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Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved. Biosensors: Properties, Materials and Applications : Properties, Materials and Applications, Nova Science Publishers, Incorporated, 2009. ProQuest

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved. Biosensors: Properties, Materials and Applications : Properties, Materials and Applications, Nova Science Publishers, Incorporated, 2009. ProQuest

Biotechnology in Agriculture, Industry and Medicine Series

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BIOSENSORS: PROPERTIES, MATERIALS AND APPLICATIONS

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BIOTECHNOLOGY IN AGRICULTURE, INDUSTRY AND MEDICINE SERIES Agricultural Biotechnology: An Economic Perspective Margriet F. Caswell, Keith O. Fuglie, and Cassandra A. Klotz 2003. ISBN: 1-59033-624-0

Biotechnology, Biodegradation, Water and Foodstuffs G.E. Zaikov and Larisa Petrivna Krylova (Editors) 2009. ISBN: 978-1-60692-097-8

Biotechnology in Agriculture and the Food Industry G.E. Zaikov (Editor) 2004. ISBN: 1-59454-119-1

Industrial Biotechnology Shara L. Aranoff, Daniel R. Pearson, Deanna Tanner Okun, Irving A. Williamson, Dean A. Pinkert, Robert A. Rogowsky, and Karen Laney-Cummings 2009. ISBN: 978-1-60692-256-9

Governing Risk in the 21st Century: Lessons from the World of Biotechnology Peter W.B. Phillips (Editor) 2006. ISBN: 1-59454-818-8

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Biotechnology and Industry G.E. Zaikov (Editor) 2007. ISBN: 1-59454-116-7 Research Progress in Biotechnology G.E. Zaikov (Editor) 2008. ISBN: 978-1-60456-000-8 Biotechnology and Bioengineering William G. Flynne (Editor) 2008. ISBN: 978-1-60456-067-1

Industrial Biotechnology: Patenting Trends and Innovation Katherine Linton, Philip Stone, Jeremy Wise, Alexander Bamiagis, Shannon Gaffney, Elizabeth Nesbitt, Matthew Potts, Robert Feinberg, Laura Polly, Sharon Greenfield, Monica Reed, Wanda Tolson, and Karen Laney-Cummings 2009. ISBN: 978-1-60741-032-4 Biosensors Properties, Materials and Applications Rafael Comeaux and Pablo Novotny (Editors) 2009 ISBN: 978-1-60741-617-3

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Biosensors: Properties, Materials and Applications : Properties, Materials and Applications, Nova Science Publishers, Incorporated, 2009. ProQuest

Biotechnology in Agriculture, Industry and Medicine Series

BIOSENSORS: PROPERTIES, MATERIALS AND APPLICATIONS

RAFAEL COMEAUX AND

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

PABLO NOVOTNY EDITORS

Nova Science Publishers, Inc. New York

Biosensors: Properties, Materials and Applications : Properties, Materials and Applications, Nova Science Publishers, Incorporated, 2009. ProQuest

Copyright © 2009 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA Biosensors : properties, materials and applications / [edited by] Rafael Comeaux and Pablo Novotny. p. ; cm. Includes bibliographical references and index. ISBN H%RRN 1. Biosensors. I. Comeaux, Rafael. II. Novotny, Pablo. [DNLM: 1. Biosensing Techniques--instrumentation. 2. Biosensing Techniques--methods. 3. Microchemistry--methods. 4. Nanostructures. QT 36 B61646 2009] R857.B54B5638 2009 610.28'4--dc22 2009015103

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CONTENTS

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Preface

vii

Chapter 1

Enzyme Modified Screen Printed Electrodes M.A. Alonso-Lomillo, O. Domínguez-Renedo and M.J. Arcos-Martínez

1

Chapter 2

Biosensors in Food Safety Control: An Update Spiridon Kintzios

53

Chapter 3

Properties and Choice of Material Used for Microbial Biosensor Mimma Pernetti, Denis Poncelet and Gerald Thouand

87

Chapter 4

Non-Conventional Strategies for Biosensing Elements Immobilization Christophe A. Marquette, Kévin A. Heyries Benjamin P. Corgier and Loïc J. Blum

127

Chapter 5

Electrochemiluminescent Sensors: Fabrications and Applications Hui Wei and Erkang Wang

Chapter 6

An Overview of Selected Lux-Marked Biosensors and Its Application as a Tool to Ecotoxicological Analysis Mwinyikione Mwinyihija

191

A Yellow Fluorescent Protein Variant as an Intracellular Iodide Biosensor in Thyroid Cells Kerry J. Rhoden, Stefano Cianchetta and Giovanni Romeo

215

Chapter 7

Chapter 8

Chapter 9

Microbial Biosensors and Biofuel Cells Based on Acetobacter and Gluconobacter Cells Juraj Svitel , Jan Tkac , Igor Vostiar , Marian Navratil and Peter Gemeiner Catalase Immobilized on Nanohybrid Materials for Electrochemical Hydrogen Peroxide Sensors: A Review Arun Prakash Periasamy, Yogeswaran Umasankar and Shen-Ming Chen

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161

247

265

vi Chapter 10

Chapter 11

Contents Gold Nanoparticle Labelled DNA Hairpin Grafting on Transparent and Conductive Oxide (TCO) Films: Characterization of Grafting and Hybridization V. Stambouli , V. Lavalley, A. Bionaz, P. Chaudouët , L. Rapenne , H. Roussel , A. Laurent, R. Jones and P. J. Pigram Biosensors Based on Ionic Liquids and Carbon Nanotubes; A Wide Potential of Applications in the Development of Third-Generation Biosensors José S. Torrecilla

Chapter 12

Human Olfactory System and Olfactory Biosensor Tai Hyun Park and Eun Hae Oh

Chapter 13

Development of Whole-Cell Biosensors Harboring the CarotenoidConverting Repoter Genes Isamu Maeda and Kazuyuki Yoshida

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Index

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285

321 331

347 363

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PREFACE A biosensor is an analytical device which converts a biological response into an electrical signal. It consists of 3 parts: the sensitive biological element, the transducer and the associated electronics or signal processors that are primarily responsible for the display of the results in a user-friendly way. The most widespread example of a commercial biosensor is the blood glucose biosensor. Recently, arrays of many different detector molecules have been applied in so-called electronic nose devices, where the pattern of response from the detectors is used to fingerprint a substance. There are also several applications of biosensors in food analysis. Optical sensors are used to detect pathogens and food toxins. Thus, the light system in these biosensors has been fluorescence, since this type of optical measurement can greatly amplify the signal. This book will present research on new materials, technologies as well as applications in the field of biosensors. Chapter 1 - Enzyme based screen printed electrodes respond to the growing need to perform rapid ’in situ’ analyses, being an alternative to the traditional electrodes. Screen printed technology has well known advantages of design flexibility, process automatization, good reproducibility, a wide choice of materials and reduce expense. It has become an alternative method for mass production of biosensors at low cost. This review gathers the developments in the electrochemical application of disposable screen printed sensors, according to the nature of the working electrode and its derivatization. Conductive inks are used to form conductive tracks and the electrodes of the sensors. In this way, carbon, gold and other metals have been used to the defined different SPEs configurations. Moreover, in order to improve the electroactive electrode area, micro and nanomaterials have been immobilized on the transducer prior the enzyme bounded. Taking into account that the sensing part attachment is the most significant step in biosensors development, the review is also focus on the different enzyme immobilization methods attempted. Applications are included where available. Chapter 2 - The conventional analysis of pesticide residues and/or pathogens in agricultural commodities is a labor-intensive procedure, since it is necessary to cover a wide range of different contaminants, using a single procedure. Standard analysis methods include extensive sample pretreatment (with solvent extraction and partitioning phases) and determination by gas chromatography (GC), high-pressure liquid chromatography (HPLC) and mass spectrometry (MS) to achieve the necessary selectivity and sensitivity for the different classes of compounds under detection. Pathogen identification usually requires culture-based methods or immunological assays, such as the enzyme-linked immunosorbent assay (ELISA). As a consequence, current methods of analysis provide a limited sample

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Rafael Comeaux and Pablo Novotny

analysis capacity, on a day/instrument basis. In a region-specific pattern, this results to a general lack of resources for implementation and enforcement of environmental and consumer safety regulations.Therefore, novel, rapid testing are needed. The present report is a thorough review of current biosensor-based methods and instrumentation of food quality control. Presented methods are associated with diversified techniques, such as impedance/conductivity measurements, cellular biosensors, microcalorimetry, flow cytometry, nuclease/lysate tests, adenosine triphosphate (ATP) measurement, luminescence/fluorescence, molecular biological tests and immunoassays. Particular emphasis is given on commercially available methods and products, as well as their market evaluation so far. Chapter 3 - Microbial biosensors are promising tools for the detection of specific substances and “global parameters” in different fields, such as environmental, food, biomedical and pharmaceutical. Immobilization of bacteria is a key feature in order to enhance the handling, the miniaturization, the storage and the stability of the biosensor. As many immobilization methods exist, a careful study must be carried out in order to select the best one for the application to a biosensor. No paper has ever been published on the systematic characterization of the immobilization systems in view of the application to a microbial biosensor. As a matter of facts, the performances of an immobilization method are usually evaluated indirectly, through the signal emitted by the biosensor. This work is intended to propose some guidelines to select the most appropriate immobilization system for a microbial biosensor. A survey of immobilization techniques and materials recently employed for microbial biosensors is provided. The selection criteria are finally applied to all the systems illustrated, on the basis of literature data, in order to provide a preliminary screening to be followed by the experimental characterization. Chapter 4 - The present article draws a general picture of non-conventional methods for biomolecules immobilization. The technologies presented are based either on original solid supports or on innovative immobilization processes. Polydimethylsiloxane elastomer will be presented as a popular immobilization support within the biochip developer community. Electro-addressing of biomolecules at the surface of conducting biochips will appear to be an interesting alternative to immobilization processes based on surface functionalization. Finally, bead-assisted biomolecules immobilization will be presented as an open field of research for biochip developments. Chapter 5 - Over the past decades, electrochemiluminescence (ECL) of tris(2,2’bipyridyl)ruthenium [Ru(bpy)32+] has received considerable attention from many researchers and been widely used to detect a variety of analytes that range from metal ions and small molecules to DNA, peptides, and proteins. Among all the areas of Ru(bpy)32+ ECL, the Ru(bpy)32+ based ECL sensors are of great active. In this chapter we will review the state of the art of Ru(bpy)32+ ECL sensors. After a brief introduction of Ru(bpy)32+ ECL and its mechanisms, the fabrications and applications of the Ru(bpy)32+ ECL sensors are discussed in details. It also indicates the future outlook in this field. Chapter 6 - Biosensors are based on incorporation of reporter genes from eukaryotes (luc) and prokaryote (lux). Lux based bacterial biosensors have gained increasing acceptance as rapid, relevant and reliable indicators of toxicity in a range of environmental samples such as ground water, soil and effluents entering rivers. Toxicity assessment in the environment can be attained by the use of lux reporter genes from Vibrio fischeri incorporated in to selected

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Preface

ix

terrestrial bacteria. The production of light (bioluminescence) is used to determine the level of toxicity by allowing the impact of the environmental contaminant on a bacteria population be measured. A reduction in bioluminescence (visible light emission in living organisms) means an effect on metabolic activity, which has a direct effect on energy production. Bioluminescence accompanies the oxidation of organic compounds (luciferins) mediated by an enzyme catalyst (luciferase). These reactions are important in biogeochemical analytical applications. The possible combinations of any bioreceptor (ensures molecular recognition) with any transducer (transforms an analytical signal) lead to a number of biosensors. Therefore the aim of a biosensor is the detection of an environmental signal, usually the presence or absence of a xenobiotic. The type of biosensor selected should be able to assess the toxicity of primary contaminant or pollutant be it organic or inorganic. Chapter 7 - Iodide is an essential trace element playing a vital role in the synthesis of thyroid hormones. Circulating iodide is accumulated in the thyroid gland thanks to a specific transporter, the Sodium Iodide Symporter (NIS). NIS-transported radioisotopes are clinically used to diagnose and treat thyroid cancer, and are being evaluated for radiotargeted cancer therapy and nuclear imaging following NIS gene transfer. Current techniques to measure the cellular accumulation of iodide via NIS in vitro include radiotracers and electrophysiological techniques. Fluorescent proteins are gaining popularity as genetically-encoded biosensors of intracellular events. Yellow Fluorescent Proteins (YFPs) are halide-sensitive, and have been used to monitor intracellular chloride concentration and chloride channel activity. In our laboratory, we have evaluated YFP-H148Q/I152L, a YFP variant with a high affinity and selectivity for iodide, as a potential biosensor of intracellular iodide concentration and NISmediated transport. YFP-H148Q/I152L can be transiently or stably expressed in cells by transfection with a cDNA-containing plasmid, resulting in a uniform cytoplasmic and nuclear distribution. Live cell imaging techniques permit dynamic changes in YFP-H148Q/I152L fluorescence to be monitored in small groups of cells or single cells. Iodide uptake can be quantified through calibration in a cell-free solution, or in intact cells permeabilized with ionselective ionophores. Exposure of FRTL-5 thyroid cells to extracellular iodide produces a rapid and reversible decrease in YFP-H148Q/I152L fluorescence consistent with iodide uptake. Iodide is concentrated up to 60-fold with respect to its extracellular concentration. Fluorescence changes are characterized by a (i) high affinity for extracellular iodide in the micromolar range, (ii) inhibition by the NIS inhibitor perchlorate, (iii) dependence on extracellular Na+, and (iv) regulation by thyroid stimulating hormone (TSH), suggesting that they are mediated by NIS. Iodide also induces a perchlorate-sensitive decrease in YFPH148Q/I152L fluorescence in COS-7 cells expressing ectopic NIS, but has no effect in cells lacking NIS. These results demonstrate that YFP-H148Q/I152L is a sensitive biosensor of iodide uptake in cells expressing endogenous and ectopic NIS. Intracellular iodide detection with YFP-H148Q/I152L may be a promising tool to study NIS function in thyroidal and nonthyroidal cells, to investigate the mechanisms underlying defective iodide transport in thyroid disease, and to identify compounds that augment the therapeutic and imaging potential of NIS-transported radioisotopes. Chapter 8 - Acetobacter and Gluconobacter bacteria are known to be able to efficiently oxidize multiple saccharide and alcohol substrates. This capability has been used to produce several industrially important products. There is also a growing trend of utilizing these bacteria to construct microbial cell-based biosensors and fuel cells. These biosensors are able to analyze oxidizable substrates including monoalcohols, polyalcohols, monosaccharides,

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several disaccharides, triglycerides and total utilizable saccharides. This review covers applications of gluconosensors to the construction of amperometric and thermal biosensors to analyze analytes important in foods and food industry, in beverages, and for the monitoring of fermentations and other biotechnological processes. Potential new applications for gluconobacter biosensors, future trends, and the development of robust high-throughput assays are discussed here. Chapter 9 - The studies of heme protein’s electrochemistry play a vital role because it provides vast information about the mechanism of metabolic process and biocatalytic pathway. This vast information helps the development and design of biosensors for various medical and clinical applications. In most heme protein’s electrochemistry, the electron transfer is almost impossible between heme protein and the underlying bare electrode. This is because of the prosthetic group buried deeply inside the polypeptide chain. Catalase (CAT) is one such heme protein which belongs to oxidoreductases family with ferriprotoporphyrin IX at its redox centre. CAT present in almost all aerobic living organisms, where it plays a major role by catalyzing disproportionation of H2O2 in to oxygen and water without forming free radicals. In order to study the electrochemistry of CAT, it has been immobilized on various substrate modified electrodes. Earlier attempts were made by researchers to immobilize CAT over pretreated glassy carbon electrode, graphite impregnated electrode, polymers, didodecyldimethylammonium bromide liquid crystals, protein agrose, silica sol-gels and methyl cellulose. CAT immobilized on various nanomaterials composed of carbon nanotubes (CNTs), metal nanoparticles, clay nanoparticles, metal oxide nanoparticles exhibit a dramatic improvement in the H2O2 reduction. Moreover, CAT immobilized on nanomaterial modified electrodes greatly enhances the electron transfer between heme group of the enzyme and electrode surface. The studies show that this heme protein is highly biocompatible and stable. This review discuss mainly upon the various nanohybrid materials immobilized with CAT and their application in H2O2 sensors. Chapter 10 - Biosensors and biochips can be hybrid nanobiosystems involving different kinds of components, i.e., solid surface, bio-molecules and nanoparticles. These components are confined in a very small area (nanometre range). It is expected that interactions are produced between both components due to their close proximity. Therefore, to optimize the performance of these biosensors, it is very important to get a deeper insight into their surface characteristics. In this context, nanoparticles linked to DNA strands (in a ratio of 1:1) immobilized on a solid surface provide the opportunity to combine complementary techniques to characterize the hybrid system. A typical example will be illustrated in this study. We have grafted DNA hairpins at their 3′-end via a silanisation process using aminopropyltriethoxysilane (APTES) on different transparent and conductive (TCO) oxide film surfaces. DNA hairpins comprise a stem in which both strands are complementary and a loop. These molecules exhibit a particularly high sensitivity for the detection of mismatches compared to the corresponding linear strands. They have been monolabelled at their 5′-end by a 1.4 nm gold nanoparticle. Because of the hairpin conformation, the label is close to the surface. Upon hybridization with a complementary target, the formation of a linear duplex structure with relative rigidity forces the label away from the surface. Due to their conductive properties, TCO films are attractive materials for biochips. They can advantageously replace the classical gold electrodes as working electrodes for direct electrochemical detection of DNA hybridization. As for silica, their surface chemistry allows

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Preface

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the covalent and strong binding of DNA. Here, we used different TCO films: ITO films, doped SnO2 films as well as insulating SiO2 films. Thanks to the presence of gold nanoparticles bound to DNA probes, the effects of grafting and hybridization of DNA could be studied on both conductive oxide surfaces. Particularly, we studied the modifications of surface morphology and chemistry as well as fluorescence results. By coupling AFM with SEM-FEG analyses, dispersed and well-resolved groups of gold nanoparticles linked to DNA were emphasized on the SnO2 films. Their surface density is 2.1 ± 0.3 x 1011 groups.cm-2. TEM images obtained after silver enhancement of gold nanoparticles on ITO films revealed round spheres corresponding to silver coated gold nanoparticles. Their density was in agreement with the data obtained by AFM on SnO2 films. The evolution of the chemical state of the modified oxide surfaces was monitored using XPS and ToF-SIMS. As expected, the XPS N 1s peak intensity increased after grafting and hybridization of DNA. The Au 4d peak was detected only on samples modified with Au labelled hairpin probes. Its intensity decreased with probe concentration. From the ratio Au/Si (Si belonging to APTES), the surface DNA density was estimated to be 9.6x1011 cm-2 and 3.7x1011 cm-2 for SnO2 and ITO films, respectively. The P 2p peak was observed only after hybridization with a weak intensity. Its presence was essentially correlated to phosphate residues originating from the hybridization solution. Positive and negative fragments of sugar, bases and phosphates from DNA probes were identified by TOF-SIMS. Positive and negative ions from Au nanoparticles were detected only in the case of Au labelled hairpin probes before and after hybridization. After hybridization of Au labelled hairpin probes with complementary Cy3 targets, quenching of the Cy3 fluorescence by gold nanoparticles was evidenced using fluorescence microscopy. This phenomenon was obtained for both oxides and is in agreement with the Nanometal Surface Energy Transfer (NSET) theory. Chapter 11 - Biosensors based on carbon nanotubes (CNTs) have gained considerable attention in recent years because of their novel properties such as their high surface area, electrical conductivity, good chemical stability and extremely high mechanical strength. In the last two decades, it has been found that many proteins and enzymes retain biological activity in other types of compounds called room temperature ionic liquids (ILs) and they show direct electrochemistry and environmentally benign media. They have been widely recognized and accepted in electrochemistry for their very broad electrochemical window, high electrical conductivity and high thermal stability. The possibility of joining carbon nanotubes and ionic liquids gives us the possibility to take advantage of the important characteristics of both groups and to open a door to another generation of biosensors, made up of ILs and CNTs composites. There are great expectations that these composites will have a wide range of potential applications in the development of third-generation biosensors. A summary of the state of the art is presented in this chapter. Chapter 12 - Although research on olfaction initially began about a hundred years ago and electrophysiological experimental techniques have been used in olfaction research since 1950's, olfaction has not received as much attention as the other senses. However, since Dr. Buck and Dr. Axel received the Novel prize for identifying the olfactory mechanism in 2004, various studies have examined olfactory sensors due to its potential commercial applications. In the olfactory system, the first event for smell sensing is the binding of the odorant molecules to olfactory receptors, which initiates electrical signals. These electrical signals

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then travel along the axons to the olfactory bulb at the front of the brain. The olfactory receptor can be effectively used as a biological element in olfactory biosensors. Olfactory biosensors can be classified into cell-based and protein-based biosensors. The cell-based biosensor uses living cells that express olfactory receptors as the biological sensing elements and the protein-based biosensor uses the olfactory receptor protein as the biological sensing elements. The signals derived from the binding of odorant molecules to the olfactory receptors can be measured using various devices such as QCM, SPR, microelectrodes, and carbon nanotubes. As a result of the development of various assay systems, the number of deorphanized olfactory receptors is increasing. The ability to use olfactory biosensors in a wide range of different scientific and medical fields for sensing odors is dependent on the development of highly sensitive and selective biosensors. This chapter describes the human olfactory system and olfactory biosensors. Chapter 13 - The changes in color from yellow or green to red in carotenoidaccumulating photosynthetic bacteria have been applied to reporter events in whole-cell biosensors. The first-generation photosynthetic bacterial sensors were genetically engineered to express crtA, the gene encoding spheroidene monooxygenase (CrtA), as the reporter gene under a control of specific transcriptional switches. Rhodovulum sulfidophilum CDM2 is a yellow mutant in which crtA was removed from its genome DNA. To construct the biosensor strains in order to detect dimethyl sulfide (DMS) or arsenite, the crtA gene was placed downstream of the DMS dehydrogenase gene promoter of Rvu. sulfidophilum or the operator/promoter region of the ars operon and arsR gene from Escherichia coli, and reintroduced into Rvu. sulfidophilum CDM2. The biosensors changed their colors from yellow to red in response to the analytes as a result of biochemical conversion of demethylspheroidene/spheroidene to demethylspheroidenone/spheroidenone by CrtA. The second-generation photosynthetic bacterial sensor uses crtI, the gene encoding phytoene dehydrogenase (CrtI), as the reporter gene. The green crtI-deleted mutant, Rhodopseudomonas palustris no.711, accumulated the colorless carotenoid, phytoene, and bacteriochlorophyll a which made bacterial color greenish. By plotting the colors of the green mutant (no.711), the yellow mutant (CDM2), and their wild-type strains in the CIE-L*a*b* color space, the differences in hue angle (ΔHAs) between a mutant and its wild type were calculated. The ΔHA value was higher in Rps. palustris no.711 than in Rvu. sulfidophilum CDM2, the host strain of the first generation biosensors. This indicates that a change in bacterial color from green to red is more distinguishable than that from yellow to red as a reporter signal of carotenoid-based whole-cell biosensors. A whole-cell arsenite biosensor has been developed using the green mutant, no.711, and characterized. A sensor plasmid containing the ars operator/promoter, the arsR gene, and the crtI gene of Rps. palustris was introduced into no.711. The biosensor changed the color in response to 10 and 50 μg/Lppb As(III), and this change was obvious to the naked eye after 24 hrs without further manipulation. In the crtI-biosensor, the relative levels of lycopene that was a CrtI-catalyzing reaction product and rhodopin that was a downstream product of lycopene increased in response to 50 μg/Lppb As(III). The hue angle in the color space after the reporter event clearly shifted from greenish yellow toward the red. The colorimetric whole-cell biosensors, by which toxic metal contaminations could be detected without additional chromogenic reagents and instruments, might be useful to monitor quality of ground water and well water especially in developing countries.

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Chapter 1

ENZYME MODIFIED SCREEN PRINTED ELECTRODES M.A. Alonso-Lomillo*, O. Domínguez-Renedo* and M.J. Arcos-Martínez* Department of Chemistry. Faculty of Sciences. University of Burgos. Plaza Misael Bañuelos s/n, 09001 Burgos, Spain.

ABSTRACT

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Enzyme based screen printed electrodes respond to the growing need to perform rapid ’in situ’ analyses, being an alternative to the traditional electrodes. Screen printed technology has well known advantages of design flexibility, process automatization, good reproducibility, a wide choice of materials and reduce expense. It has become an alternative method for mass production of biosensors at low cost. This review gathers the developments in the electrochemical application of disposable screen printed sensors, according to the nature of the working electrode and its derivatization. Conductive inks are used to form conductive tracks and the electrodes of the sensors. In this way, carbon, gold and other metals have been used to the defined different SPEs configurations. Moreover, in order to improve the electroactive electrode area, micro and nanomaterials have been immobilized on the transducer prior the enzyme bounded. Taking into account that the sensing part attachment is the most significant step in biosensors development, the review is also focus on the different enzyme immobilization methods attempted. Applications are included where available.

*

[email protected] [email protected] * [email protected] *

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2

M.A. Alonso-Lomillo, O. Domínguez-Renedo and M.J. Arcos-Martínez

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1. INTRODUCTION Nowadays, there is increasing demand for fast, reliable, low-cost methods that may be applied in situ in many areas of chemical analysis. Moreover, great interest is currently expressed in continuous monitoring of different analytes in a range of areas (environment, foodstuffs, medicine, sports medicine control, etc). Amongst the various analytical methods, electrochemical ones have already proven themselves to be convenient, exact and accurate for different analytical determinations. In fact, they are now used to replace time-consuming and expensive extraction steps, which are more commonly used in conventional analytical processes. Together with the acknowledged advantages of their versatility and the relatively low cost of electrochemical instrumentation, the high sensitivity and selectivity of some of these methods should also be considered. Amongst other reasons, the versatility of such techniques resides in the great number of substances that undergo redox processes, and in the wide range of electrodic systems that can act as substrates in those processes. The current tendency is the development of miniaturised systems, with the aforementioned characteristics, that enable determination in situ and on line. The advent of screen printing technology has added new improvements to the already well-established advantages of electrochemical methods. In fact, this technology has made it possible to mass-produce inexpensive, ready-to-use, disposable electrodes for use with electrochemical devices. Furthermore, screen printed electrodes (SPEs) increase the versatility of these kinds of techniques due to the wide range of their possible modifications. The search for selectivity in analytical determination has led to the development of a great range of devices, known as biosensors. They function on the basis of the specific reactions of an analyte in the presence of biological substances. According to the IUPAC [1], a biosensor is an integrated device that is able to supply analytical information of a quantitative or semi-quantitative nature, using a biological recognition element that is in contact with an appropriate transduction system. Among the different types employed in the construction of biosensors, optical, piezoelectric, acoustic, thermal and electrochemical transducers figure prominently in the literature. The last-mentioned technique now constitutes the basis of commercial biosensors, such as those widely used by diabetic patients to detect glucose levels. Although sensitive biological elements such as antibodies, nucleic acids, and receptors have been frequently used in the development of electrochemical biosensors, enzymatic biosensors have emerged over recent decades as one of the most sensitive and rapid analytical devices in environmental monitoring, and quality control systems, notably in the food industry. They have the potential to complement or even to replace conventional analytical methods by simplifying or eliminating sample preparation protocols, and by making field testing easier and faster with a significant decrease in per-analysis costs. Enzymatic immobilization in SPEs has given rise to the development of simple, low-cost, disposable biosensors that present potential advantages over conventional electrochemical biosensors. This is borne out by the great number of works in which the use of these devices is prescribed for the analysis of a great variety of analytes. In this chapter, we describe the scope, the importance and the enormous breadth of possibilities that these types of devices contribute to the current state of chemical analysis.

