gratitude to the Series Editor, Prof. Dr. Martin Hof, for his kind

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invitation and for our fruitful discussions. Finally, my sincere appreciation to Dr. Steffen Pauly, Dr. Andrea Schlitzberger, and Ms. Ramya Venkitachalam from Springer, for their collaboration and constant support. Lisboa, Portugal February 2019

Bruno Pedras

Contents

Luminescence-Based Sensors for Bioprocess Applications . . . . . . . . . . . Idoia Urriza-Arsuaga, Guido Ielasi, Maximino Bedoya, and Guillermo Orellana

1

Fluorescence in Pharmaceutics and Cosmetics . . . . . . . . . . . . . . . . . . . . Maurício da Silva Baptista and Erick Leite Bastos

39

Instrumentation for Fluorescence Lifetime Measurement Using Photon Counting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 David J. S. Birch, Graham Hungerford, David McLoskey, Kulwinder Sagoo, and Philip Yip Applications of Submersible Fluorescence Sensors for Monitoring Hydrocarbons in Treated and Untreated Waters . . . . . . . . . . . . . . . . . . 135 Vadim B. Malkov and Jeremy J. Lowe Luminescence in Photovoltaics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 José Almeida Silva, João Manuel Serra, António Manuel Vallêra, and Killian Lobato Time-Gated Luminescence Acquisition for Biochemical Sensing: miRNA Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Emilio Garcia-Fernandez, Salvatore Pernagallo, Juan A. González-Vera, María J. Ruedas-Rama, Juan J. Díaz-Mochón, and Angel Orte Thermally Activated Delayed Fluorescence Emitters for Light-Emitting Diodes and Sensing Applications . . . . . . . . . . . . . . . . . . 269 João Avó, Tiago Palmeira, and Fernando B. Dias Explosives Detection: From Sensing to Response . . . . . . . . . . . . . . . . . . 293 Liliana Marques Martelo, Lino Forte Marques, Hugh Douglas Burrows, and Mário Nuno Berberan-Santos

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Contents

New Trends in Fluorescent Reporters in Biology . . . . . . . . . . . . . . . . . . 321 L. Della Ciana Application of Fluorescence in Life Sciences for Basic Research and Medical Diagnostics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 Gerhard Hawa Photoluminescent Glasses and Their Applications . . . . . . . . . . . . . . . . . 365 César A. T. Laia and Andreia Ruivo Luminescence-Based Sensors for Aeronautical Applications . . . . . . . . . . 389 Bruno Pedras, Guillermo Orellana, and Mário Nuno Berberan-Santos Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413

Luminescence-Based Sensors for Bioprocess Applications Idoia Urriza-Arsuaga, Guido Ielasi, Maximino Bedoya, and Guillermo Orellana

Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Luminescent Sensors for Molecular Oxygen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 O2 Sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Fiber-Optic Probe-Type O2 Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Oxygen Sensor Layers and Spots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4 Oxygen Sensing Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Luminescent CO2 Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Carbon Dioxide Sensing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Luminescence-Based CO2 Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Marketed Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Luminescent pH Sensors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 4 4 5 6 13 14 14 15 21 22 30 30

Abstract Reliable robust sensor systems are essential to monitor in situ and allow controlling of the evolution of bioprocesses in order to maximize the product yields and guarantee their quality. The more chemical and biological information is collected, the better strategies can be applied to generate highly productive cell lines and successfully determine the target production profiles. While the most common sensors employed in cell cultivation processes are those that measure (in-line) physical parameters (temperature, liquid level, conductivity, redox potential, etc.), ruggedized chemical monitors such as pH and gas phase/dissolved O2 and pCO2 are essential to determine the status of the cultured microorganisms. Luminescent chemical sensors for these three parameters have demonstrated in the last

I. Urriza-Arsuaga, G. Ielasi, M. Bedoya, and G. Orellana (*) Chemical Optosensors & Applied Photochemistry Group (GSOLFA), Department of Organic Chemistry, Faculty of Chemistry, Complutense University of Madrid, Madrid, Spain e-mail: [email protected] © Springer Nature Switzerland AG 2019 B. Pedras (ed.), Fluorescence in Industry, Springer Ser Fluoresc (2019) 18: 1–38, https://doi.org/10.1007/4243_2019_10, Published online: 21 March 2019