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Enzyme Modified Screen Printed Electrodes

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2. SCREEN PRINTED ELECTRODES Screen printing technology, a direct printing method otherwise known as penetration printing, has been successfully used in the construction of disposable, miniaturised electrodes since 1990. It consists of layer-by-layer depositions of ink upon a substrate, through the use of a screen or mesh, in such a way that the ink passes through the open spaces in the screen to be printed on the substrate, thereby defining the geometry of the sensor. At the same time as enabling the mass production of electrodes at an extremely low cost, screen printing is above all a simple technology that can be applied in any laboratory, and it is highly appropriate for the production of disposable electrodes. SPEs developed with this technology go a long way to meeting the requirements for sensitivity, selectivity, stability, precision, ease of use, low cost and robustness that are currently pursued in the development of analytical sensors. Their low cost, and easy use, coupled with an almost infinite number of possible modifications that can be made to SPEs, have all played a decisive role in the development of the commercial versions of these electrodes. This is driven by the high levels of demand mentioned earlier, principally because of the need for analysis in the food industry, and in clinical and environmental sectors of the economy [2]. The problems of reproducibility associated with the use of solid electrodes, which require the regeneration of the electrode surface after usage, constitute the greatest drawback to the development of solid commercial electrodes. Physical and chemical-electrochemical treatments capable of efficiently regenerating a surface are inappropriate for sensors that are designed for commercial use, as they depend to a great extent on the operating mode. Disposable SPEs have undoubtedly provided an answer to this problem, as they eliminate the need for regeneration of the surface. One of the principal aspects of SPEs, which makes them highly attractive when seeking to develop commercial sensors, is the possibility of total automatization in the fabrication of a complete system containing the working electrode, and also the auxiliary and the reference electrodes, all of which are printed on the same substrate. Furthermore, the ease with which these electrodes may be miniaturised means that they are suitable to be integrated in lightweight, portable equipment, which enable readings to be taken in situ. All of the above features, associated with their ease of preparation, flexible design, and the great range of modifications to which SPEs may be subjected, mean that they possess great advantages in comparison with other devices.

2.1. General Aspects of SPEs Production The process of fabricating an SPE basically consists of passing ink through a screen mesh to deposit it upon a flat substrate, so as to print the electrode (Figure 1). A schematic drawing of the electrode that is defined by the pores of the substrate is reproduced on the same substrate. Normally, repeated combinations of different screens and inks are employed for the manufacture of the three electrodes (working electrode, reference electrode and contraelectrode), which constitute the system to be introduced into the electrochemical cell and connected to the potentiostat (Figure 2). In general, the final stage of the printing consists

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in applying a partial insulator layer, in order to define the area of electrical contact at one end, and the surface of the electrode at the other.

Figure 1. Screen printing process. An air compressor is used for the generation of a negative pressure.

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There is a series of basic stages in the process of fabricating SPEs: selection of the screen, selection and preparation of the inks, selection of the substrate, and the printing, drying and curing stages. The process is repeated for each of the successive layers.

Screen 1

Screen 2

Screen 3

Screen 4

Figure 2. Schematic representation of various screens for the construction of SPEs.

Drying and curing are sometimes performed in a single step in the printing process, whereas at other times they need to be carried out before the next layer is printed. Suitable temperatures at which to carry out these processes differ in each case and can fluctuate from room temperature up to 1000º C.

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The design of the SPE is, of course, a preliminary stage in its fabrication, as is the selection of the materials to be used, such as the substrates, inks and the sensor material, the latter material only where it is necessary to modify the working electrode. Screen printed techniques are used solely in the construction of the working electrode [3, 4] or in the construction of the working electrode with the reference electrode [5-8], or the three electrodes may be jointly manufactured (working, reference and counter) [9-13]. Furthermore, it is possible to construct systems in which more than one working electrode is used (array system) [11, 14-18]

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2.1.1. Substrate The substrate is the material upon which the functional elements of the sensor are printed. In general, the substrate should be robust, low cost, show good compatibility with the other elements of the sensor and be made of an inert material. The most commonly used substrates are made of aluminum-ceramic materials [5, 9-11, 19-28], PVC [3, 29-34], enamelled steel [35], polycarbonate [12], or cardboard coated with acrylic paint [36]. 2.1.2. Inks There are a great variety of inks available from specialized commercial companies that offer a wide range of physicochemical properties (viscosity, conductivity, thermal resistance, water resistant, etc.). However, modifications are usually made to the majority of applications, in order to adapt them to the desired analytical objective. The pastes usually contain a thickening agent (ground glass, resins, etc.), solvents (terpinolene, ethylenglycol, cyclohexanone, etc.) and additives, which give them their functional features. Thus, the first state of the manufacturing process consists in the deposition of a conductive layer of carbon, silver, gold, platinum or other material, so as to assure the electrical conductivity of the electrodes. Following which, the successive layers of ink are deposited on the substrate. Where necessary, the use of dielectric pastes should be included in the metallic-oxide-based additives or thickening agents with insulating properties, such as aluminum or silica. 2.1.3. Sensing Element The sensing element, which is the analyte-specific part of a biosensor, may be introduced directly into the paste. It is also possible to fix it to the surface of the electrode using other immobilization processes such as adsorption, cross-linking, covalent bonding, etc. In both cases, it is preferable to apply these materials during the final stages of the fabrication process, to avoid any potential loss of sensitivity and stability when the recognition elements are applied in extreme conditions [11].

3. SCREEN PRINTED ENZYMATIC BIOSENSORS One of the most important objectives of screen printing technology is, without a doubt, the preparation of disposable modified electrodes. The use of SPEs represents an extremely

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attractive alternative, when compared to conventional electrodes that use mercury, glassy carbon, noble metals and carbon paste. The enzymatic modification of these devices, however, has proved to be one of the modifications to have stimulated the greatest interest [37]. Enzymes were the first bioreceptors used, and they are still used more than any others in the fabrication of biosensors. An enzyme is a protein that is able to catalyze a chemical reaction. They react in a selective way with an analyte or family of analytes, accelerating the chemical reaction and without being consumed by it. The basic mechanism of enzymatic catalysis is as follows: E + S ↔ E-S → E + P where S is the substrate, E the enzyme, ES the enzyme-substrate complex, and P is the product. Enzymatic activity is regulated by the pH and the ionic strength of the medium, as well as the temperature. In some cases it requires the presence of a cofactor, which is a non-proteic chemical product needed for the enzymatic reaction to take place, for example NAD+ or oxygen. There are different types of enzymes classified according to the reaction type; hydrolases and the oxidoreductases being the most widely used in the fabrication of enzymatic biosensors.

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3.1. Immobilization of Enzymes One of the key stages in the design and manufacture of enzymatic biosensors is the immobilization of the enzyme on the electrochemical transducer. Characteristics as important as their working life and their sensitivity depend in great measure on the immobilization methodology that is used. The confinement of the biological material on the electrodic surface can lead to an increase in its stability, as well as to the possible reuse of the biosensor, reducing the cost of the process. Nevertheless, it may also lead to the enzyme losing its catalytic activity or to an alteration in its structural conformation with respect to its native state. Immobilization of enzymes on SPEs is usually performed in one of a variety of separate steps: the adsorption of the enzyme on the electrode surface, or on a membrane in disposable sensors; its entrapment in a carbon ink or in a polymer matrix; the formation of either a crosslinked enzyme layer, or a covalent bond between the enzyme and the electrode surface; and, finally, by screen printing an enzyme-containing ink onto the working electrode. The aforementioned approach results in a biosensor manufactured with only one technology. The enzyme layer is screen printed by the same process as the electrode pads, conductor lines and insulation [10]

3.1.1. Adsorption Adsorption is the simplest immobilization method, which is why it has been wisely used in the construction of disposable biosensors, as described in the bibliography [38-42]. It is based on the fact that many substances, such as alumina, clays, graphite and silica gel share

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the property of adsorbing biological material on their surfaces. In the process, the enzyme bonds with the surface without functionalization, solely due to ionic interactions, Van der Waals forces and hydrogen bridges. Factors such as the pH of the medium, ionic forces, the temperature or the presence of other ions can significantly affect the processes of enzymatic adsorption. The greatest drawbacks of this method are its limited mechanical stability and the weak bond that is formed with the substrate.

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3.1.2. Entrapment Entrapment consists of the physical retention of the enzyme in the inner cavities of the porous matrix, which are generally constituted by photocurable monomers or polyethylenimine-type polymers [43], Nafion [44], polyvinyl alcohol [45], polyvinyl alcohol containing stilbazolium groups (PVA-SbQ) [46] or polypyrrole [47, 48]. Hydrogels are also commonly used [49], such as poly(carbomyl sulphonate) [50-52] and sodium carboxymethyl cellulose [53, 54]. It is a simple technique from the experimental point of view, and it does not require a great amount of biological material. An additional advantage is that the enzyme undergoes no structural alteration. One of the drawbacks is the creation of a significant barrier that prevents the diffusion of the analyte towards the active centre of the enzyme, increasing the response time of the sensor. Entrapment requires rigorous control of the polymerization conditions, as they can alter the reactive groups of the protein, which can reduce enzymatic activity. 3.1.3. Microencapsulation In this technique, the enzymes are surrounded by semi-permeable membranes that allow substrate molecules and products to pass through them, but which block the enzyme [55-61]. This method allows the biological material to enter into direct contact with the transducer, and, in turn, maintains the high selectivity of the enzymes given that they are not affected by the changes in the pH, the temperature or the ionic strength of the medium [62]. 3.1.4. Cross-Linking This technique has been widely used in the stabilisation of enzymes [63]. The method uses bifunctional reactives (dialdehydes, diiminoesters, diisocyanates, diazonium salts, and even diamines activated with carbodiimide, that give rise to intermolecular bonds between enzyme molecules. Among the most widely used bifunctional reagents for the immobilization of enzymes on SPEs is GA [26, 64-68]. A very common mixed immobilization procedure consists in immobilizing the enzyme by adsorption in a polymeric matrix. In this way, a prominent enzymatic face is constructed, which is subsequently modified with bifunctional reagents [69-71]. Protein-rich residues of lysine, such as seralbumin, are frequently added so as to prevent loss of enzymatic activity. 3.1.5. Covalent Bonding Covalent bonding as a methodology is based on the activation of chemical groups in the substrate so that they react with the nucleophiles of the proteins [72]. To form covalent bonds, the most often used aminoacids are lysine, cysteine, tyrosine and histidine. This type of immobilization requires close control of parameters such as the pH or the ionic strength of the solution. It also requires a perfectly clean transducer surface. The great

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advantage of this method is that it achieves very long working lives of between 4 and 16 months. This procedure has been used to immobilize cholesterol oxidase. A thiol, in this case 3mercaptopropionic acid, was self-assembled onto a gold screen printed working electrode. Subsequently, the covalent immobilization of the enzyme was assured through carbodiimide coupling between the carboxyl groups of the self-assembled layer and the amino groups of the enzyme by using 1-ethyl-3(3-dimethylamino propyl) carbodiimide methiodide [73].

3.1.6. Screen Printed Enzymes When using SPE-based electrochemical biosensors, the immobilization of the enzyme may be performed during the process of constructing the device [74-76]. To do so, the enzyme to be immobilized is mixed with the conductive ink. This ink is usually composed of activated carbon, a polymer and silicone oil. A polymeric solution is first prepared by dissolving the polymer in a buffer solution, and filtering the resulting solution. The filtered solution is thoroughly mixed with silicone oil and finally, with a solution containing the required amount of the chosen enzyme. Other biosensor components such as cofactors or mediators can be added to the ink together with the enzyme [10, 77-79]. Many enzymes are denatured by the organic solvents themselves or by the elevated temperatures required in the curing process, which makes the bulk incorporation of enzymes into the ink matrix problematic. Consequently, water-based inks have been used to incorporate enzymes [17, 78, 80]. Screen printing offers a possible route for application of membranes, and has the added attraction of enabling sensors to be completed using a single machine method [80, 81]. This membrane acts as a protective layer for the integrated water soluble enzyme matrix, preventing dissolution of the electrode material by the test solution and potential interferents [81].

3.2.Types of Screen Printed Enzymatic Biosensors With respect to the electrochemical technique used for the transducer, enzymatic biosensors can either be amperometric [64, 82], potentiometric [61], or conductimetric [26, 83], amperometry being the most widely used method. An amperometric biosensor combines the simplicity of an amperometric transducer with the heightened selectivity of the enzymes. The analyte to be determined diffuses through the solution until it enters into contact with the active centre of the enzyme, where it reacts to produce a product that generally has redox properties. This is oxidized or reduced on the electrode to generate a product that once again diffuses through the solution. The recorded intensity is proportional to the concentration of analyte. This is not the rate-limiting step in the global process, bearing in mind that the electrochemical reaction is usually much more rapid than the enzymatic one. Different methods of electronic transfer between the enzyme and the amperometric transducer exist, on the basis of which three generations of biosensors may be distinguished. First-generation electrodes based on the measurement of either one of the products, or of the

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Enzyme Modified Screen Printed Electrodes

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cofactor of the enzymatic reaction, meaning that the analyte under study has to be electroactive. Second-generation electrodes incorporate a mediator, which performs the electronic transfer between the active centre of the enzyme and the surface of the electrode. The mediator has to react quickly with the active centre, thereby minimizing competition with the natural cofactor of the enzyme [84]. It is advisable that its redox potential be close to zero, in order to avoid redox reactions between possible interferents [85]. The most frequently used mediators are the ferri/ferrocyanide couple [53, 54, 64, 65, 86-88], Meldola’s Blue (MB) [68], Prusian Blue (PB) [69, 70, 89-94] and Cobalt Phthalocyanine (CoPC) [50, 58, 69, 70, 82, 95-97]. In the third generation of biosensors, electronic transfer between the active centre of the enzyme and the electrode surface takes place directly. This type of biosensor shows greater selectivity, given that they work at potentials that are very close to the intrinsic potentials of the enzyme itself, reducing their exposure to possible interferents [98]. One of the greatest difficulties in the construction of this type of biosensor is the way in which the electronic transfer takes place between the active centre of the enzyme and the electrode surface. The great majority of enzymes have their active centres in their interior, which means that direct contact between the active centre and the surface of transducer is impossible [99].

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4. RECENT TRENDS, NANOMATERIALBASED ENZYMATIC BIOSENSORS Nanotechnology may be defined as the study, design, creation, synthesis, manipulation and application of materials, apparatus and functional systems through nanoscale control of the materials, and the exploitation of their phenomena and properties. In order to understand the potential of this technology, it is essential to understand that the physical and chemical properties of a material change at a nanometric scale, due to quantum effects. Electric conductivity, heat, resistance, elasticity and reactivity, among other properties, behave in a different way than the same elements at a larger scale. Thus, when materials are manipulated at a nanometric scale, they exhibit totally new phenomena and properties, which allows low-cost novel materials, apparatus and systems to be created with unique properties. A high-surface area is a fundamental factor that brings with it improvements in catalytic processes or on certain surfaces such as those of electrodes. In addition, a high-surface area allows a great number of interactions between the nanomaterials and the matrix in those known as nanocomposites, which also provide them with special properties. The use of nanomaterials plays an increasingly important role in the preparation of enzymatic biosensors for the reasons that have previously been outlined. In addition to these aforementioned properties, the nanomaterials deposited on the electrodes provide a more stable surface for the immobilization of the enzyme and on many occasions the use of mediators is no longer necessary [100]. Thus, a new area of investigation is opened up, which is only just beginning to be explored, creating new and important expectations for mass production of low-cost, disposable, fast, sensitive and selective sensors that are able to respond to the cutting-edge needs of contemporary chemical analysis.

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Metallic nanoparticles (NPs) have been the subject of intense research due to their novel material properties. Amongst these, gold NPs (AuNPs) display electronic, chemical and physical properties that may be employed in optical and electronic devices, and catalysis and sensor technology. In the biological sciences, the affinity of AuNPs for proteins has resulted in their being used as biomolecular labels. A recent paper [101] has validated the applicability of AuNPs in the construction of disposable biosensors for the determination of cholesterol. The biosensor developed in the paper is based on electron transfer between the enzyme and AuNPs immobilized on rhodium-graphite SPEs, which were shown to have high sensitivity and low detection limits for cholesterol. Another method recently employed for the improvement of the properties of electrochemical sensors and biosensors, such as their response magnitude, the reversibility of electrode processes and their long-term stability, is to modify the working electrode surface with carbon nanotubes (CNTs) [102]. CNTs are a type of inorganic material with a nanostructure that displays promising characteristics as an immobilization material, due to its significant mechanical strength, high-surface area, excellent electrical conductivity and good stability. CNTs can be considered the result of folding graphite layers into carbon cylinders. There are therefore two groups of CNTs: multi-walled (MWCNTs) and single-walled (SWCNTs). CNTs have been acknowledged as one of the most promising electrode materials and research has focused on their electrocatalytic behaviour in the oxidization of biomolecules. Their performance has proven far superior to other SPCEs in terms of reaction rate, reversibility and detection limits. Some attempts to modify a working electrode of SPCE with CNTs and carbon nanofibres (CNFs) have been undertaken using both commercial and laboratory preparations. CNF, SWCNT and MWCNT suspensions can be directly deposited on the working electrode surface [102]. Guan et al. [42] have shown that the catalytic ability of an immobilized enzyme is improved when MWCNTs are used to modify SPCEs. In this case, a MWCNT suspension was mixed with a solution of the enzyme, and the mixture was applied to the surface of the working carbon electrode and then dried at room temperature. The disposable biosensor constructed in this way demonstrated that the electrons could transfer more easily between the electrode surface and bioactive center of the enzyme, because of the special structure of the MWCNT-enzyme complex. MWCNTs have also been used by different authors in the modification of SPCEs for the analysis of cholesterol. Li et al. [54]. developed a disposable biosensor. The SPCE was first modified by dropping a MWCNT solution on the working electrode surface. Then, the enzyme and the mediator were immobilized using a hydrogel. Carrara et al. [103] proposed a cholesterol-sensing system, with an improved sensitivity based on rhodium-graphite SPEs, modified with MWCNTs and using cytochrome P450scc as the catalytic enzyme. In this case, the presence of nanotubes not only increased the sensitivity, but also the linearity of the detection response.

5. APPLICATIONS OF ENZYMATIC SPES In the following sections, applications of disposable enzymatic biosensors based on SPEs are described. Fields such as food, clinical and environmental analysis have been reviewed.

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5.1. Food Analysis The conformity of the industrial products regarding maintenance and warranty of their main features has a great economic importance. Foodstuffs quality control can be considered a key objective in food industry. Demands of sensitivity, specificity, speed and accuracy of analytical measurements have stimulated considerable interest in developing biochemical sensors as diagnostic tools in food industry [37, 104, 105]. Enzyme biosensors have been established as standard analytical methods, which simplify the performance of the tests and, therefore, reduce costs [105]. In this way, miniaturization of the sensor reduces the amount of enzyme and reagents. Enzymatic biosensors based on SPEs have been used the last two decades in the determination of important analytes in food industry [10]. Next, some examples of enzyme electrodes for food analysis are briefly presented.

5.1.1. Glucose The selection of a particular foodstuff by a consumer is largely based on sensory perceptions with taste, which is influenced by diverse factors including saltiness, sweetness, bitterness and acidity as perhaps the most important factors [45]. Glucose, which is very important for the final quality control, has been intensively investigated on foodstuffs process control (Table 1). It is well-known the determination of glucose with Glucose oxidase (GOX), according to the following three reaction steps, Glucose + GOX (FAD)

Glucono-δ-lactone + GOX (FADH2)

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Glucono-δ-lactone + H2O GOX (FADH2) + O2

Gluconic acid GOX (FAD) + H2O2

The overall reaction is expressed as,

Glucose + O2 + H2O

GOX

Gluconic acid + H2O2

Thus, glucose can be monitored amperometrically by measuring either the fall in O2, concentration, the production of H2O2, or by facilitating electron transfer from the enzyme to the electrode by using, for example, mediators [74, 105, 106]. The choice of the material for the working electrode influences the electrochemical behaviour of the set-up, and Pt, Au and various carbonaceous pastes are used for screen printing the working electrode depending on the sensor principle. The amperometric detection of enzymatically generated hydrogen peroxide at an electrode potential of about + 600mV (versus Ag/AgCl), using Pt or Au electrodes, has been developed as glucose biosensors [18, 74]. Immobilization of enzymes on SPEs is usually

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done by a separate step in a polymer matrix, such as polyvinyl acetate [18], polymethacrylate [74], or by the formation of a cross-linked enzyme layer with GA [10, 11]. The high overpotential needed for hydrogen peroxide oxidation at which many electroactive substances (i.e. ascorbic acid, uric acid, etc.), which are usually present in real samples, could be reduced using mediator-based electrodes [107] or bienzymatic biosensors [38, 64, 107]. Physical adsorption is the simplest method to confine both enzyme and mediator (pyocyanin, PdO, OsO2, IrO2 or PtO2) over a carbon working electrode [38, 39, 55]. Different protocols considering the simultaneous printing of both mediator, Prussian Blue (PB) or iron hexacyanoosmate (Osmium purple), and the carbon working electrode and the subsequent GOX immobilization by cross-linking with GA or in a polymer matrix, polyethylenimine [43] or nafion [44], respectively. Nafion [91] and Hydroxyethyl cellulose (HEC) [55, 56] have been used as outer membranes after the immobilization step to protect from potential interferents. The application of screen printing technology also for enzyme immobilization simplifies the production process. To achieve sufficient adsorption of the enzyme, enzyme printing pastes contained a high proportion of graphite particles, acting as graphite electrodes, and hence a mediator is normally used to avoid the high overpotential for H2O2 detection. Tetrathiafulvalene (TTF) [10] and manganese dioxide [77] have been used. The use of a bienzymatic-screen printed carbon electrodes (SPCEs) for glucose determination in grape juice has also been reported. Horseradish peroxidise (HRP), which catalyses the reduction of H2O2, can be immobilized together with GOX [107] by crosslinking with GA [64]. The enzymatic reaction was monitored by the redox reaction of a mediator, ferrocyanide (Fe2+), at the electrode surface, which reduces the potential of electrochemical oxidation of H2O2, according to the scheme,

Fe2+

HRPox

H2O

Fe3+

HRPred

H2O2

GOXox

Glucose

O2

GOXred

Gluconic acid

e-

The effective control of biotechnological processes requires the measurement of as many significant parameters as possible as frequently as possible; therefore, the measurement of glucose, which is often not only the main carbon source but also the growth-limiting substrate, is of particular interest. Operationally, excess glucose concentration can be responsible for such problems as overproduction of lactic acid which can have detrimental effects such as decreasing pH. Furthermore, the concentration of glucose can be used to switch cell metabolism between glycolytic and oxidative states and can affect uptake and utilization of alternative carbon sources such as amino acids [106]. Analytical systems for monitoring glucose concentration in biotechnological processes such as fermentation have been described. Mersal et al. [74] developed a GOX based Pt SPE using a polymethacrylate membrane for the enzyme immobilization step for the glucose determination in E. coli cultivation samples.

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Three SPE amperometric configurations, whereby hydrogen peroxide was oxidised at + 350 mV (Ag/AgCl) were designed to be used in a flow injection analysis (FIA) system [81, 106]. GOX ink, which also contained rhodinised carbon powder and HEC, was printed over the carbon working electrode, as well as a cellulose acetate outer membrane.

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Table 1. GOX based screen printed biosensors for glucose determination in foodstuffs Working electrode Au

Mediator

Immobilization method Polyvinyl acetate membrane Polymethacrylate membrane

-

Pt

-

Pt

-

C C C

Pyocyanin Ferrocenedimethanol Ferrocenedimethanol

C

PB

C

PB

C C

Fe2+ Osmium purple

Cross-linking with GA + nafion Polyethylenimine membrane cross-linking Nafion membrane

C C Rhodinised C

TTF MnO2 -

Printed Printed Printed

Cross-linking with GA Adsorption Adsorption Cross-linking, GA

Real sample analyzed

Reference

Fruit juice

[18]

Juice samples and E. coli cultivation samples Fruit juice, wine

[74]

Soft drinks Starch samples (rice, corn, green pea, potato, sweat potato and wheat) beverage

[39] [55] [56]

wine

[43]

Grape juice Honey, vodka, plum brandy Fruit juice, wine Wine and beer Microbial fermentations

[64] [44]

[10, 11]

[91]

[10] [77] [81, 106]

5.1.2. Fructose, Sucrose Lactose, and Lactulose The concentration of carbohydrates in food may change throughout the process of food production. For example, the concentrations of sucrose, glucose and fructose in wine are dependent on the time when sugar is added (prior or post fermentation) and on the degree of fermentation [11]. Also, lactose concentration is an important parameter indicating the quality of milk. Lactulose (4O-P-D-Galactopyranosyl-D-fructofuranose), the epimerized lactose, is a synthetic disaccharide consisting of galactose and fructose and is not present in natural sources. Lactulose, which is absent in raw milk, is formed in alkaline lactose solutions or by heating of milk due to the epimerization of lactose. Therefore, it can be used as an indicator for the severity of heat treatment of milk and to distinguish between pasteurized, ultra-heat treated (HI-IT) and sterilised milk [65] Fructose, sucrose and lactose have been analyzed in different samples using a Fructose dehydrogenase [108], GOX-Invertase-Mutarotase [11] and β-galactosidase-GOX [11, 109] based Pt SPEs, respectively. Sucrose determination requires a multi-enzyme system,

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Sucrose + H2O

Invertase

Fructose + α-D-glucose O2 , H2O GOX Gluconic acid + H2O2

The combination of Invertase and GOX with a third enzyme, Mutarotase, can be utilized in order to convert α-D-glucose to its β-isomer on which glucose oxidase is specific [105]. Lactose is also determined using a multi-enzymatic sensor,

Lactose + H2O

β-galactosidase

D-galactose + glucose

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β-galactosidase can be combined with GOX [11, 109] or galactose oxidase to produce the lactose enzyme electrode. The use of galactose oxidase resulted in the construction of a glucose-non-interferning lactose sensor [105]. Lactulose, which is hydrolysed to fructose and galactose by β-galactosidase, has been determined through the amount of fructose by using cross-linked fructose dehydrogenase based Pt SPE sensor and ferricyanide as mediator [65].

5.1.3. Lactate and Acetaldehyde The electrochemical determination of lactate using SPEs modified with enzymes has received much attention due to its importance in food fields. Lactate has been extensively analyzed in many fields such as the dairy products or wine industry. Various enzyme electrodes have already been described in literature and different configurations have been proposed taking into account the working electrode nature and the enzyme immobilization procedure. Platinised carbon has been utilized as working electrode in order to reduce the operating potential. Ink containing lactate oxidase (LOX) was printed onto the Pt doped graphite SPEs [14, 15, 110]. A cellulose acetate [110] or a PVC copolymer [14] outer membrane was used. The lactose determination in dairy products, buttermilk and yoghurt, has been carried out in batch [14] and FIA [15] mode. Polymer matrices (HEC, GafQuat/lactitol and HEC/polyethylenenimine) for LOX and diffusion-limiting membranes (polyvinyl chloride co-polymer/cellulose acetate butyrate), were integrated onto the Pt doped graphite SPEs to produce lactate sensors for cattle meat [17]. A disposable lactate biosensor, based on Pt SPEs, able to operate in FIA was described and characterized by Palmisano et al. in untreated milk and diluted yoghurt samples [66]. The biosensing layer, obtained by GA co-cross-linking of LOX with bovine serum albumin (BSA), was cast on an underlying electropolymerized layer of overoxidized polypyrrole.

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Disposable SPCEs modified with both HRP and LOX system operating on direct electron transfer have been described for the determination of L-lactate in dairy product [75]. Both LOX and HRP were incorporated in the graphite ink (SPCE–HRP/LOX),

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e-

O2

LOXox

Lactate

HRPred

H2O2

LOXred

Pyruvate

HRPox

H2O

The concentration of glucose and lactate in food products has been shown to be an indicator of bacterial activity, which may result in a drastic alteration in the quality of the final product. Hence, the simultaneous measurement has been attempted using two layered GOX and LOX modified electrodes by a mediator, ferricyanide ions [86]. Concentrations of glucose and lactate in lactic fermenting beverages were measured using the upper- and lowerlayer electrode, respectively. In wines, after the alcoholic and during the malo-lactic fermentation, some heterolactic bacteria are able to transform the remaining sugars into acetic acid, d-lactic acid and mannitol. These irreversible transformations lead to a bad sour taste of the wine [8]. Several biosensors have been developed for the determination of lactate in wines. A simple two-electrode system was used in the assays with MB or MB-Reinecke’s saltmodified SPCE being the working electrode and the Ag/AgCl electrode playing the role of both reference and counter electrode. This simplification of the classic three-electrode configuration, required in amperometry experiments, was made possible by the negligible ohmic drop expected for the described screen printed two-electrode system [6-8]. The sensing layer of the working electrodes was prepared by simply depositing on the working area of the electrodes a mixture containing, lactate dehydrogenase (LDH) and NAD+ [6, 7] LDH, β-NAD+, polyethyleneimine and Nafion [8] PVA-SbQ, LDH, polyethyleneimine and Nafion [7] LDH and PVA-SbQ [6] The detection of lactate is based on the sequence,

e-

MBred

NAD+

Lactate LDH

MBox

NADH

Pyruvate

Acetaldehyde is commonly found in alcoholic beverages as a result of the oxidation of ethanol by alcohol dehydrogenase (ADH). It is also produced during fermentation by Biosensors: Properties, Materials and Applications : Properties, Materials and Applications, Nova Science Publishers, Incorporated, 2009. ProQuest

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decarboxylation of pyruvate by pyruvate decarboxylase. Because of its toxic effects, it plays a central role in the manifestation of alcohol intoxication [111]. Acetaldehyde is a very important parameter because of its capacity of blocking sulphur dioxide in a very stable combination, thus preventing it from exercising its antioxidant and antiseptic function. Additionally, acetaldehyde is one of the earliest notable parameters when malfunction occurs during wine production. Enzyme sensors based on SPEs for the acetaldehyde detection during wine production have been described according to the procedure described by Avramescu et al. for lactate, using aldehyde dehydrogenase (ALDH) as sensing element [6, 7, 111],

e-

MBred

Acetaldehyde ALDH

MBox

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NAD+

Acetic acid

NADH

5.1.4. Malic Acid L-malic acid and citric acid are major organic acids in most fruits and vegetables, contributing greatly to taste. Significant increases in L-malic acid concentration have been shown to serve as a primary indicator of fruit maturity [45]. Malic acid determination is also of great value in the wine industry, since it enhances wine flavor, acts as a flavor blender and prevents turbidity [43]. Therefore, measurement of L-malic acid provides a more objective means of determining the ripeness and hence ‘shelf life’ of horticultural produce, as well as has an important role during the malolactic fermentation in wine production. There is a procedure for the malic acid determination during winemaking based on the adsorption of a mediator, MB, over a SPCE. Malic acid enzyme (ME) was then immobilized by a three steps technique, using consecutively polyethylenimine, GA and ME [43]. The measurement principle was based on the following scheme,

H+ e-

MBred

Malic acid

NADP+ ME

MBox

NADPH

Pyruvate

The monitoring of the malolactic fermentation of wines can also be performed using an amperometric malate quinone oxidoreductase (MQO) - SPCE [112],

e-

Mox

MQO-FAD+

Malic acid

Mred

MQO-FADH2

Oxaloacetic acid

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SPCEs coupled with appropriate mediators were used as transducers for this novel biosensor in which MQO was immobilized by physical entrapment in a photo-cross-linkable poly(vinyl alcohol) polymer on the surface of the working electrode. In the same way, SPEs based on adsorbed ME with amperometric measurement of NADPH oxidation have been proved suitable for the simple, low-cost and rapid determination of L-malic acid in apple, potato and tomato samples [45]. A rhodinised carbon working electrode, which favoured the oxidation of NADPH at a lower operating potential, was used, so addition of mediator was not required.

5.1.5. Ethanol The determination and control of ethanol in different beverages is important in the brewing and distilling industries at quality control level during the production and then, to test for adulteration. For the analysis of ethanol from a complex sample, biosensors based on ADH or alcohol oxidase (AOX) are reported in the literature. Sprules et al. [57] developed a MB modified SPCE that was coated with a mixture containing ADH and NAD+, using a cellulose acetate membrane, to produce a sensor that would respond to alcohol in commercial gin samples. The determination is based on the exploitation of catalytic currents resulting from the oxidation/reduction of the modifier.

e-

MBred

ADH MBox

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NADP+

C2H5OH C2H4O

NADPH

The method developed by Koztian et al. [44] for glucose determination based on Osmium purple as mediator has been applied to determination of ethanol in beverages (Plum brandy and Vodka), using ADH. The measurement of ethanol has been carried out using an oxidase enzyme. AOX adsorbed onto SPCEs doped with 5% CoPC (CoPC-SPCE) was coated with a permselective membrane, nitrocellulose acetate or polycarbonate membrane, on the surface, which acts as a barrier to interferents. The measurement of ethanol is based on the signal produced by H2O2, the product of the enzymatic reaction [58],

e-

Co2+

O2 AOX

Co+

H2O2

C2H5OH C2H4O

The ethanol destined only for industrial purposes is adulterated / transformed into products unsuitable for human consumption. Traditionally, the ethanol is adulterated by the addition of 10% methanol, which is difficult to remove. The fast, inexpensive and user

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friendly analysis of the methanol–ethanol mixtures is important to protect the public health from alcoholic beverages falsifications [46]. A dual biosensor analysis system that is able to simultaneously quantify and discriminate ethanol and methanol has been attempted [46]. The proposed system is based on the use of complex analytical information provided by both ADH and AOX, immobilized by entrapment in a photocross-linkable PVA-SbQ polymer. The detection principle is based on the fact that the ADH is able to oxidize only the ethanol from a methanol–ethanol mixture, while AOX catalyzes preferentially the methanol, but also the ethanol. Analyzing the same sample with two biosensors and using complex calibration curves, it is possible to simultaneously quantify both methanol and ethanol without analytes separation.

5.1.6. Pesticides Pesticides are used in agriculture to increase yield, control microorganisms, which might produce toxic or carcinogenic metabolites, and reduce the costs of food production. The disadvantages of their usage are drinking water and food contamination. Their toxicity is based on their inhibitory effect on acetylcholinesterase (AChE), an important enzyme of the nervous system in higher organisms. Especially infants are supposed to have an increased susceptibility. AChE-inhibiting insecticides, organophosphates and carbamates, are extensively used in agricultural and forestry industries, because of their insecticidal activity and their relatively low persistence in the environment. The choice of cholinesterase (ChE) as a biorecognition element enables to detect simultaneously a wide group of related toxic compounds: organophosphate and carbamate pesticides [95]. Selected carbamates (aldicarb, carbaryl, carbofuran, methomyl and propoxur) were characterized by screen printed biosensors. The sensors were prepared by SP using silver- and platinum-based pastes for reference and working electrodes. The working electrode was modified by depositing another graphite-based composite layer containing CoPC as a modifier and acetylcellulose as a binder. Cross-linked AChE and butyrylcholinesterase (BChE), with BSA and GA was immobilized over the working electrode. The suitability of the biosensor detection of carbamates was evaluated on samples of potatoes and carrots in the original state and after fortification with either carbofuran or propoxur [95], using acetylcholine iodide as substrate. The same measurement principle and immobilization procedure were followed by Albareda-Sirvent et al. [113], who designed devices integrating a photolithographic conducting copper tracks and a transducing graphite paste printed on. Paraoxon and carbofuran were determined in tap water and fruit juice samples. The assay is based on the inhibition of AChE by the pesticides,

e-

RCOthiocholine + H2O

Thiocholineox Thiocholinered

ChE RCOOH

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Enzyme Modified Screen Printed Electrodes

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The enzyme converts acetylthiocholine into its electroactive product thiocholine, which is detected at the electrode surface. In the presence of a pesticide, the enzyme is inhibited, which leads to a decrease in thiocholine production and a corresponding decrease in anodic current. This decrease is proportional to the logarithm of the pesticide concentration. ChE sensors based on SPCEs modified with polyaniline, 7,7',8,8'tetracyanoquinodimethane (TCNQ), PB and CoPC have also been developed and tested for detection of anticholinesterase pesticides in infant food [69, 70], spiked grape juice [89], wheat or apple extracts [16], grapes [114], grapes juice [115], milk [71] and apples [116]. The enzyme immobilization procedure attends to different methods,

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An enzyme printing ink containing BSA, AChE and HEC was printed on the top of the working electrodes an then, was cross-linked with GA vapour for 15 min at room temperature [69-71]. AChE solution can be directly dropped on the working electrodes and, once it is completely dried, BSA is added for enzyme stabilization and GA for cross-linking [89, 115]. Six individual biosensors were fabricated by depositing wildtype (WT) AChE from Drosophila melanogaster or one of five mutant forms (B02, B03, B04, B421, and B65) of this enzyme, onto the surfaces of CoPC-SPCEs constituting the amperometric biosensor array [16]. Purified AChE from electric eel or engineered yeast were immobilized on a nylon membrane for the measurement of residual ChE activity after inhibition by the organophosphorus insecticide Paraoxon [114]. AChE solution and PVA-SbQ were deposited manually over the surface of the working electrode. The electrodes were dry at room temperature and then, exposed for 3 h under neon light at 4 ºC to allow photopolymerization [116]. Bi-enzymatic sensors have also been used for this purpose. PB as a mediator in assembling the ChE-choline oxidase (ChO) bi-enzyme sensor was developed for the detection of organophosphorus and carbamic pesticides [89]. Both enzymes were co-immobilized on the working area of the SPE covered with PB. The cathodic current of the PB mediated H2O2 reduction was recorded in an amperometric mode as a measure of the acetylcholine concentration and activity of ChE,

PBred

OH-

PBox

H2O2

eChO O 2 + H 2O

Acetylcholine + H2O

Betaine Choline

ChE CH3COOH

AChE inhibition based systems lack a sufficient sensitivity towards phosphorothionates. These substances are weak AChE inhibitors, due to the low reactivity of the P = S group caused by minor electronegativity of sulfur compared to oxygen. The toxicity of phosphorothionates is based on their in vivo biotransformation by microsomal P450

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monooxygenases into the corresponding, strongly AChE inhibiting, oxones [117]. It has been described bienzymatic biosensor for the integrated metabolization and highly sensitive detection of phosphorothionates, which renders any further sample pretreatment unnecessary. Nippostronglylus brasiliensis AChE and a triple mutant of P450 BM-3 were encapsulated by a sol–gel process, establishing a method for screen printing the sol–gel on thick film electrodes.

5.1.7. Phenolic Compounds Oxidation of low density lipoproteins (LDLs) is a crucial step in the pathogenesis of atherosclerosis, because of their fundamental role in the health of human organisms. Many studies reported the inhibition of this oxidation by phenolic compounds in red wine in vitro, demonstrating also that the consumption of beverages rich in polyphenols is associated to the increase in plasma antioxidant potential and therefore to the prevention of coronary heart diseases. An exhaustive quantitative correlation between concentration of several phenols and toxicological effects on human health has not yet been achieved. Biosensors represent an interesting alternative way to usual toxicological and spectro-photometric assays in on-field evaluation of total polyphenolic effect [118]. Graphite-based printed electrodes are fabricated for the determination of phenolic compounds in lager beers. The enzyme tyrosinase is immobilised on the electrode using a straightforward polymerisation step, which could be adapted for mass production purposes. This immobilisation procedure involves the entrapment of the enzyme in one step during the electrosynthesis of a polypyrrole film issued from the electrochemical oxidation of an amphiphilic substituted pyrrole monomer. The biosensor was used to indicate the phenolic status of beer and brew samples [47, 48]. In order to overcome transport limitations due to its dissolution, which occur in commonly used immobilization procedures, the suitable mediators can be added directly into the conductive pastes. Ferrocene modified SPCEs have been used on whose surface the enzyme tyrosinase was immobilized in a GA cross-linked matrix of BSA for the determination of phenols and polyphenols in wine [118]. Virgin olive oil (VOO) is obtained from olives by employing mechanical processing only, and without further refinement steps these oils become available for consumption. VOO is associated with health benefits, such as protection against coronary heart diseases and cancer, and is recognized by its characteristic sensory properties: aroma, bitterness, astringency, and pungency. Bitterness and pungency are related to the presence of phenolic compounds [119]. The applicability of enzyme-based biosensors for the rapid prediction of these sensory properties of VOO has been investigated. The response of tyrosinase based SPCEs, built by using a Nafion membrane, to olive sample and HRP based SPCEs, fabricated by cross-linking with GA, has been compared, o-quinone e-

Catechol

Monophenol

e-

Phenol

HRPox

H2O

Phenolox

HRPred

H2O2

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5.1.8. Amino Acids L- and D-amino acids are common constituents of many fermented products such as beers, cheese and yoghurt. D-Amino acid levels are a useful indicator of microbial contamination of milk and of cheese age. Amino acid levels can also be used to monitor the state of fermentation processes and amino acids are a major nutrient source for yeast cell growth [120]. Biosensors, incorporating L-and/or D-amino acid oxidase (AAO), coupled to SPEs have been developed for monitoring mild ageing effects [102, 120], protein solutions [72],

H2O e-

H2O2 O2

Enzyme-FAD+

Amino acid

Enzyme-FADH2

2-Oxoacid + NH4+

To facilitate hydrogen peroxide oxidation at a decreased operating potential, different procedures have been attempted such as the incorporation to the working electrode of rhodinised carbon [72, 120] and PB printed [91] or in a Nafion membrane [102].

e-

PBox

OH-

PBred

H2O2

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O2

Enzyme-FAD+

Amino acid

Enzyme-FADH2

2-Oxoacid + NH4+

Enzyme immobilization has been carried out by cross-linking with GA and BSA [91, 102, 121] or by using a membrane, which involves the covalent binding of amino groups on the component to be immobilised with activated carboxyl groups on the membrane support [72]. As a result of the mediator addition and because of the multi-layer construction of the biosensor, including a polymer layer to avoid the interferences, the limit of the detection of the developed biosensor was two orders of magnitude improved in comparison to other screen printed biosensors, as far as the determination of amino acids is concerned. Additional modification of the graphite electrode with carbon nanotubes led to a significant enhancement of the signal magnitude [102].

5.1.9. Freshness Indices Biogenic amines are indicators of the freshness of fish. They are generated during protein decomposition via amino acid decarboxylation following the slaughter of the fish, their concentration increasing with storage time. Different approaches have already been reported to use immobilized polyamine oxidases for polyamine determination in fish and tissue samples.

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Pt SPEs were modified with 3-aminopropyltriethoxysilane prior to the immobilization of Putrescine oxidase by cross-linking with GA. The biosensors were applied for the detection of putrescine, cadaverine and spermidine as an indicator of fish freshness [10]. An enzyme sensor array for the simultaneous determination of three biogenic amines (histamine, tyramine and putrescine) by pattern recognition using an artificial neural network and its application to different food samples (from fish to meat products, sauerkraut, beer, dairy products, wine and further fermented foods) was also described. A combination of oxidase, a tyramine oxidase and a diamine oxidase were immobilised each on a separate SPE via transglutaminase and GA to compare these cross-linking reagents with regard to their suitability [122]. Freshness has been determined on the basis of indicators such as ATP-related compounds which normally do not exist in the living tissues of fish. Adenosine triphosphate (ATP) is decomposed in fish meat, adenosine diphosphate (ADP), adenosine monophosphate (AMP) and related compounds are formed by autolysis and/or microbial actions following the death of fish. ATP is then degraded to uric acid (AU) through the following pathway,

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ATP

ADP

AMP

IMP

HxR

Hx

X

AU

Hypoxanthine (Hx) and inosine (HxR) concentrations depend upon the species of fish. Inosine monophosphate (IMP) is one of the major contributing factors to the pleasant flavour of fresh fish. The accumulation of Hx and/or xanthine (X) during the storage results in an 'offtaste'. The concentration of Hx, one of the intermediates of these reactions increases with prolonged storage and thus can be used as an indicator of fish meat freshness [123]. Simultaneous determination of these compounds has been carried out for a rapid estimation of freshness by using a Xanthine oxidase (XO) immobilized on SPCEs with GA and BSA. Hx has also been determined using RuO2 modified SPCEs with XO immobilized in a Nafion membrane [124].

5.1.10. Antibiotics The widespread administration of antibiotics to dairy cattle for therapeutic purposes raises significant food safety issues since antibiotic resistance can be transferred to man on ingestion of affected meat and milk products. Further, reported problems include increasing instances of allergic reactions to antibiotic residues and the inhibition of starter cultures used in the production of fermented milk products. The β-lactam antibiotics are the most commonly identified antimicrobial group found in milk since a large proportion of mastitis treatments rely on their usage. SPEs incorporating a rhodinised- carbon working electrode were modified by adsorption of penicillin binding protein (PBP) [40]. The simplicity of the approach should ultimately allow usage of the assay on remote sites, such as fields and dairies, whilst the electrochemical transduction method facilitates the generation of quantitative data.

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5.2. Clinical Analysis The potential of screen printing technique in the development of disposable biosensors for clinical analysis is exemplified by the personal glucose biosensor widely used by diabetic patients. However, the use of this technique is getting extensive to other diverse areas of the clinical analysis as the great number of published works demonstrate [125]. In the following sections are described the different designs and applications of screen printed devices used in the development of disposable biosensors for clinical analysis.

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5.2.1. Glucose Accurate and rapid determinations of glucose are indispensable in the diagnosis and management of diabetes [80]. In fact, the control of blood glucose level can avoid or reduce the development of complications related to this disease (blindness, kidney failure, nerve damage) [94]. For this reason, the detection of glucose in human blood is one of the most widespread and important applications of biosensors and it has been the main focus of numerous articles related to amperometric biosensors, including those based in SPEs, in the last three decades [126]. Enzymatic screen printed biosensors for blood glucose analysis are based on the first enzymatic biosensor developed by Clark and Lyons [127] in which the enzyme GOX is the biocomponent of the biosensor. This enzyme catalyses the oxidation of glucose in the presence of oxygen, resulting in the generation of hydrogen peroxide at the electrode surface [80]. The quantification of glucose concentration can be then carried out by the electrochemical oxidation of the H2O2 enzymatic-generated according to the following scheme,

e-

2H+ + O2 H2O2 O2

GOX(FAD)

Glucose

GOX(FADH2)

Gluconolactone

where FAD/FADH2 are the oxidized and reduced prosthetic group, flavin adenine dinucleotide, the active center of GOX [42, 82]. This method presents some problems in the analysis of whole blood and serum samples. In this kind of samples physiological biochemicals such as uric acid, ascorbic acid and acetaminophen are often presented resulting to be interferents in the analysis of glucose because they are oxidized at the same potential range as H2O2 [128]. Different strategies have been used to resolve this problem and among them metalised SPCEs have demonstrated to be very useful. In this way, Kumar et al. [129] have developed a copper-plated carbon SPE in which GOX enzyme was immobilized. The developed biosensor allows performing measurements at low potentials avoiding interferences problems. Following this strategy, the modification of SPCEs with rhodium has been found to be a good alternative in the construction of selective glucose biosensors. The electrodeposition of rhodium over the

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M.A. Alonso-Lomillo, O. Domínguez-Renedo and M.J. Arcos-Martínez

carbon electrode provides an excellent catalyst surface in which GOX can be immobilized [41]. One practical solution to partially reduce the biochemical interferences is to introduce a low-oxidation potential mediator that acts as an electron acceptor. Glucose biosensors normally employ two major groups of mediators, hydrogen peroxide redox mediating reagents and enzymatic glucose oxidation mediating reagents [130]. In the first case the sequence of reactions giving rise to the analytical signal can be seen in the following scheme,

e-

Mred

2H+ + O2

Mox

H2O2

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

O2

GOX(FAD)

Glucose

GOX(FADH2)

Gluconolactone

where Mox and Mred are the oxidized and reduced forms of the mediator. Several substances have been used as mediators in this kind of biosensors being CoPC one of the most utilized. Gilmartin et al. developed an amperometric biosensor based on CoPC-SPCE coated with cellulose and GOX [82]. The same organometallic mediator has been used by Crouch et al. [78, 80] in the construction of disposable glucose biosensors for the analysis of glucose in serum using a water-based carbon ink mixed with the enzyme and the mediator which allows the preparation of the biosensor in one-step fabrication procedure. In all cases the organometallic redox mediator permits the oxidation of H2O2 at markedly reduced overvoltages, which enhances the biosensor selectivity. Another H2O2 redox mediating reagent frequently used in the construction of amperometric screen printed biosensors is PB. In this case, PB is able to catalyze the reduction of the enzymatic-generated H2O2 allowing its electrochemical detection at very low applied potentials. Pravda et al. [92] developed a biosensor based in the immobilization of a mixture of PB and GOX microparticles on the SPCEs surface by cross-linking obtaining good results of reproducibility. The biosensor developed by Ricci et al. [93, 94] is based in the same system and has demonstrated good performance in the continuous monitoring of glucose with high degree of reproducibility jointly with a low cost and the possibility of mass production. The mediator-based biosensors described above still present several problems in the analysis of glucose in real samples. They usually require high operating potentials where unwanted reactions of coexisting electroactives constituents in blood complicate the interpretation of the signal. Moreover, they are also oxygen-depended suffering from low sensitivities [130]. The sensitivity of the measurements can then be improved by replacement of oxygen by synthetic enzymatic glucose oxidation mediating reagents [128, 130]. This kind of biosensors is based in the following reactions scheme,

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Enzyme Modified Screen Printed Electrodes

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

e-

Mred

GOX(FAD)

Glucose

Mox

GOX(FADH2)

Gluconolactone

25

One of the most used redox molecules that can mediate direct oxidation of GOX in the fabrication of disposable glucose biosensors is ferricyanide. In this way, a glucose disposable biosensor has been developed by Cui et al. [87] based in the immobilization of GOX and ferricyanide with a nitrocellulose membrane in SPCEs. The most important contribution of this last work is the new measurement method proposed. It is based on the differential measurements between two different cells: the glucose-sensing cell which incorporates GOX and the reference cell with BSA. In the described system response signals toward different redox molecules from glucose are canceled out. In recent years the incorporation of nanomaterials to the construction of biosensors has presented several important advantages due to their unique properties. Multi-wall carbon nanotubes immobilized with GOX over a SPCE surface have been used in the development of a glucose biosensor using ferricyanide as the redox mediator which shows higher sensitivity and wider linear response range than the typical glucose electrochemical biosensor [42]. Besides of the described mediators, there are other enzymatic glucose oxidation mediating reagents cited in the literature, which provides the fabrication of selective screen printed glucose biosensors among them, ferrocene [131], chromium and manganese complexes [132], osmium complexes [133] and other polymeric mediators [130]. Several other techniques have also been reported to eliminate the cited biochemical interfering substances. For example, Cui et al. [134] have found that glucose biosensors based on the immobilization of GOX on SPCEs on a nitrocellulose strip with a strong oxidation layer of PbO2, are insusceptible to interfering species showing also high degree of reproducibility. However, the same authors propose the incorporation of hexamineruthenium (III) chloride ([Ru(NH3)6]3+) to be used as a mediator in order to improve the performance of the constructed biosensor [126].