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few years their superiority over the traditional electrochemical sensors for in situ continuous real-time bioprocess monitoring, particularly those based on disposable analyte-sensitive patches attached to single-use bioreactors combined with emission lifetime measurements. This chapter reviews the progress achieved in luminescent sensing of O2, CO2, and pH for bioprocess applications that have led them to be the devices of choice for many manufacturers and customers in most situations. Keywords Bioprocess monitoring · Bioreactors · Carbon dioxide · CO2 · Fluorescence · Luminescence · O2 · Oxygen · pH · Probes · Sensors

1 Introduction Reliable analytical systems are essential to monitor and allow controlling of the evolution of a bioprocess’ conditions in order to maximize yields of the biologic products and guarantee their quality [1]. The more chemical/biological information is collected, the better optimization strategies can be applied to generate highly productive cell lines and successfully determine the target production profiles [2]. Cell cultivation (“upstream process”) in bioreactors is a complex three-phase system comprising a gas phase (aerated head space), a liquid phase (the culture medium), and a solid phase (suspended biomass) [3]. Therefore, sensors must monitor chemical, physical, and biological parameters in all the three phases during the bioprocess. Biological variables such as suspended cell density, microorganism viability, cell morphology, and protein or DNA contents are difficult to measure so that they are predominantly used in research environments because many of them require sampling and laboratory analyses. The most common sensors employed in cell cultivation processes are those that measure (in-line) physical parameters such as temperature, liquid level, conductivity, pressure, redox potential, foaming, etc. [4]; ruggedized versions of these sensors have been available for many years [1]. Essential chemical parameters such as pH and gas phase/dissolved O2 and pCO2 are measured in-line with dedicated electrochemical or optical sensors; dissolved relevant species such as glucose, lactate, or metabolites are measured in-line with biosensors (electrochemical or optical) or at-line with liquid or gas chromatographs with absorption, fluorescence, or mass spectrometric detectors [5]. In addition to being able to measure under the harsh conditions of the culture media, particularly related to sensor biofouling, external contamination by the monitoring devices must be avoided by all means. Therefore, disposable (singleuse) chemical and biological sensors are currently preferred fitted to disposable bioreactors [6, 7], and this is one of the leading advantages of optical sensors: window patches or spots adhered to pre-sterilized transparent (also disposable) plastic or glass bioreactors can be interrogated from outside, minimizing the risk

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of contamination in automatic (robotic) optimization systems (50–250 mL) and production bioreactors (>25 L) alike. Single-use bioreactor technology offers higher flexibility with lower investment and energy costs and, particularly, a streamlined production under the Good Manufacturing Practice (GMP) requirements because cumbersome labor-intensive cleaning procedures are avoided [6]. Not surprisingly, molecular oxygen, pH, and carbon dioxide sensors have been the most developed optical sensors to realize the advantages of the latter outside the laboratory applications. If, at the onset of the applications of optical sensing to bioprocess monitoring, pH was the most tested sensor due to the long-term availability of a large number of colorimetric indicator dyes, luminescent sensors quickly took over. This was due to the superior sensitivity and selectivity (plus stability, if emission lifetime-based) of the latter [8, 9], together with the significant technological advances in low-cost long-life light sources (light-emitting diodes or LEDs and diode lasers), photon detectors (e.g., avalanche photodiodes or APDs), fiber optics (multimode polymer-coated silica or PCS, low-attenuation plastic optical fibers or POF), digital electronics, and fast computing. This chapter focuses exclusively on the luminescence-based O2, CO2, and pH sensors for bioprocess applications, because these three parameters are arguably the most important in that field and because the corresponding luminescent sensors have attained enough maturity to reach the market [1, 3–7]. The term luminescence used throughout this chapter encompasses both the shortlived fluorescence emission (typically up to 50 ns) after a short excitation pulse and the longer-lived phosphorescence emission (typically above 200 ns) that display some molecules after photoexcitation with light of a shorter wavelength. Luminescent sensors can operate in the intensity mode, where the emission intensity at a selected wavelength is monitored, or in the lifetime mode, by which the average time that the photoexcited indicator molecule spends in its lowest-lying (electronic) excited state is measured. The luminescence lifetime is the reciprocal of the firstorder (or pseudo-first-order, in the case of quenching) kinetic rate constant of the excited-state deactivation process. The latter can be measured by the pump-probe method (the luminescence decay curve is rapidly sampled while returning to the ground state) or, more commonly, by the phase-resolved method using a sinusoidally modulated excitation light and measuring the phase shift of the (sinusoidally) modulated emission with respect to the excitation phase [10]. The intensity mode is always subject to drift due to the excitation light source or detector aging, luminescent indicator dye bleaching and leaching, or fiber-optic bending. Some of these factors can be corrected with intensity-ratiometric measurements at two-excitation/ single-emission or single-excitation/two-emission wavelengths; however, all of them are eliminated if the luminescence lifetime mode is used instead. The latter is nowadays readily implemented thanks to the performance of the LED sources (that can be modulated up to 20 MHz) and to the power and small size of computing microprocessors.