5.2.2. Cholesterol The literature is full with methods describing the determination of cholesterol in blood due to the relationship between elevated cholesterol levels and clinical disorders, including arteriosclerosis, lipid metabolism dysfunction, cerebral thrombosis, coronary heart disease and hypertension. Consequently, cholesterol is one of the most frequently determine analytes in clinical analysis [68, 135]. Pioneer works in the development of screen printed cholesterol biosensors are based in the immobilization of the enzyme cholesterol oxidase (ChOX). This enzyme catalyzes the oxidation of cholesterol to ketone in the presence of oxygen producing hydrogen peroxide. Therefore it is possible to carry out the indirect determination of cholesterol through the monitoring of H2O2 oxidation by amperometry according to the following scheme,

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e-

2H+ + O2 H2O2 O2

ChOXox

Cholesterol

CHOXred

Cholest-4-en-3-one

Au SPEs based on the above described system have been developed by means of the covalent immobilization of ChOX on the electrode surface with thiols and carbodiimide giving good results in terms of reproducibility, stability and selectivity for cholesterol analysis [73]. As H2O2 oxidation requires very high detector potentials at usual electrodes it is subjected to overpotential effects reducing the selectivity of the biosensor. Thus, mediator modified biosensors are an interesting alternative. In this way, CoPC has been used as a mediator in the development of screen printed cholesterol biosensors based in the immobilization of ChOX by adsorption on the carbon electrode surface retained with a solvent-cast cellulose acetate membrane [135, 136]. Cholesterol can be present in blood in two different forms: the free form of cholesterol (cholesterol) and the sterified form (cholesterol ester). Therefore, the term total cholesterol refers to the total amount of these two forms [73]. In order to determine the total cholesterol concentration in blood, several disposable biosensors have been developed. They are based on the jointly use of two enzymes: Cholesterol oxidase and cholesterol esterase (ChEs) and the electrochemically determination of H2O2 according to the next system:

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ChEs

Cholesterol ester + H2O Cholesterol + Fatty acid

e-

2H+ + O2 H2O2 O2

ChOXox

Cholesterol

CHOXred

Cholest-4-en-3-one

The use of redox mediators is also possible in order to improve the selectivity of the above described biosensor. In the literature can be found two different works describing the use of ferrocyanide as the electrochemical mediator allowing electron transfer between the reaction product and the electrode [54, 88]. In both cases, the detection of cholesterol is based on the peroxidase oxidation of ferrocyanide by hydrogen peroxide produced as a result of the cholesterol oxidase/esterase enzymatic reactions using SPCEs. In the work described by Li et al. [54] the modification of the working electrode with carbon nanotubes significantly improves the analytical performance of the biosensor. Another proposed enzyme for the analysis of cholesterol is cytochrome P450scc due to its specific interaction with cholesterol. This enzyme catalyses the cholesterol side chain cleavage reaction through a complex mechanism which allows the amperometric determination of cholesterol. Good results in the determination of this analyte have been

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found when cytochrome P450scc was immobilized on rhodium SPCEs by cross-linking with GA and BSA using rivoflavin as electrochemical mediator [68]. Better detection limits are obtained when nanomaterials are used in the construction of cholesterol biosensors. Recent works propose the modification of SPEs with gold nanoparticles [101] and carbon nanotubes [103] in order to improve the sensibility of the biosensors based in the enzyme cytochrome P450scc.

5.2.3. Lactate Lactate is a very important analyte due to its participation in muscle action, following which its concentration in blood rises. The clinical analysis of lactate is obliging for monitoring respiratory insufficiency, shocks, heart failure and metabolic disorder. It is also useful in sports medicine, particularly for detecting damage tissues, thrombosis and physical condition of racing animals and athletes [50]. There are four enzymes that can be used in the development of lactate biosensors: LOX, LDH, lactate monooxidase (LMO) and cytochrome b2 (Cyt b2) [50, 62]. In the case of screen printed biosensors only two of these enzymes have been used for clinical analysis of lactate: LOX and LDH. In the case of LOX the measurement method is based in the amperometric monitoring of the hydrogen peroxide enzymatically generated according to,

Copyright © 2009. Nova Science Publishers, Incorporated. All rights reserved.

e-

2H+ + O2 H2O2

LOXox

L-lactate

O2

LOXred

Pyruvate

This system is the base of the disposable SPCE biosensor developed by Patel et al. [50] in which LOX was immobilized with polyethyleneimine and poly(carbamoyl)sulphonate hydrogel together with CoPC used as an electrochemical mediator which confers selectivity to the biosensor in the analysis of human whole blood and serum. LDH biosensors used in the analysis of lactate are based in the electrochemical oxidation of NADH:

NAD+ e-

L-lactate LDH

NADH

Pyruvate

LDH has been used as the biorecognition element in the biosensor developed by Yoon and Kim [79], which allows the sensitive clinical analysis of lactate using a mixture of activated carbon, LDH, NAD+, polyvinylpyrrolidone (PVP) and silicone oil as the printing ink for the working electrode.

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5.2.4. Uric Acid The analysis of uric acid is of exceptionally interest in the detection of disorders associated with purine metabolism, including gout and hyperuricaemia [59]. Additionally, elevated levels are observed in a wide range of conditions, such as leukaemia, pneumonia, cardiovascular diseases and kidney damage [60, 136, 137]. Moreover, the activity of chemotherapeutic drugs can be valued by monitoring ureate levels, as increased nucleoprotein degradation results in elevated purine excretion [59]. Disposable biosensors for the analysis of uric acid in urine samples are described in the bibliography. They are based on the immobilization of the enzyme uricase in SPCEs using a cellulose acetate membrane. The monitoring of uric acid was carried out following the amperometric oxidation of the hydrogen peroxide enzymatically generated employing CoPC as a mediator according to the following scheme [59, 60, 136],

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e-

Co+

2H+ + O2

Co2+

H2O2

Uricaseox

Uric acid

O2

Uricasered

Allantoin + CO2

5.2.5. Urea The determination of urea in body fluids is one of the most frequent analyses in routine clinical laboratory. In human liver, nitrogen derived from amino acid breakdown is converted to urea via a special catabolic pathway, the urea cycle, and is readily excreted by the kidneys. An increased concentration of urea in blood and a reduced level in urine is therefore a strong indication for a renal dysfunction [26]. Disposable biosensors for urea analysis are based on potentiometric [61] and conductiometric [26] measurements. In both cases the enzyme used is urease which decomposes urea into ammonia and carbon dioxide: Urease Urea + H2O

2NH3 + CO2

NH3 + H2O

NH4+ + OH-

CO2 + H2O

HCO3- + H+

This sequence of reactions demonstrates that the analysis of urea can be carried out using a potentiometric biosensor due to the change in the potential that takes place as a consequence of the change in pH. In this way, Karyakin et al. [61] have constructed a disposable biosensor based on the immobilization of urea on a polyaniline modified SPCE using a Nafion membrane. In the other hand, the apparition in dissolution of NH4+, HCO3-, H+ and OH- ions gives rise to a change in conductivity. This fact is the base for the biosensor developed by Bilitewski and coworkers [26] where urease was immobilized by cross-linking with GA on a thick film platinum electrode. Another conductometric biosensor for the analysis of urea is

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described by Lee and coworkers [83]. In this case, a sol-gel-immobilized urease on a Pt SPE is developed for the determination of urea in serum.

5.2.6. Drugs A few works can be found in the literature describing the analysis of drugs using disposable enzymatic electrodes. The fast and sensitive analysis in blood of the analgesic acetaminophen has been carried out using an aryl acylamidase modified SPCE [76]. In this case, the enzyme was directly incorporated into an aqueous carbon-based paste used for working electrode generation. The quantification of the acetaminophen concentration in blood samples was carried out by means of the amperometric detection of the 4-aminophenol by oxidation at the working electrode: 4-aminophenol e-

Aryl acylamidase Quinoleine +

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Acetaminophen + H2O

2H+

Acetic acid

Other enzymes tested as possible biological elements for disposable biosensors construction are tyrosinase and HRP. Chang et al. [51] have developed a bi-enzymatic SPCE biosensor based on both enzymes for the analysis of phenolic compounds. The biosensor constructed represents a simple approach for the possible analysis of acetaminophen for clinical objectives. Theophylline is a member of the xanthine family of drugs, which is used for controlling the symptoms of chronic asthma. Measuring of serum theophylline levels is necessary in order to establish the therapeutic range of the drug [138]. A disposable electrochemical biosensor has been developed for the analysis of this drug in whole blood using alkaline phosphatase (AP) as the biological element immobilized in SPCEs. AP catalyses the generation of p-aminophenol from p-aminophenyl phosphate. The p-aminophenol generated can be quantified by its electrochemical oxidation at the SPCEs. Theophylline is an uncompetitive inhibitor of this process, therefore it can be analyzed by measuring the decrease in the electrochemical response of p-aminophenol [138].

5.2.7. Creatinine The analysis of creatinine in blood is utilized to evaluate the presence of renal insufficiency [52]. SPCE modified with poly(methyl vinyl ether)/maleic anhydride have been used for the construction of creatinine biosensors based in the immobilization of the enzyme creatinine deiminase [139]. The analysis of creatinine in serum samples was made by Ac Impedance measurements. In a more recent work disposable biosensors have been developed for the analysis of this substance based in a multi-enzyme sequence (creatininase, creatinase and sarcosine oxidase) and the amperometric measurement of the enzymatically-generated hydrogen peroxide [52]. The enzymes were immobilized on a Pt SPE surface using a poly(carbamoyl sulphonate) hydrogel.

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5.2.8. Glutathione Altered levels of glutathione in plasma have been implicated in a number of pathological conditions, including Alzheimer’s, Parkinson’s diseases, diabetes, macular degeneration, and HIV disease [140]. In the literature it is possible to find different disposable biosensors based on SPCEs modified with the enzyme glutathione peroxidase and CoPC as a redox mediator for the analysis of glutathione in human whole blood [3, 141, 142]. 5.2.9. Other Analytes of Interest in Clinical Analysis In addition to the already described analytes in the previous sections, there are other substances which quantification has a special interest from a clinical point of view. Among these analytes, the determination of blood ketones amount provides an interesting way of identification of the presence of glucose metabolism disorders [53, 143]. With this aim, ketone 3-β-hydroxybutyrate (3HB) has been analyzed using disposable biosensors. The enzyme used for the analysis of this kenote is 3-hydroxybityrate dehydrogenase (3HBDH) which catalyses the transformation of 3HB in acetoacetate being NAD+ reduced to NADH. The posterior oxidation of NADH to NAD+ on the electrode surface allows the analysis of the ketone amount,

NAD+ e-

3-β-hydroxybutyrate 3HBDH

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NADH

Acetoacetate

3HBDH has been immobilized in a SPCE using a layer of hydrophilic gel together with ferricyanide used as a mediator for the rapid, sensitive and low cost analysis of 3HB in blood samples of several patients obtaining similar results to routine hospital blood analyzer [53]. In a recent work [143], 3HB is determined using a screen printed iridium-modified biosensor which allows the analysis of the target analyte using a single reaction step without any mediator. Another interesting analysis of particular clinical importance is the determination of glutamate pyruvate activity (GPT). Elevated GPT value in serum indicates myocardial and hepatic diseases [144]. Chang et al. [144] have developed a disposable biosensor for the analysis of GPT in serum. The biosensor is composed of immobilized L-glutamate oxidase (L-GLOX) in a photo-cross-linkable polymer membrane on a palladium-deposited SPCE. The GPT determination is carried out by means of the amperometric response obtained for the hydrogen peroxide generated as a consequence of the enzyme activity,

e-

2H+ + O2 H2O2

L-GLOXox

L-glutamate

O2

L-GLOXred

α-ketoglutarate + NH3

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Finally, the analysis of albumin as a potential tool for measurement of total protein in human serum has been carried out using a disposable biosensor constructed by immobilization of tyrosinase by cross-linking with GA on a SPCE. The analysis of albumin is carried out amperometrically by measuring the reduction current of the previous oxidized protein by the enzyme action [67].

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5.3. Environmental Many human activities release pollutants to the environment. Sometimes, the liberation to the waters or atmosphere is acute and drastic, leading to massive mortalities of different organisms and living things. However, more often, the liberation is by means of low and chronic concentrations which must be periodically tested. The metals and pesticides are without a doubt the pollutants whose determination has become an essential routine. Metals, particularly heavy metals, are a by-product of manufacturing and related processes that inadvertently enter the environment: soil, oil and air. Heavy metals are of great importance, since they are conservative pollutants, which can be accumulated through trophy chains. The determination of pesticides has also become increasingly important in recent years because of the widespread use of these compounds, which is due to their large range of biological activity and a relatively low persistence. The determination of both types of pollutants by means of enzymatic biosensors is based on the inhibition these types of substances provoke in the biocatalytic activity of some enzymes. In enzymology this is known as enzymatic inhibition, a phenomenon in which a compound, called an inhibitor, in most cases similar in structure to the substance (substrate) upon which an enzyme acts to form a product, interacts with the enzyme so that the resulting complex either cannot undergo the usual reaction or cannot form the usual product. The inhibitor may function by combining with the enzyme at the site at which the substrate usually combines (competitive inhibition), or at some other site (noncompetitive inhibition). In the latter, the inhibitor does not prevent binding of the substrate to the enzyme but sufficiently changes the shape of the site at which catalytic activity occurs so as to prevent it. Competitive and non-competitive inhibitions affect the enzyme kinetics differently. In the case of mixed inhibition, the inhibitor binds the enzyme and the enzyme–substrate complex with a different affinity. For irreversible inhibitors, the enzyme–inhibitor interaction results in the formation of a covalent bond between the enzyme active centre and the inhibitor. The term “irreversible” means that the decomposition of the enzyme–inhibitor complex results in the destruction of the enzyme, e.g. its hydrolysis, oxidation, etc. In recent years, electrochemical determination based on the use of disposable electrodes where enzymes have been immobilised, has had a notable development. There are many enzymes that are susceptible to being inhibited by different compounds, whilst they are active. On the other hand, the way of immobilising these enzymes in the electrodes attached to different types of inks used in the manufacture of the electrodes, as well as the modification of these enzymes, in the reason why, in the bibliography, a large number of different applications are described.

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5.3.1. Metals It is a well-known fact that some metallic ions, especially heavy metals, can inhibit the activity of various enzymes. Disposable biosensors based on the principle of inhibition have to date been applied for a wide range of heavy metals. Some reviews collect these applications [145, 146]. The most widely employed enzyme in the inhibitive detection of heavy metals ions using SPEs is urease. The urease enzyme catalyzes the hydrolysis of urea and the reaction produces ammonium:

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Urea + H2O + urease → CO2 + 2NH3 The development of an amperometric sensor for the monitoring of urease activity was feasible by coupling the urea breakdown reaction, catalysed by urease, to the reductive ammination of ketoglutarate catalysed by glutamic dehydrogenase (GLDH). The ammonia provided by the urea conversion is required for the change of ketoglutarate to glutamate with the concomitant oxidation of the NADH cofactor. NADH oxidation is monitored amperometrically after urease immobilization, onto the screen printed three-electrode configuration. Rodriguez et al. [147, 148] use a screen printed three-electrode amperometric biosensor based on urease and NADH–glutamic dehydrogenase system in the screening of Hg(II), Cu(II) and Cd(II) in environmental samples. Analysis of water and soil samples with the developed nafion-based sensor produced inhibition on urease activity, according to their metal contents. Application of the assay system to leachate samples gave reliable and accurate toxicity assessments when compared to atomic absorption spectrometry (AAS) and inductively coupled plasma atomic emission spectroscopy (ICP-MS) analysis. This approach proves to be a simple and rapid (15 min, including enzyme inhibition time) method for metal ions detection. As a consequence of the ammonium liberation in the urease reaction, a variation in the pH value takes place. This change might cause a decrease in the potential of an internal pHsubsensor. Thus, for example, the presence of ruthenium dioxide in the biosensing film in the SPEs causes pH-dependent potentiometric sensitivity. Ogonczyk et al. [149, 150] use this fact to determinate silver and copper ions at sub-ppm levels. The biosensing thick-film is prepared using biocomposite screen printable material composed of ruthenium dioxide, urease, graphite and organic polymer. Electric circuit and electrode insulation are also fabricated in the same thick-film technology using inexpensive commercially available pastes. In the case of other strong inhibitors (copper and mercury ions), similar results were obtained. Other tested heavy metals (Ni, Co, Fe, Cd and Pb ions) were detected at millimolar levels. It is worth noticing, that the described procedure was free from pseudointerferences caused by pH and buffer capacity of samples, because inhibition and detection steps were separated. The biotests seem to be useful for toxic metal ions detection in the course of in-field environmental screening. Inhibition of oxidase enzymes activity, immobilised SPEs are also used to determinate pollutant metals [151]. The activity of the oxidase enzymes has been measured following H2O2 production in the presence of the substrate with a Ru/graphite working electrode polarized at +700 mV vs. Ag/AgCl. Inhibition of the activity of enzymes such as AOX, sarcosine oxidase and glycerol-3-P oxidase by the metal ions Hg(II), V(V), Cu(II), Ni(II) resulted in the construction of calibration curves for the metals in the low ppm range. Total

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analysis time was 10 min (5 min incubation + 5 min measuring) with a residual standard deviation of 10-17 %. It is showed that heavy metals also inhibit ChEs enzymes. AchE and BChE are inhibited by Cu, Fe, and Cd ions by using acetylthiocholine chloride as substrate. The linear range of detection for the tested heavy metals with the AchE or BchE modified electrodes were comprised beteween 0 and 10 mM [152].

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5.3.2. Pesticides A pesticide may be a chemical substance, biological agent (such as a virus or bacteria), antimicrobial, disinfectant or device used against any pest. Pests include insects, plant pathogens, weeds, molluscs, birds, mammals, fish, nematodes (roundworms) and microbes that compete with humans for food, destroy property, spread, are a vector for disease or cause a nuisance. Although there are benefits to the use of pesticides, there are also drawbacks, such as potential toxicity to humans and other animals. Pesticides are often referred to according to the type of pest they control. Another way to think about pesticides is to consider those that are chemical pesticides or are derived from a common source or production method. Other categories include biopesticides, antimicrobials, and pest control devices. Most frequently pesticides are classified as: Organophosphate pesticides: These pesticides affect the nervous system by disrupting the enzyme that regulates acetylcholine, a neurotransmitter. Most organophosphates are insecticides. They were developed during the early 19th century, but their effects on insects, which are similar to their effects on humans, were discovered in 1932. Some are very poisonous (they were used in World War II as nerve agents). However, they usually are not persistent in the environment. Carbamate pesticides affect the nervous system by disrupting an enzyme that regulates acetylcholine, a neurotransmitter. The enzyme effects are usually reversible. There are several subgroups within the carbamates. Organochlorine insecticides, such as Dichlorodiphenyltrichloroethane (DDT) and chlordane, were commonly used in the past, but many have been removed from the market due to their health and environmental effects and their persistence. Pyrethroid pesticides were developed as a synthetic version of the naturally occurring pesticide pyrethrin, which is found in chrysanthemums. They have been modified to increase their stability in the environment. Some synthetic pyrethroids are toxic to the nervous system. Organophosphorous and carbamate pesticides are widely used in agriculture due to their high activity, low bioaccumulation and moderately rapid degradation in the environment. Nevertheless, some of the applied pesticides may remain in the soil and in agrofood products for some time [153]. There is, therefore, a need for fast and inexpensive testing devices for pesticide detection. Electrochemical biosensors have been successfully used in the determination of pesticides in different samples and in the recent decade numerous biosensing elements have been integrated, giving rise to enzymatic biosensors and immunosensors. Enzymatic determination of pesticides is most often based on inhibition of the activity of selected

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enzymes such as ChEs, organophosphate hydrolase, alkaline and acid phosphatase, ascorbate oxidase, acetolactate synthase, tyrosinase and aldehyde dehydrogenase (ADH). Enzymatic biosensors were developed using various electrochemical signal transducers, different methods of enzyme immobilization and various measuring methodologies. Application of single-use screen printed biosensors in batch measurements and flow-injection analysis with enzyme biosensors are most intensively developed procedures. An improvement of detectability level can be achieved by the use of recombinant enzyme mutants, while multicomponent determinations by the use of biosensor matrices and data processing with artificial neural networks. In some cases the determined pesticide can be also a substrate of enzymatic reaction [154]. Some of the most interesting applications for the pesticide determination are described below. They have been classified according to the enzyme used.

5.3.2.1. Acetylcholinesterase Because most pesticides exert an admitted effect in the function of the neurotransmitter acetylcholine, over the last decades, ChE biosensors have emerged as a sensitive and rapid technique for toxicity analysis in environmental monitoring, as well as food, and quality control. These systems have the potential to complement or replace the classical analytical methods by simplifying or eliminating sample preparation protocols and making field testing easier and faster with significant decrease in costs per analysis. These biosensors are based on measurement of the current intensity produced through electrochemical oxidation of thiocholine, which is obtained as a by-product of the hydrolysis of acetylcholine catalysed by AchE, in the following reaction:

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e-

Dithio-bis-choline Thiocholine

AChE

Acetylcholine + H2O RCOOH

The presence of pesticides in the sample for analysis inhibits the activity of the enzyme that leads to a drop in the current intensity, which is then measured. Amperometric determination of pesticides based on inhibition of AChEcan be also accomplished using bienzymatic systems involving AChE and ChO. In this case, the choline, obtained by hydrolysis of acetylcholine in the presence of the AChE enzyme, is oxidised by ChO to produce betaine and hydrogen peroxide as it is described by the following reaction: Choline + H2O + ChO  Betaine + H2O2 Hydrogen peroxide obtained in this way can easily be oxidised using electrochemical techniques and the current produced by the oxidisation process can be measured amperometrically.

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Enzyme Modified Screen Printed Electrodes H2O2 + Betaine

H2O2 e-

ChO

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O2 + H2O

35

Choline + H2O

The presence of an agent inhibiting the enzymatic activity of AChE leads to a decrease in choline concentrations that leads to a decrease in the current intensity that is measured. These bienzymatic electrodes provide better detection limits than those only using immobilised AChE, but have the disadvantage of requiring a more drawn out analysis time. The bibliography lists many studies based on this inhibitive action for the determination of pesticides using screen printing technology and since 1997, many papers that describe these types of electrodes have been published with the aim of developing low cost, disposable electrodes [155-160]. Domínguez et al. [146] have carried out an exhaustive review of this. Nowadays, a wide variety of modifications of these methods are still being used in order to increase the possibilities of pesticide determination. Laschi et al. [161] had been developed an AChE based biosensor by using CoPC modified SPCEs as transducers to chronoaperometric determination of carbofuran. A detection limit of 4.9 x 10-10 M for carbofuran was found with an analysis time of 15 min. The study of the immobilisation layer composition, enzyme units and cross-linker amounts, was emphasised. On the other hand, the use of nanoparticles can play an interesting role for the preparation of enzymatic biosensors. They can provide a stable surface for enzyme immobilization, and allow the electrochemical sensing to be performed without the need for external electrontransfer mediators. The conductivity properties of such materials would enable the design of simple, sensitive and stable electroanalytical procedures based on enzyme immobilization and this is an important challenger to develop in the future. A simple method to immobilize AChE on silica sol-gel (SiSG) film assembling AuNPs was proposed by Du et al. This method has been applied in the determination of monocrotophos, a non-specific systemic insecticide and acaricide, used to control common mites, ticks and spiders with contact and stomach action, which quickly penetrates plant tissue. The inhibition of monocrotophos was proportional to its concentration ranging from 0.001 to 1 µg/ml and 2 to 15 µg/ml, with the correlation coefficients of 0.9930 and 0.9985, respectively. The detection limit was 0.6 ng/ml at a 10% inhibition. The developed biosensor exhibited good reproducibility and acceptable stability, thus providing a new promising tool for analysis of enzyme inhibitors [162]. The determination of Methamidophos, a highly active, systemic, residual organophosphate insecticide/acaricide/avicide with contact and stomach action was carried out by using an amperometric biosensor based on enzyme AChE. Biosensor consists of four Pt SPEs on a ceramic strip. In this case, AChE was physically adsorbed onto the electrode surface. The limit of detection was 2.45 nM [163]. Another organophosphorous and carbamate pesticides as monocrotophos, malathion, metasystox and lannate over the concentration range 0-10 ppb were determinate electrochemicaly by using SPEs containing immobilized AChE enzyme. AChE was attached to the SPE by in situ bulk polymerization of acrylamide to ensure efficient adherence within the membrane with minimal losses in enzyme activity [164]. The determination of pesticides by enzymatic inhibition of acetylcholine has been also carried out in flow systems. Among the several fabrication techniques used to construct

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microflow systems, the low-temperature cofired ceramics (LTCC) technology, taking advantage of its multilayer approach, is one of the most versatile ones. Moreover, due to its perfect compatibility with screen printing techniques, it also permits the integration of electronic components used to control the whole system setup. The miniaturized system was characterized and successfully evaluated by determining carbofuran at the nanomolar level [165]. Because of the excellent electrocatalytic activity of the carbon nanotubes, these nanomaterials have been used in order to develop the carbon nanotubes-modified SPCE. These electrodes have been used to detect organophosphate pesticides and chemical nerve agents in biological samples such as rat saliva samples. Certain saliva enzymes may be useful biomarkers for detecting exposures to enviromental pollutants. In this regard, saliva biomonitoring offers a simple and noninvasive approach for rapidly evaluating pesticides exposures in real time. An electrochemical sensor coupled with a microflow injection system was developed for a simple, rapid, and sensitive characterization of ChE enzyme activities in rat saliva. The AChE enzyme activity was further investigated using the CNT-based electrochemical sensor with commercially available purified AChE and ChE in saliva obtained from nave rats. It is found that the calibration curve is linear over a wide range of AChE concentrations from 5 pM to 0.5 nM, and the sensor is very sensitive with the detection limit down to 2 pM [166]. The development of a portable biosensor for screening neurotoxic agents in water samples has been recently described [167]. The system consists of (i) a SPE with AChE immobilized on it, (ii) a self-developed portable potentiostat with an analog to digital (A/D) converter and a serial interface for transferring data to a portable PC and (iii) an own designed software, developed with Lab-Windows CVI, used to record and process the measurements. The design is specially suited for screening purposes, does not need sample preconcentration, is totally autonomous and suitable for the field detection of neurotoxic agents in water. The system has been developed to perform high precision amperometrical measurements with low drifts, low noise and a good reproducibility.

5.3.2.2. Butyrylcholinesterase As with AChE, BChE have also been used in the construction of bionsensors. Potentiometric as well as amperometric bionsensors have been developed. In the first case, a pH sensitive electrode detection system has been used. These bionsensors are based on the following reaction catalysed by the BChE enzyme: Butyrylcholine + BChE  Choline + butyric acid As in the case of AChE, bienzymatic biosensors have also been developed for BChE by immobilising both BChE as well as ChO. Trichlorfon and Coumaphos are organophosphate insecticides used to control cockroaches, crickets, silverfish, bedbugs, fleas, cattle grubs, screw-worms, lice, scabies, flies, ticks, leafminers and leaf-hoppers. Both insecticides have been determined by using a biosensor based on BChE from horse serum, immobilised onto the nafion layer by crosslinking with GA vapours on a SPE. The detection limits were found to be 3.5 x 10-7 M for trichlorfon and 1.5 x 10-7 M for coumaphos [168].

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BChE biosensors have shown a higher affinity towards paraoxon (50% inhibition with 4 ppb) and chlorpyrifos-methyl oxon (50% inhibition with I ppb). A comparative study using mediators such as CoPC and PB modified SPEs, has been performed by Arduini et al. [96]. Sarin, is an extremely toxic substance whose sole application is as a nerve agent. Sarin, discovered by two German scientists attempting to create stronger pesticides, is a fluorinated phosphonate and is similar in structure and has a similar mechanism of action as some commonly used insecticides, such as malathion. It is similar in biological activity to carbamates used as insecticides. An inexpensive and portable system to be used by first responders and military personnel is of interest owing to the continuing threat of possible terrorist attacks. Amperometric biosensors based on BChE inhibition show such potentialities. The enzyme was immobilized onto SPEs modified with PB and the nerve agent detection was performed by measuring the residual activity of enzyme. The optimized biosensor was tested with sarin and VX standard solutions, showing detection limits of 12 and 14 ppb (10% of inhibition), respectively. The enzymatic inhibition was also obtained by exposing the biosensors to sarin in gas phase [169].