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2 Luminescent Sensors for Molecular Oxygen 2.1

O2 Sensing

Molecular oxygen (O2) plays an essential role in all aerobic bioprocesses if we consider that it is the key substrate for most microorganism and cell growth, maintenance, and metabolite production. A large variety of aerobic processes are driven in viscous aqueous media in the presence of salts and organic substances, in which oxygen is sparingly soluble. When a temporary depletion of dissolved O2 takes place, an irreversible cell damage and a reduction in production yield may occur [11]. Therefore, a properly designed bioreactor must ensure an adequate O2 supply, regulated by continuous O2 monitoring, independently of the bioreactor dimensions (laboratory, pilot, or industry scale) and the operation mode (batch, semicontinuous, or continuous), in such a way that optimal growth conditions and product formation are guaranteed. As a result, O2 monitoring is an issue of utmost importance in different industrial bioprocesses. For instance, in the pharmaceutical sector where the production of high-quality products, such as biotechnology drugs, vaccines, or antibiotics, is extraordinarily common, O2 levels play an important role in the final product yield and quality. The O2 content is also essential for beer-brewing companies. In the wort fermentation process, where the sugars are transformed into alcohol and carbon dioxide, the O2 concentration plays a vital role in the yeast effectiveness. Both O2 excess and deficiency can result in undesirable clarity, color, and taste properties of the final beer [12]. In the environmental field where biological systems have been used to transform objectionable materials into more environmentally friendly substances or high-value-added products, dissolved oxygen (DO) concentration is also relevant. For instance, water is cleaned in various steps in municipal wastewater treatment plants (WWTPs). Organic matter is digested by bacteria and other microorganisms through the biological process for water purification, where DO is essential to improve plant efficiency (energy consumption) and to prevent undesirable odors (https://www.emerson.com/documents/automation/application-data-dissolved-oxy gen-measurement-in-wastewater-treatment-en-68468.pdf. Accessed 10 Sept 2018). When it comes to the generation of high-value-added products, energy (biogas) can be produced by the anaerobic fermentation of residual organic matter. This process takes place in bioreactors in the presence of anaerobic organisms, whose activity and growth levels must be sustained by an accurate control of many variables including O2 (https://www.barbenanalytical.com/-/media/ametekbarbenanalytical/downloads/ application_notes/biogas_an_reva.pdf. Accessed 10 Dec 2018). The O2 concentration in the generated biomethane final product must also be monitored continuously, in real time and online, regardless if the sustainable biofuel is intended for injection into the natural gas distribution grid or for automotive purposes [13]. So far, in O2 determination, the traditional electrochemical devices (galvanic for measurements in the gas phase and amperometric for DO determinations) have been the most widely employed, in spite of suffering from drawbacks such as the short life

Luminescence-Based Sensors for Bioprocess Applications

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of the electrolyte or the membrane that limits the long-term stability and the regular need for recalibration and technical inspection that increase both the cost and the maintenance time. In contrast, optical sensors have become an advantageous alternative, especially when applied to small-scale reactors, since they are sterilizable, easily miniaturized, inexpensive, rugged, resistant to biofouling, reproducible, and easy to handle with almost no maintenance (only change of the sensitive film). Moreover, they can be interrogated contactless through the glass reactor wall or through a window. Over the last decade, many optical O2 sensors have been developed and commercialized, and their performance has been tested in different applications and conditions [14]. Most of them are based on the dynamic quenching of the luminescence of a photoexcited indicator dye by the O2 molecules. Collisions between them lead to an energy transfer that decreases the indicator luminescence quantum yield (approximated by the intensity at a particular wavelength) and lifetime [8]. This collisional quenching is described by the Stern-Volmer equation [10], where L0 (τ0) and L (τ) are the luminescence intensities (lifetimes) in the absence and the presence of oxygen, KSV is the so-called Stern-Volmer constant, kq is the bimolecular quenching constant, and [O2] is the oxygen concentration (Eq. 1). L0