5.3.2.3. Aldehyde Dehydrogenase Dithiocarbamate fungicides inhibit ADH irreversibly. Measurement of these pesticides was accomplished by inhibition of this enzyme while monitoring the reduction of the cofactor, according to the following reaction: Propionaldehyde

NAD+ e-

ADH

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NADH

Propionic acid

By measuring the reduction in current intensity produced due to the oxidization of the NADH that is formed, it was possible to measure the concentration of pesticide present in the sample. However, oxidization potentials for NADH are very high, and many interferents were present. Maneb is an ethylene(bis)dithiocarbamate fungicide used in the control of early and late blights on field crops and ornamentals. Zineb are dithiocarbamate fungicides used to prevent crop damage in the field and to protect harvested crops from deterioration during storage or transport. Maneb and zineb were determinate by using sensors based on SPEs modified with the MB-Reinecke’s salt combination. Two enzyme immobilization procedures were tested: entrapment in PVA-SbQ and cross-linking with GA. Chronoamperometry was employed to observe the biosensor responses during enzymatic hydrolysis of propionaldehyde. Detection limits, calculated using 10% inhibition were 31.5 ppb and 35 ppb for maneb and zineb, respectively [170, 171]. Nabam (disodium ethylene-bis-dithiocarbamate) is an agricultural fungicide formerly used in Australia for the control of a wide variety of fungal diseases. A disposable bienzymic sensor for the reliable determination of nabam was developed by Noguer et al. ADH and a highly stable NADH oxidase were immobilized on the surface of a disposable Pt-sputtered SPCE by using photocross-linkable PVA-SbQ. As low as 8 ppb of pesticide has been detected

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in these first experiments, whereas the detection limit of the recommended spectrophotometric method is about 400 ppm [172]. Metam sodium is a broad spectrum soil fumigant that can be used to control plant parasitic nematodes, weeds, germinating weed seeds, and soil-borne plant pathogenic fungi affecting a variety of economically important fruit and vegetable crops. Once in the soil, this pesticide degrades rapidly to methylisothiocyanate (MITC), the product's primary bioactive agent. Biosensors developed for the determination of metam sodium and its main metabolite MITC has been developed based on ADH inhibition. ADH was immobilized on the surface of a disposable SPCE by using a photocross-linkable PVA-SbQ. It was shown that metamsodium did not inhibit ADH while MITC could be detected at levels of 100 ppb. The sensor thus allowed to discriminate between metam-sodium and its toxic metabolite [173].

5.3.2.4. Acid Phosphatase Enzymatic bionsensors constructed using acid phospatase (AP) as their biological component are based on hydrolytic inhibition of glucose-6-phosphate catalysed by AP as it is described in the following reaction: Glucose-6-phosphate + H2O + AP  Glucose + HPO42In order to detect the inhibition process, the presence of a second enzyme, for example GOX, is required. In the presence of oxygen, this enzyme catalyses oxidization of the glucose formed in the preceding reaction into gluconic acid:

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Glucose + O2 + GOX  Gluconic acid + H2O2 The H2O2 obtained is reoxidised on the electrode producing a current intensity that gives amperometric readings and which decreases in the presence of pesticides.

e-

Glucose-6-phosphate + H2O

O2 AP

H2O2

GOXox

Glucose

O2

GOXred

Gluconic acid

HPO42-

This system has been employed by Mazzei et al. [174] for the determination of malathion, paration and paraoxon. Amperometric monitoring can be carried out by using a Clark type dissolved O2 electrode [175]. In this case, the decrease in the quantity of oxygen on the electrode surface is quantified. The formation of gluconic acid permits measurement of any possible inhibitive action due to the presence of pesticides through potentiometric monitoring using a pH electrode. 2,4-Dichlorophenoxyacetic acid (2,4-D) is a common systemic herbicide used in the control of broadleaf weeds. It is the third-most widely used herbicide in North America and the most widely used herbicide in the world. Shyuan et al. [176] describe an electrochemical biosensor for the detection of 2,4-D. The biosensing film deposited on the SPE was a sol-

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gel/chitosan film containing immobilized alkaline phophatase enzyme (ALP). The 2,4-D herbicide was determined by the quantitative inhibition on ALP. The linear response range towards the substrate AA2P was 10-80 µM. For 2,4-D determination, the maximum inhibition of the ALP was achieved after 80 ppb (60% inhibition) 2,4-D with a linear response range of 2-80 ppb. Malathion is an organophosphate parasympathomimetic pesticide that is widely used in agriculture, residential landscaping, public recreation areas, and in public health pest control programs such as mosquito eradication. Malathion is an insecticide of relatively low human toxicity. An AP based biosensor developed by Podesta et al. shows a high sensitivity, with a lower detection limit of about 5 ppb in the malathion determination. The catalytic activity of AP was detected by means of a SPE, in presence of the substrate ascorbic acid 2-phosphate (A2P). The current change due to the electrochemical oxidation of the ascorbic acid as reaction product was monitored [177].

5.3.2.5. Multienzymatic Biosensor for Pesticides Determination It is necessary to have continuous monitoring of pesticides in waste water in order to develop discharge-monitoring systems that in a few seconds or minutes can indicate the quality of water. The sensors described above are based on univariate recording of data. However, because frequently more and more attention is being directed to the use of multivariate enzymatic biosensors, the use of enzymes from different classes in one array can provide more information about the heterogeneous composition of samples. This can be carried out using sensor arrays that record multivariate dynamic responses over time with many sensors simultaneously. Multienzyme biosensor arrays based on four or eight electrodes with different immobilised enzymes, such as peroxidase, tyrosinase, AChE, BChE, cellobiose dehydrogenase, and soybean peroxidase groups (oxidoreductases and hidrolases) were explored for wastewater quality determination [152, 178-180]. Common for the data analysis is the multivariate pattern using various linear or non linear modelling like principal component analysis (PCA), partial least square regression (PLS), artificial neural networks (ANN), etc. Most of the problems present in this kind of device came from the requirements of different co-substrates for functioning the enzymes used. However no limitations for combining several enzymes seem to exist. The detection limits for pesticides including carbaryl, heptenophos, fenitrothion, dichlovorvos and phosphamide were in the nanomolar range [180].

CONCLUSION In recent years, a great development in screen printed enzymatic biosensors for numerous analytical applications has been observed, taking into account the enormous amount of references reported. The high specificity of biological recognition processes in enzymatic reactions has permitted the development of innumerable devices, including electrochemical ones, which have become extremely useful in the determination of numerous substances. The possibilities of the enzymatic electrochemical biosensors can be increased by means of replacing the classical electrodes by disposable SPEs. SPEs present important advantages

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such us a great flexibility and they appear to be particularly attractive for in situ determinations. This review provides information about the application of screen printing technology in fields of especial interest such as environmental science, food industry, pharmaceutical and clinical assays. The great versatility and low cost of this technology is responsible for its continuous development. Their more important improvements are mainly expected from two different ways. First, the incorporation of new printed materials, as well as new support surfaces, will lead to enhance the reproducibility and sensitivity of the screen printed based sensors. With this aim, new attempts for the modification of the working electrode are also in continuous growth, focusing on new ligands, polymers and nanostructure materials.

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[151] D. Compagnone, M. Bugli, P. Imperiali, G. Varallo, and G. Palleschi, Determination of heavy metals using electrochemical biosensors based on enzyme inhibition. Biosensors for Direct Monitoring of Environmental Pollutants in Field, 1997 38, 220. [152] R. Solna, S. Sapelnikova, P. Skladal, M. Winther-Nielsen, C. Carlsson, J. Emneus, and T. Ruzgas, Multienzyme electrochemical array sensor for determination of phenols and pesticides. Talanta, 2005 65, 349. [153] F. Flores, J. Artigas, J.L. Marty, and F. Valdes, Development of an EnFET for the detection of organophosphorous and carbamate insecticides. Analytical and Bioanalytical Chemistry, 2003 376, 476. [154] M. Trojanowicz, Determination of pesticides using electrochemical enzymatic biosensors. Electroanalysis, 2002 14, 1311. [155] J.J. Rippeth, T.D. Gibson, J.P. Hart, I.C. Hartley, and G. Nelson, Flow-injection detector incorporating a screen-printed disposable amperometric biosensor for monitoring organophosphate pesticides. Analyst, 1997 122, 1425. [156] G.S. Nunes, P. Skladal, H. Yamanaka, and D. Barcelo, Determination of carbamate residues in crop samples by cholinesterase-based biosensors and chromatographic techniques. Analytica Chimica Acta, 1998 362, 59. [157] A.L. Hart and W.A. Collier, Stability and function of screen printed electrodes, based on cholinesterase, stabilised by a co-polymer/sugar alcohol mixture. Sensors and Actuators B-Chemical, 1998 53, 111. [158] Y.G. Li, Y.X. Zhou, J.L. Feng, Z.H. Jiang, and L.R. Ma, Immobilization of enzyme on screen-printed electrode by exposure to glutaraldehyde vapour for the construction of amperometric acetylcholinesterase electrodes. Analytica Chimica Acta, 1999 382, 277. [159] S. Andreescu, L. Barthelmebs, and J.L. Marty, Immobilization of acetylcholinesterase on screen-printed electrodes: comparative study between three immobilization methods and applications to the detection of organophosphorus insecticides. Analytica Chimica Acta, 2002 464, 171. [160] S. Andreescu, T. Noguer, V. Magearu, and J.L. Marty, Screen-printed electrode based on AChE for the detection of pesticides in presence of organic solvents. Talanta, 2002 57, 169. [161] S. Laschi, D. Ogonczyk, I. Palchetti, and M. Mascini, Evaluation of pesticide-induced acetylcholinesterase inhibition by means of disposable carbon-modified electrochemical biosensors. Enzyme and Microbial Technology, 2007 40, 485. [162] D. Du, S. Chen, J. Cai, and A. Zhang, Immobilization of acetylcholinesterase on gold nanoparticles embedded in sol-gel film for amperometric detection of organophosphorous insecticide. Biosensors & Bioelectronics, 2007 23, 130. [163] M. Pohanka, K. Kuca, and D. Jun, Amperometric biosensor for pesticide methamidophos assay. Acta Medica (Hradec Kralove), 2007 50, 239. [164] K. Dutta, D. Bhattacharyay, A. Mukherjee, S.J. Setford, A.P.F. Turner, and P. Sarkar, Detection of pesticide by polymeric enzyme electrodes. Ecotoxicology and Environmental Safety, 2008 69, 556. [165] X. Llopis, N. Ibanez-Garcia, S. Alegret, and J. Alonso, Pesticide determination by enzymatic inhibition and amperometric detection in a low-temperature cofired ceramics microsystem. Analytical Chemistry, 2007 79, 3662. [166] J. Wang, C. Timchalk, and Y.H. Lin, Carbon nanotube-based electrochemical sensor for assay of salivary cholinesterase enzyme activity: An exposure biomarker of

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organophosphate pesticides and nerve agents. Environmental Science & Technology, 2008 42, 2688. [167] A. Hildebrandt, J. Ribas, R. Bragos, J.L. Marty, M. Tresanchez, and S. Lacorte, Development of a portable biosensor for screening neurotoxic agents in water samples. Talanta, 2008 75, 1208. [168] E.V. Gogol, G.A. Evtugyn, J.L. Marty, H.C. Budnikov, and V.G. Winter, Amperometric biosensors based on nafion coated screen-printed electrodes for the determination of cholinesterase inhibitors. Talanta, 2000 53, 379. [169] F. Arduini, A. Amine, D. Moscone, F. Ricci, and G. Palleschi, Fast, sensitive and costeffective detection of nerve agents in the gas phase using a portable instrument and an electrochemical biosensor. Analytical and Bioanalytical Chemistry, 2007 388, 1049. [170] T. Noguer, B. Leca, G. Jeanty, and J.L. Marty, Biosensors based on enzyme inhibition: Detection of organophosphorus and carbamate insecticides and dithiocarbamate fungicides. Field Analytical Chemistry and Technology, 1999 3, 171. [171] R.S. Lima, G.S. Nunes, T. Noguer, and J.L. Marty, Enzymatic biosensor for the detection of dithiocarbamate fungicides. Kinetic study of aldehyde dehydrogenase enzyme and biosensor optimization. Quimica Nova, 2007 30, 9. [172] T. Noguer, A. Gradinaru, A. Ciucu, and J.L. Marty, A new disposable biosensor for the accurate and sensitive detection of ethylenebis(dithiocarbamate) fungicides. Analytical Letters, 1999 32, 1723. [173] T. Noguer, A.M. Balasoiu, A. Avramescu, and J.L. Marty, Development of a disposable biosensor for the detection of metam-sodium and its metabolite MITC. Analytical Letters, 2001 34, 513. [174] F. Mazzei, F. Botre, and C. Botre, Acid phosphatase/glucose oxidase-based biosensors for the determination of pesticides. Analytica Chimica Acta, 1996 336, 67. [175] M.D. Gouda, M.S. Thakur, and N.G. Karanth, A dual enzyme amperometric biosensor for monitoring organophosphorous pesticides. Biotechnology Techniques, 1997 11, 653. [176] L.K. Shyuan, L.Y. Heng, M. Ahmad, S.A. Aziz, and Z. Ishak, Screen-printed biosensor with alkaline phosphatase immobilized in sol-gel/chitosan film for the detection of 2,4dichlorophenoxyacetic acid. Sensor Letters, 2006 4, 17. [177] E. Podesta, C. Botre, R. Pilloton, F. Botre, and F. Mazzei, A screen-printed enzymatic electrode for the determination of organo-phosphorous pesticides. Sensors and Microsystems, Proceedings, 2004, 76. [178] R.W. Keay and C.J. McNeil, Separation-free electrochemical immunosensor for rapid determination of atrazine. Biosensors & Bioelectronics, 1998 13, 963. [179] E. Tonning, S. Sapelnikova, J. Christensen, C. Carlsson, M. Winther-Nielsen, E. Dock, R. Solna, P. Skladal, L. Norgaard, T. Ruzgas, and J. Emneus, Chemometric exploration of an amperometric biosensor array for fast determination of wastewater quality. Biosensors & Bioelectronics, 2005 21, 608. [180] R. Solna, E. Dock, A. Christenson, M. Winther-Nielsen, C. Carlsson, J. Emneus, T. Ruzgas, and P. Skladal, Amperometric screen-printed biosensor arrays with coimmobilised oxidoreductases and cholinesterases. Analytica Chimica Acta, 2005 528, 9.

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Chapter 2

BIOSENSORS IN FOOD SAFETY CONTROL: AN UPDATE Spiridon Kintzios Laboratory of Plant Physiology, Faculty of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece The EMBIO Diagnostics Project, Nicosia, Cyprus

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ABSTRACT The conventional analysis of pesticide residues and/or pathogens in agricultural commodities is a labor-intensive procedure, since it is necessary to cover a wide range of different contaminants, using a single procedure. Standard analysis methods include extensive sample pretreatment (with solvent extraction and partitioning phases) and determination by gas chromatography (GC), high-pressure liquid chromatography (HPLC) and mass spectrometry (MS) to achieve the necessary selectivity and sensitivity for the different classes of compounds under detection. Pathogen identification usually requires culture-based methods or immunological assays, such as the enzyme-linked immunosorbent assay (ELISA). As a consequence, current methods of analysis provide a limited sample analysis capacity, on a day/instrument basis. In a region-specific pattern, this results to a general lack of resources for implementation and enforcement of environmental and consumer safety regulations.Therefore, novel, rapid testing are needed. The present report is a thorough review of current biosensor-based methods and instrumentation of food quality control. Presented methods are associated with diversified techniques, such as impedance/conductivity measurements, cellular biosensors, microcalorimetry, flow cytometry, nuclease/lysate tests, adenosine triphosphate (ATP) measurement, luminescence/fluorescence, molecular biological tests and immunoassays. Particular emphasis is given on commercially available methods and products, as well as their market evaluation so far.

Keywords: Biosensor, Cell-based sensor, Enzyme-based sensor, Immunosensor, Food-borne pathogens, Pesticides, Commercial instrumentation.

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1. INTRODUCTION: FOOD SAFETY ON THE FRONT PAGE In recent years, a series of negative incidents concerning food safety has heightened consumer’s awareness of food commodities and their production. Apart from increasing concerns about food safety, often fuelled by mass media reports, changes in legislation created the opportunity for quality control laboratories and manufacturers of equipment for analytical assays to provide assurance along the entire food supply chain from the farm to retailer's shelf. Food safety control is a major economic activity with a volume of $2 billion and a growth rate of 12 % (BCC Research 2002). According to sector surveys, food companies dedicate up to 2% of their revenue to food quality control, although the market is segmented and, in part, immature (Alocijla and Radke 2003). The detection and identification of food-borne pathogens and/or pesticide residues continues to rely on conventional culturing techniques. These are very elaborate, time-consuming and expensive. The already existing food quality control laboratories use conventional techniques, providing a limited range of analyses. For that reason they are unable to cover the needs of the domestic market, thus the offer to demand ratio is extremely low. Consequently, there is a major requirement for simple, rapid, specific, sensitive and economically feasible methods. The term sensor has been defined as a device or system - including control and processing electronics, software and interconnection networks - that responds to a physical or chemical quantity to produce an output that is a measure of that quantity. A biosensor comprises two distinct elements: a biological recognition element (e.g. antibodies, enzymes or whole cells) and, in close contact, a signal transduction element (e.g. optical amperometric, acoustic and electrochemical) connected to a data acquisition and processing system (Eggins 1996, Patel 2002). There is a clear trend for the development of biosensors for food safety assessment. For example, eight percent of US patent biosensor applications refer to assays and techniques focusing on the detection of chemical contaminants (3,5%), pathogen (3,1%) or water pollutants (0,2%) (figure 1). The majority (55%) of these applications describe electrochemical/enzyme-based sensors, followed by a minor participation of immunosensors, cell-based sensors and other techniques (figure 2). From yet another point of view, a recent survey (September 2008) of 5,762 publications on biosensor technologies revealed that 211 of them were describing novel food safety assays, in particular the detection of pesticides (179), toxins (109) or food-borne pathogens (13) (figure 3). The purpose of the present mini-review is to briefly present the current status of biosensor applications in two major fields of food safety, namely the detection of pesticide residues and food-born pathogens, as well as to discuss future perspectives and opportunities.

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Figure 1. Percentage distribution by area of application of US biosensor patents (source: www.patentsonline.com).

Figure 2. Percentage distribution by technology of US biosensor patents (source: www.patentsonline.com)

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Figure 3. Distribution of biosensor-related publications by area of application in food safety.

2. CLASSIFICATION OF BIOSENSOR TECHNOLOGIES FOR FOOD SAFETY

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2.1. General Traditionally, biorecognition elements are classified as enzyme-based, immunoreactive and cell/organism-based (figure 4). A further classification of biorecognition techniques within each group is schematically demonstrated in figures 5A-D. Furthermore, biosensory techniques currently used in food safety are listed in table 1. The main advantages and disadvantages of each technology are presented in table 2. In spite of an extremely large number of possible combinations between different types of biorecognition elements and transducers, a direct communication of the biologic entity and the transducer is limited only to a few examples (e.g. peroxidases or pyroloquinoline quinone dehydrogenases combined with amperometric transducers (Gorton et al. 1999).

Figure 4. Traditional classification of biosensors by biorecognition element. Biosensors: Properties, Materials and Applications : Properties, Materials and Applications, Nova Science Publishers, Incorporated, 2009. ProQuest

Biosensors in Food Safety Control: An Update

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a

b

Figure 4. (Continued).

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c

d Figure 5. Secondary classification of biosensors to subgroups: (A) Enzyme-based (B) Enzyme-base/electrochemical (C) Immunosensor (D) Cell-based sensors.

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Table 1. Biosensor techniques currently used in food safety assays Electrochemical

Enzyme-based Immunological Optical

Amperometry

Valdés-Ramírez et al. (2008) Caetano and Machado (2008)

Conductometry Electrochemical

Dzyadevych and Chovelon (2002) Andreescu and Marty (2006) Hock et al. (2002) Zacco et al. (2007) Danielsson et al. (2001)

Luminescence Surface Plasmon Resonance Bacterial cells

Cell-based

Other techniques

Microalgal cells Cardiac cells Neuronal cells Mass spectrometry-Polymerase chain reaction Quartz crystal microbalance

Muhammad-Tahir and Alocilja (2004) Chouteau et al. (2004) Natarajan et al. (2006) Ecker et al. (2006) Kim et al. (2007)

Table 2. Main advantages and disadvantages of different biosensor technologies applied in food safety testing Biosensor Technology Enzyme-based amperometric

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Immunosensors

Cell-based

Surface plasmon resonance

Micromechanical cantilever-based

Advantages Small size Short response time Easy to use Reasonable cost Low cost Short-to-medium response time Easy to use

High sensitivity Multi-catalytic systems Bioactivity-related information No labelling of the reagents required Real-time assays Reusable sensor surface Small-to-miniaturized equipment High sensitivity Miniaturized equipment

Disadvantages Low stability Electrochemical interferences with sample components Low sensitivity Low selectivity if polyclonal antibodies are used Interference due to matrix effects Not able to differentiate live/dead cells Low selectivity Low storability Not suitable for low molecular weight analytes

Interferences in liquid samples

2.2. Electrochemistry/Amperometry The most widely used biosensors are the enzyme-based amperometric electrodes, often requiring the presence of an intermediary compound, a so-called mediator, which shuttles redox equivalents between the recognition element and transducer. These biosensors, depending on the mediator’s nature and the immobilization method, can be classified in first, second and third generation, respectively (Castillo et al. 2004). First generation amperometric sensors include only the biorecognition element and a transducer and are polarized to an appropriate potential, to either reduce the molecular oxygen or to oxidise the formed hydrogen peroxide. The recorded current is proportional to the substrate concentration. In

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second generation biosensors, the natural co-substrate is replaced with an artificial redox mediator, having a proper formal potential to regenerate the redox centre of the enzyme. In third generation biosensors, the mediator is integrated together with the enzyme (or other biosensing element) and the electrode, shuttling the electrons between the redox centre of the enzyme and the polarized electrode.

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2.3. Immunosensors An immunosensor is a biosensor that uses antibodies as the biological element (Marco et al. 1995, Patel 2002). The transduction element in the case of immunosensors reported for chemical and microbial contaminants is largely based on optical, electrochemical and piezoelectric signals (Luppa et al. 2001). The availability of an antigen-specific antibody is the key to the success of immunoassays. Polyclonal antibodies (PAbs) contain an assortment of antibody molecules recognizing different antigens and epitopes, and therefore may show some cross-reactions with antigens from different microbes. PAbs can be made epitope-specific for improved detection. The quality of Pabs may vary from batch to batch, which could affect the end-result (Marquette et al. 2006, Ricci et al. 2007). On the other hand, monoclonal antibody (MAb) is homogeneous and highly specific (Bhunia 2008). For example, Hearty et al. (2006) isolated a IgG2a monoclonal antibody against Listeria monocytogenes which was further incorporated in a surface plasmon resonance biosensor (SPR-see below). Alternatively, Nanduri et al. (2007) reported the use of a phage-displayed scFv antibody to the virulence factor actin polymerization protein (ActA) for detecting the same pathogen (also by using an SPR biosensor platform). An improvement over traditional immunosensors is the bond-rupture techniques (Hirst et al. 2008). Bond-rupture provides a means to detect the attached mass and distinguish between attachments of different affinity without costly and time-consuming labelling procedures. According to the theory of operation of bond-rupture sensors, the transducer surface is coated with receptors to which the target binds. Targets bind specifically and other molecules bind non-specifically. The surface is oscillated at increasing amplitude until bond-rupture occurs. By incrementing the force, particles of lower affinity rupture from the surface first, thus the additional mass caused by erroneous non-specific bonding is separated from the mass of interest which does not rupture until higher force.

2.4. Cell-Based Sensors Cell-based sensors use, as biorecognition elements, live, intact cells and -in some casestissues, organs or whole organisms (Kintzios 2007). Whole cells provide multipurpose catalysts, particularly in processes that require the participation of a number of enzymes in sequence; therefore, the utilisation of whole cells as a source of intracellular enzymes is often a better alternative to purified enzymes in various industrial processes. Cell-based biosensors are likely to have improved stability and higher biocatalytic activity, while their greatest advantage is their ability to provide physiologically relevant data in response to an analyte and to measure the bioavailability of the analyte (Daunert et al. 2000). Most of the biosensors

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reported use bacterial cells as the sensing element (Keenan et al. 2007). Microbial biosensors based on light emission from luminescent bacteria are being applied as a sensitive, rapid and non-invasive assay in several biological systems (Matrubutham and Sayler 1998). Although bioluminescent bacteria are found in nature, many bioluminescent cell sensors have been developed using genetically engineered cells.

3. ASSAY METHODS 3.1. General

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The two most common methods of transducing biosensor responses are electrical and optical.

3.1.1. Electrical Amperometry, which is described under 2.2. above, is the most standard representative of electrochemical sensory technology, commonly coupled with enzymes or antibodies as biorecognition elements. Microphysiometry is based on the principle that electrically active cells or tissues can be interfaced with microelectrodes which allow the capture of extracellular spikes or impedance changes associated with cellular or tissue responses. Various potentiometric electrodes have been used to detect extracellular metabolites, e.g. potentiometric pH electrodes measured the acidification of the external environment caused by the production of acidic catabolites such as organic acids and CO2. Ion sensitive field effect transistors (ISFETs), can also be used to detect the acidification of the extracellular environment. A significant advance in the field of microphysiometry was contributed by Hafeman et al. (1988) who developed the light-addressable potentiometric sensor (LAPS): this system, which is very similar to a pH ISFET, allows for detecting the sensor surface potential by illuminating a small spot at any desired position with a focused pulsed lightpointer. In this way, surface potential measurements are not limited by the ability to microfabricate discretely insulated gates. Electrically active cells or tissues, can be interfaced with microelectrodes which allow the capture of extracellular spikes or impedance changes associated with cellular or tissue response. This interaction has a number of components, which can be classified in three groups: (i) direct interaction of microelectrodes with the cell membrane (ii) interaction with analytes present in the extracellular microenvironment and (iii) interaction with other parts of the probe, such as the gel and the solution gathered in to the matrix pores (immobilized cell sensors) or culture solution (suspended cell sensors). Impedance sensors are based on the changes in conductance in a medium due to the microbialbreakdown of substrates into electrically charged ionic compounds and acidic byproducts. The detection time, that is, the time necessary for these changes to reach a threshold value, is inversely proportional to the initial inoculums and the physiological state of the cells. The principle of all impedance-based systems is that they measure the relative or absolute changes in conductance, impedance, or capacitance at regular intervals (Ivnitski et al., 1999, 2000).

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There already exists a considerable number of biosensory methods based on electrophysiological effects. Some of them use cells, others use assay electric properties, or immobilized components. In its most elementary version, an electrophysiological biosensor consists of a cell part that is attached by suction to the tip of a glass microelectrode, and a highly sensitive feedback current-to-voltage converter that measures sub-picoampere currents, i.e. by employing the basic patch-clamp technique for cell membrane potential measurements (Zang and Hamill 2000, Yang and Bashir 2006). Owicki and Parce (1991) developed a silicon microphysiometer as a biosensor-based instrument that detects changes in the physiological state of cultured living cells, by monitoring the rate at which the cells excrete acidic products of metabolism. Following a similar approach, Lehmann et al. (2000) developed a biocompatible ion-sensitive field effect transistor (ISFET) array, to measure the pH close to a surface and the global extracellular acidification rate at the same time. Hediger et al. (2001) developed a microsystem for the culture and electrical characterisation of epithelial cell layers for cell-based diagnostic applications. Arndt et al. (2004) applied an electric cell–substrate impedance sensor (ECIS) in order to monitor the apoptosis-induced changes in cell shape, in an integral and quantitative fashion. The Bioelectric Recognition Assay (BERA), developed by Kintzios et al (2001a,b, 2004) is a biosensory method based on a unique combination of a group of cells, whose immobilization in a matrix preserves their physiological functions and measures the expression of the cell interaction with target molecules, through the change in electrical properties. A BERA sensor consists of an electroconductive, tube-like probe containing components of immobilized cells in a gel matrix. Cells are selected to specifically interact with the molecule under investigated. In this way, when a positive sample is added to the probe, a characteristic, ‘signature-like’ change in electrical potential occurs upon contact between the molecule and the gel matrix.

3.1.2. Optical Optical biosensors are probably the most popular in bioanalysis, due to their selectivity and sensitivity. Recently, fluorescence and surface plasmon resonance (SPR) based methods have gained momentum because of their sensitivity. The instrumentation used in order to detect visible, fluorescent, or luminescent signals from cells or tissues, includes microscopes, fiber optics, CCD cameras and other optical equipment. Due to its high sensitivity and the advantages in the measurement of high-density microtiter plates, bio- and chemiluminescence imaging is quite suitable for the development of high throughput screening (HTS) systems (Roda et al. 2005). However, quantification of the results is limited by the lack of appropriate calibration systems, the invasive nature of intracellular recording and the influence of the sample properties on the emission spectrum and intensity. 3.1.2.2. Surface Plasmon Resonance (SPR) SPR is a surface sensitive optical technique for monitoring biomolecular interactions ccurring in very close vicinity of a transducer (gold) surface, and that has given it a great potential for studying surface-confined affinity interactions without rinsing out unreacted or excess reactants in sample solutions (Shankaran et al. 2007). It allows real-time study of the binding interactions between a biomolecule (antibody) immobilized on a transducer surface with its biospecific partner (analyte) in solution without the need for labeling the biomolecules by exploiting the interfacial refractive index changes associated with any

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affinity binding interaction. In general, an SPR immunosensor is comprised of several important components: a light source, a detector, a transduction surface (usually gold-film), a prism, biomolecule (antibody or antigen) and a flow system. During the last years, the SPR technique is rapidly gaining recognition and application as a powerful tool for biomaterial characterization. The use of SPR to probe surface interactions is advantageous since it is able to monitor any dynamic process, such as adsorption or degradation, rapidly. It can give information on the rate and the extent of adsorption, enabling the determination of dielectric properties, the association/dissociation kinetics and affinity constants of specific interactions (Spadavecchia et al., 2005). In SPR imaging measurements, the investigated area is illuminated at a fixed angle. Spatially localized variations in the metal surface cause local variations in reflected intensity which can be seen as a variation of colour in a SPR image. Since SPR imaging is sensitive only to molecules in close proximity to the surface (within 200 nm), unbound protein molecules remaining in solution do not interfere with in situ measurements. This makes possible the monitoring of weak or reversible antibody or DNA–DNA complementary binding interactions. SPR-based sensors are governed by two basic principles: wavelength interrogation and angle interrogation (Homola et al., 2002 ). Wavelength interrogation uses a fixed angle of incidence but measures spectral changes, while in angle interrogation, a fixed wavelength is used but the angle of reflectance is monitored. Most of the commercial SPR systems are operated based on the angle interrogation mode.

SPR IMMUNOASSAYS Interest in the development and application of SPR immunosensors for the detection of low molecular weight compounds is growing rapidly for a variety of reasons, including sensitivity, selectivity, speed and reliability in analyses with additional emphasis on portability, miniaturization and on-site analysis (Mullett et al., 2000; Karlsson, 2004). The most notable attraction in SPR based immunosensors is the highly specific detection of small molecules with extraordinarily low detection limits for a wide variety of analytes in complex matrices (Shankaran et al., 2006). SPR DNA/RNA Assays The interaction processes taking place between oligonucleotides immobilised onto suitable photolithography patterned gold substrates and their complementary strands are beginning to assume an important role in biotechnological applications and in particular in the realization of specific biosensors for specific applications in food analysis control (Spadavecchia et al., 2005). The advantages of the technology are: (i) it is not necessary to have highly purified components, (ii) quantities below microgram can be analysed, and (iii) analysis and detection are done by one step in a real-time procedure (Miraglia et al., 2004). First results technique for the detection of Roundup Ready™ soybean have been reported by immobilising biotinylated PCR products or target oligonucleotides on the chip and hybridising them with respective probes (Feriotto et al., 2002).

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3.1.2.3. Other Optical Technologies Fiber Optic Biosensors The basic principle of the fiber optic sensor is that when light propagates through the core of the optical fiber (waveguide), it generates an evanescent field outside the surface of the waveguide. When fluorescentlabeled analytes such as pathogens or toxins bound to the surface of the waveguide, are excited by the evanescent wave generated by a laser (635 nm), and emit fluorescent signal (Bhunia et al. 2007, Taitt et al. 2005), the signal travels back through the waveguide in high order mode to be detected by a fluorescence detector in real time.

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Fluorescence Resonance Energy Transfer Fluorescence resonance energy transfer (FRET) allows the measurement of FRET during interaction of two molecules labeled with two different fluorophores whose absorbance and emission spectra overlap. One fluorophore acts as a donor (reporter) and the other one as an acceptor (quencher) (Majoul 2004, Bhunia 2008). Ko and Grant (2006) reported the development of a fiber optic portable biosensor utilizing the principle of fluorescence resonance energy transfer (FRET) was developed for fast detection of Salmonella typhimurium at 103 cells/ml in ground pork samples. Fourier Transform Infrared Spectroscopy Fourier transform infrared spectroscopy (FT-IR) spectroscopy is used to generate bacterial spectral scans based on the molecular composition of a sample. A library of spectral scans can be generated for different bacterial species and strains, which can be used for future comparison (Bhunia 2008). It is a nondestructive rapid method and sample identification depends on the available spectral library. FT-IR has been used for classification or identification of several foodborne pathogens, including Yersinia, Staphylococcus, Salmonella, Listeria, Klebsiella, Escherichia, Enterobacter and Citrobacter (Gupta et al. 2005, Mossoba et al. 2005, Sivakesava et al. 2004). FT-IR photoacoustic spectroscopy was used for the identification of spores of several Bacillus species with 100% accuracy (Thompson et al. 2003). The same system was also used for the identification of E. coli O157:H7 on an apple surface (Irudayaraj et al. 2002). Light Scattering Light scattering technology differentiates samples based on refractive index, size, shape, and composition. When an illuminated light from a polarized monochromatic light source shines on a sample (bacteria, for example), scattered light forms distinct patterns which could be used for identification and detection of bacteria (Bhunia 2008).The method is widely used for water inspection and for studying biological cells ( Hielscher et al. 1997, Jordan et al. 2002 ), but it did not produce reproducible result s when tested with bacterial colonies (Nebeker et al. 2001 ). Conversely, optical forward scattering yielded reproducible scattering patterns. Recently, a diode laser was used to gene rate light scattering images of Listeria colonies growing on agar plates for their identification and classification (Banada et al .2007, Bayraktar et al. 2006).

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3.3. Piezoelectric Sensors A piezoelectric sensor is a device based on materials such as quartz crystals, which resonate on application of an external alternating electric field. The frequency of the resulting oscillation is a function of the mass of the crystal. Thus, interaction of an analyte in a sample with the corresponding biorecognition element, previously immobilised by passive adsorption or by covalent interaction to a quartz crystal, will increase the overall mass, measured as a change in the frequency of oscillation. Salmonella enterica cells were detected at microgram quantities using a piezoelectric sensor (Harteveld et al. 1997, Lin and Tsai 2003). S. typhimurium cells were detected with a detection rang e of 9.9x105 to 1.8x108 cfu/ml (Park and Kim 1998). Pathirana et al. (2000) reported an improved method with a detection limit of a few hundreds cells. Olsen et al. (2005) reported the rapid detection of S. typhimurium in solution, based on affinity-selected filamentous phage prepared as probes physically adsorbed to piezoelectric transducers. Chen et al. (2008) used a thiolated probe as the biorecognition element for detecting E. coli O157:H7. A quartz crystal microbalance (QCM) is a variation of the piezoelectric sensor, which consists of a thin quartz disc with implanted gold electrodes. QCM has been used for the detection of staphylococcal enterotoxins (Lin and Tsai 2003), L. monocytogenes (Minunni et al. 1996), S. typhimurium (Wong et al., 2002), S. paratyphi (Fung and Wong, 2001), Bacillus cereus (Vaughan et al.,2003), and E. coli O157:H7 (Kim and Park 2003, Su and Li 2005).

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3.4. Microcantilevers Several research papers reported that microcantilevers can transduce a number of different signal domains (such as mass or stress) into a mechanical deformation (bending or a change in the resonance frequency) with a resolution which is orders of magnitude higher than that achievable with macroscopic structures (Albrecht et al. 1990, Wolter et al. 1990, Raiteri et al. 2001).

3.5. Other Methods More exotic assay methods, such as those utilizing photothermometric transducers (Pogacnik and Franko 2003) have also been reported.

4. MAIN APPLICATIONS 4.1. Pesticide Detection Biosensors based on cholinesterase enzymes (ChEs) have emerged as an ultra sensitive and selective technique for toxicity monitoring for environmental, agricultural, food or military applications. These devices are based on the inhibition of ChEs by toxicants such as

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organophosphate or carbamate pesticides and heavy metals. ChE are inhibited by both organophosphate (OP) and carbamate pesticides, but the mechanism of inhibition is different. In the case of carbamates the inhibition is slightly reversible while most OP pesticides induce an irreversible inhibition. Two types of cholinesterases are known and have been used for biosensor fabrication: acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). ChE enzymes extracted from the Drosophila melanogaster and the Electric Eel are commercially available and are the most widely used for biosensor fabrication (Andreescu and Marty 2006). Waibel et al. (2006) reported the use of AChE from Nippostrongylus brasiliensis AChE (NbAChE) for the detection of phosphorothionates. The potentiometric detection of the AChE activity is based on the measurement of the change in pH and/or in the redox potential in the enzymatic layer. (Kumaran and Morita 1995, Ghindilis et al. 1996, Hildebrandt et al. 2008). Modern potentiometric ChE sensors measure the pH shift using advanced pH-sensitive sensors such as ion-selective field-effect transductors (ISFET) or light-addressable potentiometric sensors (LAPS). Other ChE biosensors are based on 4-aminophenyl acetate as substrate (La Rosa et al. 1994, Andreescu et al. 2002) or combine three enzymes (choline esterase, choline oxidase, peroxidase) (Ghindilis et al. 1996). With few exceptions, the lowest detection limits are in the ppb range for all ChE biosensors. The practical application of immobilized ChEs has a significant limitation. The inhibition results in a decrease of the ChE activity so that repetitive use of the same biosensor without enzyme reloading for reactivation is limited. The solution to this problem is to employ single use disposable electrodes. Recently, research has been focused in the development of alternative or better systems for detection of pesticides by using different enzymes or enzyme/sensor interfaces. Various glutathione transferase (GST)-based assays have been also developed for the quantification of pyrethroids (Enayati et al. 2005) and malathion (Kapoli et al. 2008). The inhibition of tyrosinase has been also investigated for the determination of carbaryl using amperometric biosensors (Kuusk and Rinken, 2004). Other researchers reported the use of alkaline phosphatase (Mazzei et al. 2004) or organophosphate hydrolase (Simonian et al. 2001, White and Harmon 2005). Contrary to biosensors using pure enzymes, whole cells can give information on the ecotoxicological effects of pollutants as factors such as bioavailibility, environmental parameters (temperature, pH, etc.) that influence cell sensitivity will be integrated to the biosensor response. A recent example of a cell-based sensor for organophosphate and carbamate pesticides was reported by Mavrikou et al. (2008). The sensor utilized the ChE activity of neuroblastoma cells, thus allowing for the selective detection of chlorpyriphos and carbaryl in concentrations as low as 1 part per trillion (ppt). Immunosensors have also been developed for the detection of a number of pesticides (Laschi et al. 2003, McCarney et al. 2003, Zacco et al. 2006). A summary of biosensor applications in the detection of pesticides is given in table 3.

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Biosensors in Food Safety Control: An Update Table 3. Pesticides currently detected by different biosensor technologies Biosensor Technology Enzyme-based amperometric

Biorecognition element Cholinesterase

Cholinesterase

Enzyme-based optical

Cholinesterase Organophosphate hydrolase Tyrosinase Polyphenol oxidase Cholinesterase

Glutathione transferase

Immunosensor/electrochemical

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Immunosensor/optical (SPR) Cell-based

Micromechanical cantileverbased

Antibody

Synechococcus sp. Solanum tuberosum E. coli S. halepene Antibody

Compound (s) Carbamates (carbaryl, carbofuran*, aldicarb, bendiocarb, methomyl*) Organophosphates (diazinon*, dichlorvos*, methyl parathion, paraoxon*, malathion*, fenitrothion, trichlofon**, chlorpyrifos) Phosphorothionates Paraoxon, carbaryl parathion Carbaryl, atrazine, diuron atrazine Propoxur, carbaryl Paraoxon organophosphates Malathion Pyrethroids : permethrin, cypermethrin, deltamethrin, λ-cyhalothrin

Alachlor Atrazine Nicarbazin Diuron Atrazine Organophosphates Glyphosate 2,4-dichlorophenoxyacetic acid (2,4-D)

Detection limit (order) ppb/ppt*

ppb/ppt*/ppf**

ppb ppb ppm ppm ppm ppm/ppb ppm ppt ppb/ppt

ppm ppb ppt ppm nM μΜ μΜ nM ppm

4.2. Pathogen Detection and Determination The food sector represents the largest market segment within the Industrial Microbiology Market and represents almost half of the total market (Strategic Consulting 2005). The volume of the combined food and beverage global microbiology market in 2005 is approximately $2 billion. The gain in the market value for rapid methods will be even more pronounced than the testing volume in creases since the rapid methods have much higher average prices per test than traditional methods. The following figure 6 summarizes the percentage of global testing conducted in 2005 to analyze the dominant pathogens in the food sector (Patrick et al. 2007).

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Figure 6. Percentage of dominant pathogen species analyzed by global microbiological testing in 2005.

Conventional methods in microbiological analysis are used despite their long turnover times because of their high selectivity and sensitivity. Rapid methods, in particular biosensors have the potential to shorten the time span between sample uptake and results, but their future lies in reaching selectivities and sensitivities comparable to established methods at a fraction of the cost. Although not so critical, issues such as ease of use, low maintenance and continuous operation also need to be considered (Lazkcka et al. 2007). Established methods include the polymerase chain reaction (PCR), culture and colony counting methods as well as immunology-based methods are the most common tools used for pathogen detection. They involve DNA analysis, counting of bacteria and antigen–antibody interactions, respectively. In spite of disadvantages such as the time required for the analysis or the complexity of their use, they still represent a field where progress is possible. These methods are often combined together to yield more robust results. Recent developments in the field include combinations of PCR with mass spectrometry (MS) (Ecker et al. 2006). Rapid methods and automation in microbiology are dynamic fields of study that address the utilization of microbiological, chemical, biochemical, biophysical, immunological and serological methods (Fung 1995). Advanced methods are associated with diversified techniques, such as impedance/conductivity measurements, microcalorimetry, flow cytometry, nuclease/lysate tests, adenosine triphosphate (ATP) measurement, luminescence/fluorescence, molecular biological tests and immunoassays (Jay 2000). Generally, rapid methods aspire to replace (or, at least, complement) conventional assays such as ELISA tests and cell cultures. In the latter case, identification of a bacterial population requires several weeks, which can be reduced to a mere 5-48 hrs by using an appropriate advanced bioassay system. However, assay time is inversely correlated with the size of the population under investigation, with an average capacity of detecting 102-104 cells/ml within a time frame of 5-7 hrs. When designing novel microbiological methods, two issues are of major significance: the ability to specifically characterize the bacterial species under investigation and the ability to evaluate the biological activity (especially the virulence) of the investigated species or strain.

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The first required can be satisfied by appropriated molecular genetic (e.g. DNA fingerprinting, PCR) or proteomic (immunoassay) analysis tools. It is far more difficult to apply a rapid assay method for bioactivity. As a matter of fact, cell culture and/or co-culture of host cells with target human/or animal cells remains the method of choise, even though it is a very expensive and time-consuming approach. A variety of cell culture systems are employed to assess certain pathogenic properties of viable cells. The properties often assessed are invasiveness, permeability, cytotoxicity, adherence/binding and morphology/cell size. Cells employed are usually derived from culture and are mucosal, fetal intestine, HeLa, Chinese hamster ovary (CHO), Vero and other cells. Immunomagnetic beads have been used for capture of E. coli O157:H7 (Chapman and Ashton 2003, Evrendilek et al. 2001, Yu and Bruno 1996), Salmonella (Favrin et al. 2001, Jordan et al. 2004), and Listeria (Uyttendaele et al. 2000, Kaclikova et al., 2001). Sometimes, the capture efficiency is highly variable, which depends on the pathogen and the quality of the antibodies used. In recent years, application of IMS coupled with PCR assays are showing very promising results for the detection of E. coli O157:H7 (Fu et al., 2005), S. enterica (Mercanoglu and Griffiths 2005, Lermo et al. 2007), and L. monocytogenes (Amagliani et al. 2006, Ueda et al. 2006). Mujika et al. (2008) reported the development of a magnetoresistive immunosensor for the detection of E. coli O157:H7. The biosensor was able to detect and quantify small magnetic field variations caused by the presence of superparamagnetic beads bound to the antigens previously immobilized on the sensor surface via an antibody–antigen reaction. Finally, electrochemical immunosensors have been used for the detection of S. enterica (Delibato et al. 2005, 2006), E. coli, L. monocytogenes and C. jejuni (Chemburu et al. 2005). Electrochemical sensors have been also successfully coupled with immunomagnetic beads (Gehring et al. 1996, 1999, Delibato et al. 2005). Another issue is the possibility of conducting non-invasive assays, i.e. non-interfering with the investigated biological system. Rapid methods satisfying this criterion are based either on microphysiology (including impedance measurement) or fluorescence/ luminescence.

4.2.1. Biological Recognition Elements There are three main classes of biological recognition elements which are used in biosensor applications for food microbiological safety. These are (i) enzymes, (ii) antibodies and (iii) nucleic acids. In the detection of pathogenic bacteria, however, enzymes tend to function as labels rather than actual bacterial recognition elements. Enzymes can be used to label either antibodies (Ko and Grant, 2003) or DNA probes (Lucarelli et al., 2004) much in the same fashion as in an ELISA assay. More advanced techniques may operate without labelling the recognition element, such as the case of surface plasmon resonance (SPR) (Thomas et al. 2006, Waswa et al. 2007, Raz et al. 2008) piezoelectric or impedimetric biosensors (Guan et al., 2004). Impedance sensors have been used for analyzing bacterial contamination (Listeria) in milk (Capell et al. 1995, Madden and Gilmour 1995) and cheese samples (Rodrigues et al. 1995). The impedance-based assay has also been used for the detection and enumeration of Campylobacter (Falahee et al. 2003), E. coli (Upadhyay et al. 2001), Staphylococcus (Glassmoyer and Russell 2001), and Salmonella (Yang et al. 2003) from food samples. Varshney et al. (2007) and Varshney and Li (2007) reported the use of an impedance sensor

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in combination with magnetic nanoparticle-antibody conjugates (MNAC) for the detection of E.coli O157:H7. In an entirely different approach, whole-cell sensors incorporating luciferase technology and bacteriophage A, A511/AE85, TM4 or P22 have been used for tracking E. coli, Listeria sp., Mycobacterium sp. or Salmonella sp., respectively (Jacobs et al. 1993, Ulitzur and Kuhn 2000). Mammalian cells have been widely used for the analysis of the pathogenicpotential of foodborne bacteria (Bhunia and Wampler 2005). L. monocytogenes-induced severe membrane pore formations and cell death was shown to be due to the induction of apoptosis (Menon et al. 2003). A hybridoma cell-based assay was used for detection of L. monocytogenes from food samples using a two-step method of immunobead capture and cytotoxicity analysis (Gray and Bhunia 2005). Bacillus species can be detected using the same Ped-2E9 cell-based cytotoxicity assay (Banerjee et al. 2007). The sensitivity of Ped-2E9 hybridoma cells to pathogenic Listeria and Bacillus species makes this cell line a potential candidate to be employed in a cell-based biosensor device (Bhunia 2008). Finally, Immonen and Karp (2007) developed a method for determining ultralow amounts of nisin in food samples that is based on luminescent biosensor bacteria. Modified bacterial luciferase operon luxABCDE was placed under control of the nisin-inducible nisA promoter in plasmid pNZ8048, and the construct was transformed into Lactococcus lactis strains NZ9800 and NZ9000. A summary of biosensor applications in the detection of food-born pathogens is given in table 4. Table 4. Food-borne pathogens currently detected by different biosensor technologies

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Biosensor Technology Immunosensor/electroche mical

Immunosensor/optical (SPR)

Biorecognition element Antibody

Antibody

Immunosensor/optical (FRET)

Antibody

Immunosensor/optical (FT-IR)

Antibody

Immunosensor/optical (fiber optic)

Antibody

Immunosensor/ piezoelectric

Antibody

Pathogen (s) E. coli L. monocytogenes C. jejuni S. enterica Salmonella sp. Staphylococcal enterotoxin B E.coli B. cereus E.coli S. typhimurium L. monocytogenes E.coli S. typhimurium Citrobacter freundii Enterobacter cloacae S. typhimurium S. enterica Clostridium botulinum Campylobacter jejuni L. monocytogenes S. typhimurium

Detection limit (CFU ml-1) 50 10/ppt 50 106 105 ppb 102 105

103

103

104 101

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Biosensors in Food Safety Control: An Update Biosensor Technology Immunosensor/magnetic bead Microrganism-based sensor

Biorecognition element Antibody

Bacteriophage

Bacteria Mammalian cell Impedance sensors

Pathogen

Pathogen (s) E. coli L. monocytogenes S. enterica Salmonella sp. E. coli, Listeria sp., Mycobacterium sp. Lactococcus lactis monocytogenes Bacillus sp. Salmonella sp. E. coli, Listeria sp.,Campylobacter sp., Staphylococcus sp.

Detection limit (CFU ml-1) 104

103

ppb 102 103

5. CONCLUSION- COMMERCIAL STATUS AND PERSPECTIVES

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Biosensors are the fastest growing technology for pesticide and food-borne pathogen detection, as documented by the increase of the number of publications in SCI journals. Except for selectivity, performance will lie in a necessary compromise between time and sensitivity. However, and in spite of the great number of publications on biosensors applied in food analysis only a few systems are commercially available. The majority of tests associated with food safety relate to microbiological safety. Indeed, the market potential for detection and identification of bacterial and viral pathogens in the food safety area is estimated at around $150 million per year. As shown in the following figure 7, the market value for food microbiology testing since 1998 has grown significantly and has had an annual average growth rate of 9.2%, compared to a mere 7% of the total testing categories.

Figure 7. Average annual growth of the market value for food microbiology testing since 1998.

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Τraditional methods currently account for approximately 65% of the tests performed worldwide in 2005 in the food microbiology market. Rapid methods accounted for the remaining 35%, or approximately 220 million tests. It is expected, however, that by 2010 traditional methods will represent only 52% of all tests, with a total growth rate of 11%, whereas the market share of rapid methods will grow three times faster (figure 8), reaching 394.6 million tests in 2010. Optical techniques perhaps provide better sensitivity than electrochemical ones, but their cost and complexity makes them unattractive to most end users. Electrochemical techniques, on the other hand, are much easier to use but when it comes to detecting pathogens, their performance is still far from adequate. In order to become attractive, biosensors first need to show that they are capable of reaching at least the same detection levels as traditional techniques (between 10 and 100 CFU ml−1). Next, they need to do so in a fraction of the time without overlooking cost (Lazcka et al., 2007). As far as cell-based sensors are concerned, progress in three-dimensional microfabrication technology has opened new possibilities for miniaturising cell culture and analysis devices. A number of microfabricated proliferation sensors has emerged, essentially based on assaying parameters of a cell suspension culture (Matsubara et al. 2004), predominantly extracellular acidification as well as electric impedance.

Figure 8. Projected increase in market value of traditional and rapid food safety assay methods (white columns: microbiological safety, black columns: all applications).

Problems associated with the non-commercialization of novel biosensors are related to anumber of causes such as: 1. Lack of analytical data with real samples: With few exceptions (Albareda-Sirvent et al. 2001), the majority of sensors reported in literature have been tested on standard solutions and not on real samples. An example of a practical application for an AChE biosensor was described by Schulze et al. (2002). Quite recently, Mavrikou et al. (2008) reported on the successful detection of pesticides in tobacco, using a commercial variation of the Bioelectric Recognition (BERA) sensor.