=L ¼ τ0 =τ ¼ 1 þ k q τ0 ½O2  ¼ 1 þ K SV ½O2 

ð1Þ

In this type of sensors, the luminophore, which is commonly a Pd(II) or Pt (II) porphyrin, or a Ru(II)-polypyridyl complex, with long-lived (μs) luminescent properties [15, 16], is usually immobilized in a polymer matrix and integrated into bioreactors in different formats depending on the final application. Several reviews of the state-of-the-art O2 sensors applied to bioprocess monitoring can be found in the literature, from more traditional culture vessels (shake flask and bioreactors) [17] to microreactors and microfluidic chips [18–20]. Hereunder, some examples of luminescent O2 sensors applied to various biological processes are described, covering not only those sensors used in research laboratories but also those commercially available. Although they will be classified according to their format (sensor probes, sensor spots and layers, and sensor particles), examples of their application to O2 monitoring in different bioprocess vessels (bioreactors, shake flasks, well plates, or microfluidic chips) are also illustrated.

2.2

Fiber-Optic Probe-Type O2 Sensors

Early attempts to introduce optical O2 sensors in bioprocess applications started by immersing a fiber-optic probe in the cultivation media, the tip of which was coated with the sensitive fluorophore immobilized in an oxygen-permeable matrix [21]. Several examples can be found in the literature [22–24], where a luminophore (polycyclic aromatic hydrocarbon or Ru(II) polypyridyl complex) was embedded in a silicone or polystyrene membrane. Optical O2 sensing was applied to E. coli or

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yeast cultures, comparing their behavior to that of a traditional electrode but displaying particular advantages (no analyte consumption, fast response, absence of electrical interferences, resistance to biofouling). Further on, luminescent O2 sensors tended to be miniaturized, and significant efforts were made to improve their long-term stability, for example, by placing a biocompatible polymer overcoat, additionally protecting the sensor from biofouling and chemical degradation [25]. Likewise, those optical probes (“optodes”) started to be commercially available from different manufacturers for different applications (see Table 1). Generally, these devices consist of three parts: a sensor head connected to an optoelectronic unit or containing itself all the integrated optoelectronics; a dissolved O2 sensor cap, which contains the sensitive luminophore; and a sensor shaft that connects the oxygen meter and the sensing cap (Fig. 1). The diameter (1.6–40 mm) and immersion length (60–450 mm) of these commercial sensor probes vary depending on the brand. They are able to operate in a wide range of temperatures (typically 0–50 C, but up to 140 C in some models) and pressures (typically 0–10 bar, but up to 200 bar in specific models for environmental applications) and require very little maintenance (typically, just exchange of the sensor cap). Their sensitivity and accuracy are optimal for industrial applications, as well as their response time, which can vary anywhere from 2 to 60 s. The longer response time is often due to the aforementioned overcoat, used as an additional protection layer from biofouling or from mechanical abrasion. Although it was demonstrated that optical wand-type sensors improved some limitations of traditional electrodes (long-term stability, calibration drifts, CO2 interferences, and reliability at low O2 concentrations), the increasingly widespread use of small bioreactors required not only more miniaturized sensors but also contactless measurement systems.

2.3

Oxygen Sensor Layers and Spots

In this approach, the luminescent sensor film is affixed to the bottom or the side wall of a glass or plastic vessel, placing the optical fiber and the instrumentation outside the culture media, in a contactless measurement mode. In this way, by embedding the sensitive dyes in an O2-permeable polymer matrix, the sensor remains stationary during the bioprocess causing the least possible disruption and leaving valuable space for other required devices (e.g., stirrer, reagents, or gas feedthroughs). This sensor format has made miniaturization of the sensor possible, to their use not only in bioreactors and shake flasks but also in small-scale plastic vessels (typically 50–250 mL), and from microwell plates to microfluidic devices (Fig. 2). Many examples are described in the literature for different applications, and, as of today, these oxygen sensor formats are commercially available from various manufacturers. For instance, Tolosa et al. [26] have described a DO luminescent thin sensing layer, based on a platinum complex as indicator dye, immobilized in a polymer and

Pentair OGM WLO PreSens OXYBase series

Mettler Toledo InPro 6960i Hach M1100S10H

Ocean Optics FOXY probes Hamilton Visiferm DO

Sensor Mettler Toledo InPro 6870i PreSens OIM PSt3

0.1– 45 mg/L 0.02– 22.5 mg/L

15 ppb– 40 ppm

8 ppb– 25 ppm

0–62.85% 4 ppb– 25 ppm