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2. Inability to provide a spectrum of sample components: This is a major, inherent characteristic of virtually all biosensor technologies. For example, AChE-based enzyme biosensors will respond to a broad spectrum of pesticides, since all organophosphosphorus and carbamate pesticides (but also heavy metals) inhibit the ChE activity (albeit with different inhibition degree). The same is roughly true for cell-based sensors. Consequently, the simultaneous presence of heavy metals and pesticides in contaminated samples provides a challenge for their use for purely regulatory purposes where a specific analyte must be determined with a prescribed accuracy (Amine et al. 2006). Due to this important limitation, ChE or cell-based biosensors are mainly attractive for measuring the total toxicity of the sample, rather than a specific inhibitor, meaning that they can be used as front line alarm systems, rather than full analytical devices. In addition to mutant enzymes with a differential sensitivity to the toxic compound, the use of multiarray sensors coupled with an artificial neural network for data treatment and analysis could substantially improve biosensor selectivity and allow exact identification of the inhibitor present in a sample (Bachmann et al., 2000; Schulze et al., 2005). 3. Matrix interference: Most of the existing limitations could be directly related to the selectivity in multicomposite mixtures and complex matrices and the inability of identifying a specific toxic analyte. However, their sensitivity is sufficient to detect minimum level of pesticides and heavy metals imposed by regulatory agencies and they remain attractive as alarms or screening devices. For example, evaluation of cross-reactivity and matrix effect is an important aspect in designing SPR immunoassays, because they determine the selectivity and hence the reliability of the measurement (Melo and Kubota 2002). 4. Limited lifetime of the biological component: This may vary from days (cell-based sensors) to weeks or even months (enzyme-based, immunosensors). 5. Ease of handling: For example, despite impressive SPR immunoassays developed in research laboratories, miniaturized portable immunoassay devices for real-time applications are yet to play a major impact on commercial success (Shankaran et al. 2007). Regarding the availability of commercial biosensor devices for food analysis, a number of instruments has been commercially available for some years, e.g. enzyme-based biosensors for determination of saccharides (YSI), antibody-based biosensor for bacterial toxins (Research International ), an antibody-based electrochemical biosensor (Detex1) for detection of Salmonella, E. coli O157 and Campylobacter (Molecular Circuitry Inc. ), L-lactam antibiotics (Paul Scherrer Institut ) and water-soluble vitamins (Biacore AB) (Patel 2002). More recently, a flow-through immunocapture system called Pathatrix® (MatrixMicroScience Ltd., Cambridgeshire, UK) has been validated for capture and detection of Salmonella, E. coli O157:H7, and Listeria species (Prentice et al. 2006). An automated immunomagnetic capture system called BeadRetriever (Dynal Biotech Ltd., UK) has been used for the detection of E. coli (Fegan et al. 2004), Salmonella (Duncanson et al. 2003), and L. monocytogenes (Amagliani et al. 2006). Recently, Shriver-lake et al. (2007) reported a single analyte assay for E. coli O157:H7 developed for the Naval Research Laboratory (NRL) Array Biosensor. This biosensor was originally developed to detect multiple antigens in

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multiple samples with little sample pretreatment in under 30 min (Ligler et al. 2003, Sapsford et al. 2006). Fiber optic biosensors are among the first commercially available optical biosensors, marketed by Research International (Monroe, WA) for the detection of foodborne as well as pathogens of biosecurity importance. The manual version of the instrument is called Analyte 2000 and the portable semiautomated version is called RAPTORTM. Compact SPR systems with multichannel configurations are more attractive for simultaneous detection of different target analytes (Pyo et al., 2005). Examples of prototype devices include the Spreeta™ analyzer by Texas instruments (Waswa et al., 2007) and the system developed by Homola et al. (2002) for the detection of Staphylococcal enterotoxin B. There are also a few companies manufacturing SPR instruments for studying biomolecular interactions on a commercial scale (Zubritsky, 2000; Baird and Myszka, 2001; McDonnel, 2001). Each company produces different SPR systems equipped with a variety of options usable for specific applications. The most known of these companies are Biacore, Affinity sensors, Artificial Sensing Instruments, IBIS Technologies, Aviv, Bio Tul AG, Windsor scientific, Quantech, Texas, NTT and Moritex (formerly, Nippon Laser and Electronics), while market models also include the SPR spectroscope (MultiscopTM, Optrel GbR, Germany), Reichert SR7000 (Reichert Analytical Instruments, Depew, NY), and resonant mirror based IAsys (Thermo Labsystems, Cambridge, UK). The purchase price of an SPR equipment ranges from a few thousand to over 200,000 $US, depending on manufacturer, model and capabilities. Biacore and Affinity Sensors have marketed sensor chips with a carboxymethylated dextran matrix and a streptavidin-derivatized surface, which is versatile but not compatible with every type of application or sensor surface chemistry. As a result, several new commercial chips have been introduced in the past few years, permitting new SPR-based applications. Given, in addition, the relatively small number of instrumentation manufactures, an increase of the new developers of SPR-related products is anticipated in the immediate future. Other commercial biosensors utilize technologies such as impedance. Establsihed models include Bactometer® (Bio Merieux, France), Malthus AT analyzer (Malthus Instruments, UK) BacTracTM and m-Trac (SyLab, Austria) and the Rapid Automated Bacterial Impedance Technique (RABIT) (Don Whitley Scientific Ltd., UK) (Bhunia 2008, Yang and Bashir 2008). A summary of most prominent biosensor technologies already commercially applied in food safety is given in table 5. It is obvious that immunosensors are the method of choice, although this may change as alternative technologies (e.g. impedance-based sensors) are becoming the favorites of many researchers and companies worldwide. Table 5. Summary of established biosensor technologies already commercially applied in food safety Biosensor technology Enzyme-based Immunosensors (mostly SPR) Cell-based Impedance-based

Number of commercial products 2 22 2 6

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In the future, compact and portable devices specifically designed for in-field analysis and development of arrays of multiple sensors will constitute another area of intensive research. Considerable progress is expected in genetic engineering for the production of more selective and sensitive enzymes and/or cells with designed selective interaction against different pesticides or other biomolecules

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In: Biosensors: Properties, Materials and Applications ISBN: 978-1-60741-617-3 Editors: R. Comeaux and P. Novotny © 2009 Nova Science Publishers, Inc.

Chapter 3

PROPERTIES AND CHOICE OF MATERIAL USED FOR MICROBIAL BIOSENSOR Mimma Pernetti1, Denis Poncelet2 and Gerald Thouand3 1

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Present address: GlaxoSmithKline Biologicals, Site de Wavre-Nord W35, Avenue Fleming 20, 1300 Wavre - Belgium 2 ENITIAA, UMR CNRS GEPEA, Rue de la Géraudière, BP 82225, 44332 NANTES Cedex 3, France 3 University of Nantes, UMR CNRS 6144 GEPEA-CBAC laboratory, Campus de la Courtaisière-IUT, Département Génie Biologique, 18 Bd Gaston Defferre, 85035 La Roche-sur-Yon cedex, France

ABSTRACT Microbial biosensors are promising tools for the detection of specific substances and “global parameters” in different fields, such as environmental, food, biomedical and pharmaceutical. Immobilization of bacteria is a key feature in order to enhance the handling, the miniaturization, the storage and the stability of the biosensor. As many immobilization methods exist, a careful study must be carried out in order to select the best one for the application to a biosensor. No paper has ever been published on the systematic characterization of the immobilization systems in view of the application to a microbial biosensor. As a matter of facts, the performances of an immobilization method are usually evaluated indirectly, through the signal emitted by the biosensor. This work is intended to propose some guidelines to select the most appropriate immobilization system for a microbial biosensor. A survey of immobilization techniques and materials recently employed for microbial biosensors is provided. The selection criteria are finally applied to all the systems illustrated, on the basis of literature data, in order to provide a preliminary screening to be followed by the experimental characterization.

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1. INTRODUCTION A biosensor, as it is shown in figure 1, consists of a biological element, which, in contact with an analyte, produces a (bio)chemical signal, transformed in electrical signal by a transducer. Transducers can be electrochemical (Lee et al. 2002; Paitan et al. 2003), optical (Bartolome et al. 2003; Biran and Walt 2002; Polyak et al. 2001, Horry et al, 2007), calorimetric (Verhaegen et al. 1999), piezoelectric or acoustic (Yamasaki et al. 2004). The biological element may be one of the three types: bio-molecules, tissues or cells (Braguglia 1998).

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Figure 1. A simplified scheme of a biosensor. The biological element can be: bacteria, cells, antibodies, nucleic acids. The transducer can be: electrochemical, optical, acoustical, thermal, piezoelectric.

Cell-based biosensors employ whole microbial cells as sources of intracellular enzymes, thus providing multipurpose catalyst, avoiding complex purification processes and reducing risks of inactivation (Baronian 2004; D'Souza 2001b, Nakamura et al, 2008). The main advantages of these biosensors are: rapid measurements, no need for complex sample preparation nor specialised personnel, easy handling, low cost, stability in wide operating conditions (D'Souza 2001b; Osbild et al. 1995). These biosensors are mainly built with viable cells, exploiting their metabolic activity, as the analyte may be a substrate or an inhibitor and the response may be a product, respiratory activity or luminescence (Baronian 2004; Bartolome et al. 2003; Philp et al. 2003; Polyak et al. 2001). In such a case, immobilization procedure must be applied in order to maintain those activity. In order to develop a real biosensor, cells must be integrated in the appropriate device, which allows storage, access to the sample and signal detection (Belkin 2003). Immobilization of microorganisms in a support is a key feature for further development and commercialisation of biosensors, as it provides the proximity between the cells and the transducer and it enhances handling, miniaturization, storage and repetitive measurements (D'Souza 2001a). Several interesting reviews have been recently published on different kinds of microbial biosensors (Liu and Mattiasson 2002; Baronian 2004; D'Souza 2001a; D'Souza 2001b; Walmsley and Keenan 2000, Ron, 2007; Nakamura et al, 2008). The present work is not intended to be an exhaustive review of all the existing microbial biosensors nor of all the immobilization methods and materials, as literature on this subject is very wide (Junter and

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Jouenne 2004, Bjerketorp et al, 2006). This paper is intended to propose some criteria to select the most appropriate immobilization method for microbial biosensor. For this purpose, it also provides a brief survey of some applications and some immobilization techniques recently employed in microbial biosensors. The immobilization techniques are then evaluated through the selection criteria, in order to propose a screening, as the first step preceding the experimental research on immobilized biosensor. The present chapter is structured as follows: Applications : a summary of some applications of immobilized microbial biosensors, with the purpose of understanding the main problems it is necessary to face in the choice of the appropriate immobilization system. Selection criteria for immobilization : main issues to analyse before the selection of an immobilization system for a specific biosensor. Immobilization systems : the different immobilization techniques and materials employed in microbial biosensors are herein resumed and illustrated, with their advantages and drawbacks. Conclusions : the selection criteria previously proposed are applied to all the techniques and materials illustrated, on the basis of the literature. This screening would be the first step of the selection procedure, to be followed by the effective measurement and evaluation of properties.

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2. APPLICATIONS OF MICROBIAL BIOSENSOR The worldwide biosensor market is quickly growing: at the end of 2003 it was 7.3 billion dollars and it expected to grow to 8.2 billions in 2009 (Fuji-Keizai 2004, Luong et al, 2008)) because of the low cost, simple operation, rapid response and high specificity. Three main sections are specially developed: environmental, medical and food (Alocilja and Radke 2003). For environmental applications several new technologies are expected to be commercialised in the next years; biosensors provide three main kinds of analyses: global parameters such as biological oxygen demand and toxicity (MICREDOX, Pasco et al. 2004), water toxicity (Kim and Gu 2003; Philp et al. 2003, Diez-Caballero et al. 2004), gas toxicity (Gil et al. 2002), specific pollutants such as acrylonotryle (Håkansson and Mattiasson 2004), polycyclic aromatic hydrocarbons (Lee et al. 2003), organotin (Durand et al, 2003; Horry et al, 2007), broad spectrum monitoring (Roda et al. 2004, Lee and Gu 2004). The immobilization system should guarantee field-portability, ease to use and resistance to harsh conditions. In food applications, the demand for rapid analysis of nutritional parameters, additives, contaminants and olfactory characteristics is expanding, due to industry and law requirements. Numerous antibody and enzyme biosensors have extensively been studied and

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commercialised (Alocilja and Radke 2003; Mello and Kubota 2002; Thouand et al. 2001), microbial biosensors also result promising for their costs and stability. The main applications are food safety monitoring, such as pathogen contamination of poultry foods (Medina, 2004) detection of specific compounds, such as glucides (D'Souza, 2001) or sucrose (Rotariu et al. 2002) or ethanol (Rotariu et al. 2004) smell and taste evaluation, through the detection of short-chain fatty acids in milk and dairy products (Schmidt et al. 1996). The immobilization system for food biosensors should provide stability in harsh conditions, specially in continuous-mode operation, low cost and possibility of repeated measurements. Moreover, sterility is a key feature, since the risk of product contamination is high. In biomedical and pharmaceutical applications the growing concern for the use of animals for toxicity tests is boosting interest in biosensors: genotoxicity assessment (Walmsley and Keenan 2000), detection of specific compounds in blood and urine such as mono and di- saccharides (Held et al. 2002), tryptophan (Seki et al. 2003) early-stage evaluation of potential drugs (Biran and Walt 2002).

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The immobilization system for biomedical and pharmaceutical biosensors should provide above all high diffusivity in order to maximize the sensitivity and sterility, as cell leaching may be dangerous.

3. SELECTION CRITERIA FOR IMMOBILIZATION Being the market of analytical methods very competitive, immobilization method and material play a key role in the development of an innovative and successful biosensor. Immobilization should grant low cost, miniaturization, sensitivity and stability (Rogers and Gerlach 1999). The overall characteristics of sensors employing immobilized cells are determined by the characteristics of the matrix in conjunction with the physical transducer (Galindo et al. 1992). Microorganisms (bacteria, algae, bacteriophage, fungus) may be immobilized using different methods, such as adhesion, adsorption, entrapment (D'Souza 2001b; Leenen et al. 1996; Semenchuck et al. 2000; Tkac et al. 2002). All these techniques are currently used in biosensor development, the choice depends on the microorganism, on the analyte, on the transducer and on the configuration employed. Immobilization system must be accurately selected in order to ensure survival of active cells, signal detection, easy and rapid measurement, long term storage (Belkin 2003). Furthermore, immobilization should guarantee and enhance the most important characteristics of an efficient biosensor, such as specificity, sensitivity, reliability, portability and simplicity (D'Souza 2001b).

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Table 1. Biosensors recently developed for different applications. The microbial strain, immobilization method and type of response are specified APPLICATION

Analyte

BOD

Global parameter ENVIRONMENTAL

Microorganism

Immobilization

Type of response

Reference

Bacteria from sewage

Membrane adsorption

Respiratory activity

Rastogi et al. 2003b

Trichosporon cutaneum and Bacillus subtilis

PVA and silica

Respiratory activity

Jia et al. 2003

Arxula adeninivorans

PCS

Respiratory activity

Jang et al. 2004

Respiratory activity

Pasco et al. 2004 Philp et al. 2003

®

Proteus vulgaris

PVA Lentikats

Photobacterium fischeri, Pseudomonas putida

PVA

Bioluminescence

Recombinant strains

Agar

Bioluminescence

Recombinant strains

Agar Agar + glass beads

Bioluminescence

Escherichia coli

Alginate

Bioluminescence

Escherichia coli

Sol-gel

Bioluminescence

Acrylonitrile

Activated sludge

Membrane adsorption

Respiratory activity

Håkansson and Mattiasson 2004

Phosphorus Nitrogen

Synechoccus

Agar

Bioluminescence

Schreiter et al. 2001

Water toxicity

Gas toxicity

Genotoxicity

Specific pollutants

Kim et al. 2003a; Kim and Gu 2003 Gil et al. 2000 ; Gil et al. 2002 Polyak et al. 2001 Premkumar et al2002 ;Premku mar et al. 2001b ; Simpson et al. 2000a

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Table 1. (Continued).

FOOD

Polycyclic Aromatic Hydrocarbons

Escherichia coli

Agar + glass beads

Bioluminescence

Lee et al. 2003

Middle chain alkanes

Psychrotropic yeasts

Attachment on glass beads

Respiratory activity

Alkasrawi et al. 1999

Organotin Tri-butyl tin)

Escherichia coli::luxAB

Entrapment in agarose

Bioluminescence

Horry et al, 2007

Broad spectrum monitoring

Transcription analysis

Escherichia coli

Membrane + solid medium

Bioluminescence

van Dyck et al. 2001

Safety monitoring

Interaction bacteria- poultry skin

Salmonella

Covalent attachment on chip

Refractive index (SPR)

Medina 2004

Ethanol

Saccharomyces ellipsoideus

Membrane adsorption

Respiratory activity

Lobanov et al. 2001; Rotariu et al. 2004

Specific compounds

Gluconobacter oxydans Sucrose Saccharomyces cerevisiae

BIOMEDICAL AND PHARMACEUTICAL

Membrane adsorption Membrane adsorption

Respiratory activity Respiratory activity

Tkac et al. 2003 Rotariu et al. 2002 Schmidt et al. 1996

Quality

Free fatty acids

Arthrobacter nicotianae

Alginate

Respiratory activity

Toxicity

Cholanic acid

Saccharomyces cerevisiae

Agarised medium

Respiratory activity

Campanella et al. 1996

ENALAPRIL

Bacillus subitilis

Membrane adsorption

Respiratory

Fleschin et al. 1998

Mono- and disaccharides

Escherichia coli

k-carrageenan

Respiratory activity

Held et al. 2002

Cephalosporins

Escherichia coli

Agar

pH

Tryptophan

Escherichia coli

Agarose

pH

Drug candidate

Saccharomyces cerevisiae and Escherichia coli

Silica microwells, adhesion with PEI

Fluorescence

Specific compound

Drug discovery

Garcia et al. 1998 Seki et al. 2003 Biran and Walt 2002

Properties and Choice of Material Used for Microbial Biosensor

93

The following algorithm is proposed for the research preceding the development of a microbial biosensor: 1. 2. 3. 4. 5. 6.

Study of the application Identification of main issues involved Æ selection criteria for immobilization system Literature screening Choice of a set of immobilization methods / systems Characterization of the systems through application of selection criteria Development of the final biosensor Æ field test

Only the last two steps will involve experimental work, which will follow some important steps of study and characterization of the system. Firstly, the accurate study of the application, in order to point out the main properties required. Then, it is fundamental to evaluate the intrinsic characteristics of the immobilization system, in order to verify their suitableness to the specific application. The final step is the application to the biosensor and the evaluation of its performances. Though being a key feature, intrinsic characteristics of the immobilization matrix have never been systematically analyzed, since the quality and the effectiveness of different immobilization systems are usually evaluated indirectly, through the signal emitted (Davidov et al. 2000; Philp et al. 2003; Semenchuck et al. 2000). Table 2 summarizes the properties that immobilization system should provide for biosensor application and the methods proposed to evaluate each of them.

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Table 2. Summary of the properties of the immobilization system and the evaluation methods proposed Property

Evaluation method

Biocompatibility – cell activity

CFU counting, INT, ATP, respirometry, luminescence, fluorescence, signal detection (indirect)

Mechanical resistance

Compression/rupture

Chemical resistance

Compression/rupture after soaking in different mediums

Biological resistance

Biodegradation in activated sludge

Diffusion

Diffusion test, adsorption

Signal emission

Analyte measurements

Operational stability Storage stability Sterility (cell leaching)

Repeated analyte measurements- cell activity Analyte measurements – cell activity Cell counting in medium

Procedure Simplicity

Literature – number operations required, operating conditions

Cost

Literature, market survey, information from suppliers

Extra properties (ex. transparency)

Spectroscopy

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Biocompatibility In order to have an efficient biosensor, cellular activity should be unchanged after immobilization. The materials employed should be compatible with cells and the procedure for membrane formation should be carried out in mild conditions in terms of pH, temperature and concentration of solvent or cross-linkers. Cell viability before and after immobilization should then be investigated, in order to evaluate the effective biocompatibility. Cell viability may be evaluated by CFU (colony forming unit) counting, when natural gels can be dissolved in order to release the cells (Polyak et al. 2001). As not all the supports can be easily dissolved, viability of immobilized bacteria may be determined by respirometry, ATP (adenosine triphosphate is a carrier of chemical energy in cells, hence its content quantifies the active biomass concentration.), luminescence, fluorescence (Navrátil et al. 2000) and INT (Tetrazolium salt; 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride (INT) is employed to quantify the respiratory activity of microorganisms(Hatzinger et al. 2003; Smith and McFeters 1997).

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Mechanical, Chemical and Biological Resistance Mechanical resistance should be guaranteed, as supports may be exposed to shear stress and compression, depending on the biosensor configuration (membrane reactor or packed bed, in continuous or batch mode). As biosensors may be used in different environments, the immobilization support must also be resistant to chemical agents and to swelling in hypo-tonic solutions. Solubility tests should be carried out in different media, in order to compare the performances of the supports. Moreover, especially in environmental biosensors, supports containing cells are exposed to wastewater, seawater, rivers and soils, which are rich in microbial population. The immobilization support should be neither biodegraded by such organisms, nor be covered by thick biofilm, which would hinder analyte diffusion and signal emission. In general, natural gels can be degraded, while no biodegradation of synthetic gels has ever been reported (Leenen et al. 1996). In order to compare different polymeric materials, it is necessary to assess their mechanical properties. For biosensor application, an elastic membrane is required, since rupture during handling, storage and transportation must be avoided. Dynamical Mechanical Analysis (DMA) measures the mechanical properties of materials while they are subjected to a periodic stress, usually applied sinusoidally. For most materials the stress and the strain are out of phase. DMA measures the amplitudes of the stress and the strain and the phase angle between them. The instrument is schematised in figure 2. For bacteria survival in the matrix, substrates and oxygen must easily diffuse through the support; in the wide literature of cell immobilization, metabolite diffusion through the matrices has been investigated and modelled (Beuling 2000; de Alteriis et al. 2001; Dworecki et al. 2003; Leenen et al. 1996). For example, diffusion coefficient oxygen in Ca-alginate and k-carrageenan is about 2·10-9m2/s, while in PVA it is about 1·10-9m2/s. Diffusion coefficient of glucose in the same matrices is about 0.6·10-9m2/s. If the specific consumption rate is known, it is possible to calculate the profiles of metabolites through the membranes, in order

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to compare different matrices and to optimize the membrane thickness and cell concentration (Laca et al., 1998).

Figure 2. Schematic illustration of Dynamical Mechanical Analysis (DMA) with sample repetitive deformation.

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Diffusion of Substrates and Analytes For biosensor development diffusional limitation is a crucial feature, as it affects two phenomena: diffusion of nutrients and by-products diffusion of analytes For bio-sensing application, the immobilization support must also ensure a high diffusivity of the analyte, in order to minimize the response time and the detection limit. Diffusion tests with different analytes should be carried out, in order to verify the suitability of the material for the sensing system to be developed. As a matter of facts, the flux of analyte may be limited due to chemical bounds between the analyte and the polymeric matrix. This was observed for metal ions, which are adsorbed by alginate (Nurbas et al. 2002) and complexed by polyacrylate (Leth et al. 2002). In this case, adsorption and desorption of analytes on the matrix should be investigated to better understand the phenomena. However, analyte diffusion through the matrix should be studied separately from detection by immobilized bacteria, as the latter is influenced also by bacterial uptake and efflux system (Rensing and Maier 2003).

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Signal Emission Signal intensity, detection limit and response time of free cells depend on the analyte bioavailability, on its diffusion through the cellular membrane, on the metabolic reaction involved and on the transducer. For immobilized cells, these values are influenced also by stress on bacteria due to immobilization procedure and diffusion limitation in the matrix. Once compared the performances before and after immobilization, the bottle-neck can be identified, then operating conditions and support characteristics may be optimized in order to maximize the response. For example, preconditioning and induction time, together with membrane thickness, affect the biosensor performance. Signal emission is obviously evaluated using the specific detection system employed in the biosensor, after immobilization of sensing cells.

Stability

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In order to be widely employed and commercialized, biosensors should show high operational stability and storage stability. They are both evaluated by analyte measurements or viability analysis. Operational stability gives the maximal time during which a reliable response can be obtained from a biosensor. This depends on the biosensor configuration and on the operation mode, such as continuous or batch. Between each batch measurement, the biosensor could be kept at low temperature and in buffer solution, in order to preserve its properties and cellular activity. Operational stability is influenced by bacterial metabolism and physiology and by support resistance. If the signal should slightly change between different measurements, the sensor may be calibrated again (Chan et al. 1999); if the signal keeps decreasing in time, the biosensor could not be employed for multiple measurements and would rather be a disposable sensor (Ito et al. 2002). This may depend on sensing mechanism and on irreversible adsorption of the analyte on the cellular membrane or on the support. Storage stability, or shelf-life, gives the maximal time a biosensor can be kept inactive before being employed for measurements. Depending on the configuration, the membranes, the polymeric beads, the coated probes or the microtiter plates containing cells can be stored separately until required. These sensing elements can be stored in different conditions, such as dried, frozen, sealed or immersed in buffer solution at -20°C, 4°C or room temperature, which affects final costs. During storage, bacteria should be inactive and non-growing, but they must be able to recover their activity with a preconditioning treatment in diluted medium (Chee et al. 1999). Compared to "freshly prepared" ones, stored biosensors generally require a longer incubation time in analyte medium than fresh biosensors (Schreiter et al. 2001).

Sterility Sterility requirements involve different issues, which depend not only on the immobilization system, but also on the sensor configuration.

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Immobilization conditions: the procedure must be carried out in sterile conditions, in order to avoid other bacteria contamination in the matrix. The supports and polymer solutions must be sterilized by heat or filtration and the operations must be carried out under laminar flow. Cell leaching: sensing microorganisms may be released in the medium during the measurements. Cell leaching occurs especially in the case of natural gel membranes (Heitzer et al. 1994). This must be prevented, as the biosensor may be in contact with the environment and with alimentary products; therefore a good stability of the membrane should be guaranteed. Cell leaching can be assessed measuring the optical density or colony forming unit counting of the medium kept in contact with cell containing membranes. Contamination: the medium to analyze may be contaminated with other bacteria. Medium contamination may occur specially in flow injection analysis sensors, if long-term analysis are carried out. Moreover, bacteria may form a biofilm on the membrane surface, thus reducing the signal (Heitzer et al. 1994). As the presence of free bacteria may interfere with analysis, contamination should be prevented adding low concentrations of sodium azide (Held et al. 2002), which could also inhibit the bioelement. Contamination may be indirectly identified through signal detection.

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Procedure Simplicity The immobilization procedure should be easy to carry out, also for large scale production. Complex procedures increase the risk of errors, which reduces immobilization reproducibility and cell distribution homogeneity. Moreover, long operations for the preparation of biosensor may inhibit cell activity. Procedure simplicity is evinced from literature and during protocol preparation. For example, the number of steps and the variation of some parameters (such as temperature, pH and ionic force) in different techniques should be compared. From this point of view, agarose is simpler than alginate and PVA, and all of them are by far less "aggressive" than sol-gel.

Costs The global cost of immobilization for a biosensor depends on the raw materials and on the procedure. The economical interest of a biosensor is also indirectly related to the material stability, as a disposable biosensor would be more likely less cost-effective than a re-usable one. A rapid cost assessment should be carried out through literature and company inquiries, in order to understand the feasibility and the commercial interest of an immobilization technique.

Extra Properties Biosensors may require supplementary characteristics for immobilization, depending on the specific transducer, on the reaction and on the kind of cell. For example, bioluminescent biosensor will require a transparent support, in order to ensure the complete signal

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transmission (D'Souza 2001b). Biosensor detecting specific compounds from a complex solution, may need a support with a particular cut-off, in order to enhance its selectivity (Tkac et al. 2002). Pernetti et al. implemented the characterization schema herein proposed using agarose as model for gel entrapment in a preliminary study (Pernetti et al. 2003). The method was also applied to other hydrogels, such as Polyvinyl-alcohol, alginate and silicone (Pernetti, 2004). Some papers investigated the performances of different immobilization procedures methods on microbial biosensors performances (Corbisier et al. 1999; Leth et al. 2002; Liu and Mattiasson 2002; Peter et al. 1996; Semenchuck et al. 2000), many other papers employed one or two immobilization methods. It is difficult to compare the different works presented in literature, as they differ in microbial strain, type of response, biosensor configuration and operating conditions. However, before applying the characterization system, measuring all the properties, a screening may be carried out on the basis of literature.

4. IMMOBILIZATION SYSTEMS

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The classification of immobilization systems is controversial, as definitions are not unequivocally fixed. However, as it is schematised in table 3, two main classes of immobilization may be distinguished, depending on the support and on the procedure employed: -

superficial immobilization: cells are retained on the surface of an inert support, due to: - chemical reactions, as adhesion (or attachment), - physical bound, as adsorption on porous membranes or carriers

-

entrapment: cells are included in a matrix.

Table 3. Scheme of the different supports availablefor the main immobilization methods Immobilization

Method

Support Direct binding

Scheme

Microorgan i

Adhesion Mediated binding Superficial Membrane Adsorption Porous carrier

Entrapment

Encapsulation / entrapment

Matrix

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4.1. Superficial Immobilization 4.1.a. Adhesion Adhesion (or attachment) includes all forms of immobilization in which cells are bound to the surface of a solid support (Mulchandani and Rogers 1998). The bound between cells and support may be: direct, without the addition of any reagent, or mediated, due to the add of reactive components. Direct binding may be formed by cells on cellulose, glass, cotton fabric, synthetic membranes, by taking advantage of their property to naturally adhere to surfaces. Although easy to perform and mild on cells, this bound is very weak and cells can be easily released in the medium. Reversible immobilization through hydrophobic interaction has also shown promise (D'Souza and Deshpande 2001).

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Mediated binding: in order to enhance the stability and to prevent cell leaching, bacteria may be bound to the support, employing some specific reagents, such as cross-linkers, proteins, polymers and receptors.

Cross-Linkers Attachment by cross-linkers involves the linkage of any reactive component of cell membrane, generally the amine groups, to the support, by covalent binding. This technique employs cross-linking agents, such as glutaraldehyde, which results in a loss of cell viability. Being efficient but not biocompatible, this technique may be employed to immobilize nonviable cells, source of intracellular enzymes. The main advantages of this technique are its stability and the absence of diffusion limitation; nevertheless, since covalent bindings are irreversible, the matrix cannot be regenerated. Cell adhesion is successfully employed in BIACORE, a surface plasmon resonance (SPR) biosensor, detecting the changes of refractive index in the proximity of a chip (Medina 2002; Medina 2004). Cells can be mixed to gelatin and then bound to different supports by cross-linking preserving a good bio-sensing activity, for detection of sugars (Svitel et al. 1998), phenolic compounds (Timur et al. 2003), cadmium ions (Chouteau et al., 2004). Non viable cells can be immobilized as well in hen egg white, by glutaraldehyde cross-linking (D'Souza 2001a). Proteins and Polymers Bacteria may be attached on different surfaces using polyethylenimine (PEI) (D'Souza et al. 1986). PEI is a weak poly-basic aliphatic amine with good anion-exchange properties. It is employed to impart poly-cationic characteristics to several surfaces, such as cellulose, glass, plastics, in order to strongly bind the cells, without affecting their viability (D'Souza 2001a). This technique was employed for immobilization of yeasts on glass beads, for detection of middle-chain alkanes (Alkasrawi et al. 1999), giving stable response for more than 17 days. The method is simple and produces a stable biofilm with no cell leakage and no detectable effect on the viability of the cells. PEI attachment was also applied to the development of a microbial biosensor, detecting phenolic compounds (Nandakumar and Mattiasson 1999b).

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Receptors Cells can be immobilized on a support by receptor-mediated specific adhesion. The main advantages of this technique are the specificity and the reversibility of the binding for regeneration of the support. Several non-covalent adhesions such as biotin-avidin and antibody-antigen have been reported for immobilization of proteins. Immobilization of biotinylated bacteria onto biotin/avidin modified electrodes (Da Silva et al. 2004) proved to preserve bacterial activity, in spite of the strong linkage constraining bacterial cell division. Another interesting attachment method employs antibodies: non-specific anti-Escherichia coli antibodies are glutaraldehyde-anchored on aminosylilated gold or silica glass surfaces and bacteria are subsequently attached. This method was used for several Escherichia coli strains, expressing luciferase in response to different physiological stress conditions including heat shock, DNA damage, fatty acid availability, peroxide and oxidative stress. 5 months storage stability and continuous operation were obtained with this configuration, together with sensitivity and detection limits comparable to that of non-immobilized bacteria (Premkumar et al. 2001a). Nonetheless, as these supports are relatively expensive, their commercial application for cell immobilization may be economically infeasible. Finally, an interesting technique was proposed by Wang and co-workers (Wang et al. 2001): recombinant cells with surface-expressed cellulose-binding domain were bound tightly and rapidly to cellulose fibers, thus providing a very simple and economical way of wholecell immobilization. The main advantages of adhesion are the simple procedure, the absence of diffusion gradient; on the other hand, the drawbacks are the harsh conditions for cell immobilization, with consequent low biocompatibility and the difficult regeneration of used biosensor. No systematic characterisation of these immobilization techniques is available in literature; storage, operational stability and cell survival have rarely been monitored for microbial biosensors based on adhesion. 4.1.b. Adsorption Adsorption techniques are commonly used to retain viable cells directly on a porous support. Immobilization procedure is extremely simple and it does not require skilled personnel; it is generally performed in mild conditions, in order to preserve bacterial activity. Nevertheless, this sort of immobilization is relatively unstable, affected by pH, temperature and ionic strength of the medium, so that cell leaching may occur. Moreover as cell distribution is not controlled, a poor reproducibility and diffusion limitation through the cell layer may be observed. The support may be of different nature, such as polymeric membranes or porous carriers (Ball 1999). Membranes Membranes are loaded with cells simply by suction or soaking. Once loaded with bacteria, membranes are dried and generally mounted directly on the transducer, then covered with resistant membranes, having a low pore size (0.1-1μm), to avoid cell leaching and to ensure sterile conditions, yet allowing oxygen diffusion (Liu et al. 2000). The different sorts of membranes already employed for microbial biosensors are listed in table 4, where their operational stability and storage time are also specified. Operational stability could be enhanced with substrate injections, in order to regenerate bacterial activity (Heim et al. 1999).

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Table 4. Summary of the performances of different membranes for microbial immobilization in biosensors. As applications and operating conditions were different, only operational stability and storage could be compared Membrane

Microorganism

Transducer

Filter paper

Bacillus from soil

Potentiometric

Rhodococcus erythropolis and Issatchenkia orientalis

Electrochemical

Bacillus subtilis

Clark electrode

Cellulose acetate

Polycarbonate

Nitrate cellulose

Nylon

Operational stability

Reference

1 month after drying

Verma and Singh 2003

14 days continuous

Heim et al. 1999

5 days at 25°C

Fleschin et al. 1998

at 4°C (not studied)

Mulchandani et al. 2002 Ikarriama et al. 1997

five months

Chee et al. 1999

Moraxella sp.

Clark electrode

Escherichia coli Pseudomonas putida Saccharomyces ellipsoideus

Fiber optic

14 days at 4°C 20 measurements Non reusable

Clark electrode

10 days

Potentiometric oxygen electrode

4-5 days at 4°C 20-25 measurements

Mixed culture from a degradation reactor

Storage

200 measurements

Rotariu et al. 2002; Rotariu et al. 2004 180 days in buffer at 4°C

Rastogi et al. 2003a; Rastogi et al. 2003b;

Clark electrode

Chromatographic paper

Gluconobacter oxydans

Clark electrode

Teflon membrane

Pseudomonas aeruginosa

Chloride electrode

6 days

Håkansson and Mattiasson 2004

8 days at 20°C – 25 measurements

Reshetilov et al. 1998a; Reshetilov et al. 1997; Reshetilov et al. 1998b; Reshetilov et al. 2001

35 days storage at 4°C and –10°C

Han et al. 2002

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Porous Carrier Porous carriers must be loaded with bacterial suspension in order to encourage cell growth: loading procedure may take from a few hours to some days, culture medium is then removed and particles are rinsed. Porous dimethyl silicone discs were employed to immobilize Rhodococcus ruber whole cells for the detection and quantification of acrylonitrile (Roach et al. 2003). The supports were cultured with cells for two weeks, then stored at 4°C up to six weeks. The biosensor could be used for 5 days, after which it was not re-usable. Porous borosilicate glass beads and silicon foam beads were employed to immobilize Rhodococcus erythropolis and Issatchenkia orientalis for a column-reactor FIA biosensor (Heim et al. 1999). One night culture was sufficient to load the beads, but the column-reactor configuration did not prove suitable for reliable analysis, both for washing-out problems and for pressure drop. An innovative technique is the preparation of a sort of sandwich: bacteria are placed on glassy carbon electrodes, then coated with a cellulose acetate membrane, having a remarkable size exclusion effect and enhancing the selectivity (Tkac et al. 2002; Tkac et al. 2003). The electrodes thus prepared, stored at 20°C, could be used for six days.

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4.2. Entrapment Cell entrapment consists in inclusion within a matrix. This technique offers the best potentialities in terms of stability, biocompatibility, mechanical and chemical resistance, cell leaching, transparency and reproducibility (Tkac et al. 2002). The major drawback of entrapment is the low diffusivity of substrates and analytes through the matrix, which can be minimized by increasing the porosity or minimizing the membrane thickness. The entrapment materials already used for microbial biosensors are: hydrogel, sol-gel, latex, carbon paste, which will be briefly illustrated in the following section. A few papers have been published on the comparison of different matrices for microbial biosensors (Leth et al., 2002; Semenchuk et al., 2000). However, it is possible to compare different experimental works carried out with heterogeneous conditions, in order to summarize the applications of the entrapment matrices and their performances, as it is shown in table 7.

4.2.a. Hydrogel Hydrogels form a specific class of polymeric biomaterials. They can be defined as twoor multi-component systems consisting of a three-dimensional network of polymer chains and water that fills the space between macromolecules. Generally in the swollen state the mass fraction of water in a hydrogel is much higher than the mass fraction of polymer. As hydrogels must be able to hold, in equilibrium, certain amounts of water, the polymers used must have at least moderate hydrophilic character. This material proves suitable to cell immobilization, due to its biocompatibility. Hydrogels may be natural, mainly polysaccharides, or synthetic (Leenen et al. 1996). Numerous studies have been carried out with several hydrogels for immobilized microbial biosensors. Some of the most recent ones will be herein presented, with the purpose of providing a view on the multiple possibilities.

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Natural Gels Natural polysaccharides, such as alginate, k-carrageenan, chitosan and agarose, extracted from algae or seaweed, jellify in mild conditions, guaranteeing a high viability, nevertheless, mechanical properties and stability are generally poor. The structures of some of them are shown in figure 5. Table 5. Schematic structures of natural gels: alginate, agarose and k-carrageenan Alginate

Agarose

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k-carrageenan

Alginate This polysaccharide, as it is shown in table 5, contains free carboxyl groups, which can bind Ca2+ or other cations to form the typical egg-box structure. It is widely employed in immobilization cell technology (Orive et al. 2003), because of its simple jelling procedure and biocompatibility. Therefore it has always been employed to immobilize bacteria for biosensors (Corbisier et al. 1999; Davidov et al. 2000; Heitzer et al. 1994; Koler et al. 2000; Schmidt et al. 1996; Semenchuck et al. 2000). Membranes are simply formed by immersing supports covered with alginate solution containing cells in CaCl2 bath; multiple layers of immobilized bacteria can be formed directly on the electrode probe (Peter et al. 1996; Polyak et al. 2001) or on the bottom of microtiter plate wells (Koler et al. 2000). Calcium alginate gel may be dissolved by sodium citrate in order to release the cells for counting (though the yield of extraction does not reach 100%). Some studies showed that alginate solution containing cells can be stored at 4°C up to 10 days and then employed for biosensor immobilization, without significant activity loss (Schmidt et al. 1996). Alginate-coated probes can be stored for one week at room temperature, after which they must be re-hydrated for 2 hours in calcium nitrate to recover bacterial activity (Peter et al. 1996). Alginate gel shows a poor stability: calcium must be added to the medium, otherwise the matrix may easily swell and deteriorate, due to the weak polyelectrolytic bounds (Schmidt et al. 1996) or the presence of phosphate in the liquid medium. Heitzer et al. (1994) showed that after four days, membranes release bacteria, due to cell growth, and after nine days a biofilm is formed on the gel surface. Additionally, cations and heavy metals may bind to the free carboxyl groups (Jeon et al. 2002; Nurbas et al. 2002; Stoll and Duncan 1997), thus reducing the sensibility and reliability of the sensor. Mechanical resistance and stability may be improved including alginate beads in synthetic membranes (Simpson et al. 2000b). An alternative is to use chemically modified alginate: bacteria can be encapsulated in biotinalginate micro-spheres, then conjugated to streptavidin-coated optical fibers (Polyak et al. 2004); this technique allowed the encapsulation of different bacteria strains for multipurpose biosensor.

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K-Carrageenan This is another polysaccharide extracted from seaweed, containing both free carboxyl groups and charged sulfate groups; jelling involves helix formation on cooling from a hot solution and binding with gel-inducing K+ and gel-strengthening Ca2+. k-carrageenan is currently employed for cell entrapment, also for biosensors (Cassidy et al. 1997). Escherichia coli cells were immobilized in k-carrageenan on screen printed electrodes for detection of mono- and di- saccharides, with good results: cells retained their activity for at least six days (Held et al. 2002). Peter et al. (1996) found similar specific activity for Rhodococcus sp. immobilized in k-carrageenan, alginate and agarose gel discs. Agar This polymer consists of a mixture of agarose and agaropectin and it is widely employed, mixed with culture medium, as growth support, as it is not easy for microorganisms to metabolize. Cells can be immobilized in agar gel containing culture medium (agarised medium), the disks thus obtained are fixed between a nylon net and a gas permeable membrane; the sandwich thus obtained is placed on the electrode by means of an O-ring. This technique has been employed to immobilize Saccharomyces cerevisiae for the detection of cholanic acids (Campanella et al. 1996). A biosensor for detection of gas toxicity was developed with a recombinant Escherichia coli immobilized in agar medium, then connected to the end side of a fiber optic light probe (Gil et al. 2000). The membranes thus prepared could be stored at 4°C up to 20 days, without affecting their sensitivity. However diffusivity through the matrix could be enhanced by mixing glass beads to agar containing cells, in order to increase the porosity (Gil et al. 2002; Lee et al. 2003). Agar gel was employed to prepare films containing different Pseudomonas and Achromobacter strains for surfactant detection attaining an operational stability up to 6 days (Taranova et al. 2002). Cells immobilized in agarised medium may slowly grow and reach the death phase, which affects the biosensor operational stability and the possibility to re-use the membranes, with consequent cost increase. Agarose This is a linear disaccharide, a component of agar; once it is hardened, the gel network contains double helices, stabilized by the presence of water molecules bound inside the double helical cavity. It must be dissolved in water at 70 °C, then it hardens by cooling, within the range of temperature 40-20°C, resulting in transparent, firm and stable gels. It shows a high biocompatibility, transparency and good stability properties (Belmont-Hebert et al. 1998). Though agar was already often employed for biosensor immobilization, agarose seems to be more promising, as its purity guarantees a transparent gel and lower melting temperatures. Studies on microbial biosensor for heavy metal detection showed that bacteria immobilized in agarose emitted light signal twofold higher that the ones in alginate (Leth et al. 2002), probably due to metal adsorption in the latter. Agarose has good diffusion properties, as proved in some experimental studies (Tercier and Buffle 1996); moreover, being electrochemically neutral, heavy metals are generally not bound in agarose network. Some other studies employed pure low melting agarose to immobilize luminescent bacteria for pollutant detection (Corbisier et al. 1999; Pernetti et al. 2003; Semenchuck et al. 2000, Horry et al, 2007).

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Some biosensors were also realized immobilizing bacteria in other natural hydrogels, such as chitosan (Peter et al. 1997) and pectate gel (Svitel et al. 1997). The latter requires a higher polymer concentration for gelling, thus causing viscosity problems and complex procedures.

4.2.B. Synthetic Gels Synthetic matrices generally show a remarkable mechanical and chemical stability, though a high viability loss, due to the harsh conditions and aggressive reagents required for gelling or polymerisation. Although many synthetic gels are used for cell encapsulation, few of them are currently employed for microbial biosensors. The most employed are illustrated in table 6 and in the following section.

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Table 6. Schematic structures of PolyVinyl alcohol (PVA), Polyacrylamide, Polyurethane (PUR) and Poly (carbamoyl)sulfonate (PCS). PVA

Polyacrylamide

PUR

PCS

Polyvinyl Alcohol (PVA) Polyvinyl alcohol is a white soluble high-molecular compound, not soluble in ordinary organic solvents. It shows excellent tensile stress, mouldability, impact strength, wear resistance and excellent electrical insulation. Generally used as raw material for coating and adhesives, PVA shows interesting features for cell immobilization, such as biocompatibility, depending on the procedure employed, high diffusivity and good mechanical resistance (Lozinsky and Plieva 1998). PVA containing bacteria may gel through different procedures (Hall and Mc Loughlin 2000): cross-linking with boric acid (Semenchuck et al. 2000; Wu and Wisecarver 1991), repetitive freezing and thawing (Lozinsky and Plieva 1998), phosphorilization (Chen and Houng 1997; Cheng 1998), drying (Horsburgh et al. 2002), \gamma -irradiation, photo-cross-linking (Rouillon et al. 1999). Freeze-thawing is a very

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simple procedure and it proved efficient for bacteria immobilization for organo-phosphorus neurotoxins detection (Rainina et al. 1996) and toxicity assessment in wastewater (Philp et al. 2003): cell pellet and PVA solution are mixed and then frozen at 20°C and thawed at room temperature, in order to obtain transparent and elastic hydrogel. Besides its mechanical resistance, its stability over wide ranges of pH, temperature, ionic strength and salinity, PVA cryogels are macro-porous, thus increasing its diffusivity to analytes (Rainina et al. 1996). PVA was also employed for a BOD sensor coupled with an enzymatic hydrolysis system (Tag et al. 2000). Chips can be prepared by pouring a mixture of cell suspension and PVA in a silicon wafer, then covered with an epoxy resin (Konig et al. 2000); biosensors thus prepared could be used for 40 days, while shelf life was six months. Nevertheless, after storage, incubation time required before measurements increased from 4-15 hours to 50 hours. A modified polyvinyl alcohol, which allows immobilization in lens-shaped discs at room temperature, has already been commercialized (Jekel et al. 1998) and it is applied to BOD biosensors (Pasco et al. 2004), though a high loss in signal emission is observed.

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Polyurethane (PUR) Polyurethanes are a common commercial polymer, and can possess amazingly diverse properties depending on how they are manufactured: foam rubber, elastic fiber, resilient plastics, or hard coatings. The hydrogel is prepared by cross-linking and curing a terminal isocyanate-containing polyurethane resin in the presence of water. This kind of hydrogel shows high mechanical stability, but the monomers are toxic, causing activity losses up to 6070% (Sumino et al. 1992). Poly (Carbamoyl)Sulfonate (PCS) The use of PCS for cell immobilization was introduced by Vorlop et al. (1992) and Willke et al. (1994). PCS is prepared from isocyanate terminated polyurethane (PUR) prepolymer with bisulphite. Then pH may be adjusted with polyethyleneimine (PEI) and cells can be added. The polymerization procedure is less toxic than the one used for PUR, resulting in higher biocompatibility. The hydrogel membranes are easily formed at room temperature within 24 hours. Muscat and Vorlop (1997) found that the diffusivity of some organic compounds through PCS and alginate membranes were in the same range; moreover cellular activity in PCS was as good as alginate, whereas no viability was observed in PUR. Nitrifiers bacteria were immobilized in PCS and fixed on Clark electrode in order to detect inhibitors in wastewater (Konig et al. 1998): immobilized bacteria could be stored up to three weeks in continuous mode, though a biofilm of heterotrophic bacteria was inevitably formed on the surface of the membrane, reducing the biosensor sensitivity. PCS was also employed to immobilize Arxula adeninivorans for BOD measurements (Chan et al. 1999): the biosensor thus realized could be used for 40 days and it could be stored at room temperature for two months. Other biosensors have been recently developed with bacteria in PCS, for online control of wastewater treatment reactors (Jang et al. 2004). Polyacrylamide Polyacrylamide gel is formed when a dissolved mixture of acrylamide and cross-linkers polymerize into long chains that are covalently linked. The gel structure is held together by the cross-linker, such as N, N'-methylene-bisacrylamide. The polymerization of acrylamide gel can be initiated either by a chemical peroxide or by a photochemical method. The pore

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size of the gel may be varied to produce different molecular sieving effects to increase or hinder the diffusion of specific compounds. Polyacrylamide gel offers greater flexibility than agarose gels. Nevertheless, since the polymerization procedure is extremely complex and the monomer and the cross-linker are toxic, polyacrylamide immobilization may be fatal to cell survival. Rhodococcus sp. immobilized into polyacrylamide for detection of chlorinated and brominated hydrocarbons showed viability losses up to 90% (Peter et al. 1996). Polyacrylamide may be successfully used to immobilize non-viable cells, as gamma -ray polymerization can be carried out in frozen conditions and brings to a highly porous matrix (D'Souza 2001a). The immobilization procedures of some of the most common hydrogels are shown in table 7 (Pernetti, 2004).

Sol-Gel Sol-gel technique includes hydrolysis and condensation of metal alkoxides in adequate solvents followed by thermal treatment. The biological element to be encapsulated is added to sol after partial hydrolysis of the alkoxides. Sol-gel is an interesting inorganic-organic hybrid, presenting the rigidity and transparency of silicate caging. This technique has been already applied to cell immobilization (Fennouh et al. 1999; Gill and Ballesteros 2000; Jia et al. 2003). Sol-gel mixture is prepared with tetramethylorthosilicate, distilled water and HCl, it is sonicated, aged at 4°C and mixed with cell suspension. The mixture is coated on glass slides previously cleaned and dried. The slides are then dried, washed and stored in buffer solution (Premkumar et al. 2002). This system results biocompatible, even if a slight cell inactivation was observed, probably due to the drying step or the jelling (Premkumar et al. 2001b) or alcohol concentration. Sol-gel entrapment results interesting for the development of bioreporter integrated circuits, as bacteria may be directly immobilized in thin and stable layers on microchips (Simpson et al. 2000a). A study has been recently carried out, immobilizing five bioluminescent Escherichia coli strains detecting different compounds, in order to test the suitability of this technique to biosensor development (Premkumar et al. 2002). Specific luminescence responses of free and immobilized cells were quite similar for membrane thickness in the range of 100-1000µm. The biosensor thus prepared could be reused for multiple measurements after rinsing and shelf-life attained three months. However, sol-gel technique is laborious and complex and bacterial activity after immobilization should be further investigated. Latex Latex is a suspension of an insoluble substance, like acrylic vinyl acetate, wrapped in another kind of molecule, like a copolymer. This emulsion co-polymerization has been widely used in the industry for production of polymeric materials with tailored properties. Interesting studies were carried out on Escherichia coli immobilized in latex copolymer films (Lyngberg et al. 1999b). Bacteria are immobilized in commercial copolymer latex (Rovace SF091, Rohm and Hass), then coated with polyester film and with another latex topcoat. Patches thus prepared were dried and cured, resulting in strong, transparent and flexible membranes. This technique was employed for a single-use bioluminescent biosensor for mercury detection (Lyngberg et al. 1999a).

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Table 7. Immobilization procedure of some hydrogels employed for microbial biosensors as realised by Pernetti (Pernetti, 2004) Hydrogel

Immobilization procedure

Alginate

- cast the 2% polymer solution + bacteria in moulds or in microtiter plates - immerse the moulds in 1% CaCl2 solution (15 min) or add CaCl2 solution to plates (30 min)

Agarose

- dissolve 2% w/w in water at 70°C - add bacteria at 30°C - cast the polymer solution in moulds or in microtiter plates - leave hardening at room temperature (10 min)

PVA by freezing

- cast the 10% polymer solution + bacteria + 1% glycerol in moulds or in microtiter plates - seal moulds and microplates - store in freezer at -20°C (16h) - thaw in refrigerator at 4°C (30 min) - acclimate in incubator at 30°C (30 min)

Picture

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Table 7. (Continued). PVA by cross-linking

add MgCl2 concentration ?to PVA solution (2%w/w) stir by helix at 200 rpm (avoiding bubbles) add glutaraldheyde solution (concentration under hood (total 0.01%) stir for 1.5h cast the polymer solution + bacteria + glycerol 1% in moulds or in microtiter plates dry between 25-35°C, in oven or air flow stop drying when 85% of water is evaporated rehydrate membranes in distilled water to attain original weight

Silicone (7-9800 Dow Corning Chemicals, platinum catalysed silicone elastomer)

mix the two solutions A, B at 1:1 w:w ratio with double helix for 1.5h for pre-polymerization reaction add agarose beads with bacteria or glycerol + bacteria cast the polymer solution in moulds or in microtiter plates leave drying for 2h

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Table 8. Summary of the different entrapment matrices employed for different applications; operational stability and storage are also shown Matrix

Microorganism

Transducer

Pseudomonas fluorescens

Photomultiplier

Arthrobacter nicotianae

O2 electrode

Escherichia coli

Luminometer

Escherichia coli Pseudomonas and Achromobacter Recombinant strains

O2 electrode O2 electrode

Escherichia coli

Luminometer

Alcaligenes eutrophus

Luminometer

Deinococcus radiodurans

O2 electrode

Pseudomonas rathonis T

O2 electrode

Photobacterium (Vibrio) fischeri and Pseudomonas strain

Luminometer

Escherichia coli

pH electrode

PCS

Arxula adeninivorans

O2 electrode

Sol-gel

Escherichia coli

Luminometer

Latex

Escherichia coli

Luminometer

Carbon paste

Moraxella sp

Electrode

Alginate

k-carrageenan

Agar

Agarose

PVA

Luminometer

Operational stability < 4 days

Storage

Heitzer et al. 1994 7 days in buffer + CaCl2 6 days at 4°C in NaCl

5 days 6 days in buffer at room temp 2 weeks at 4°C

< 6 days

Schmidt et al. 1996 Corbisier et al. 1999 Held et al. 2002 Taranova et al. 2002

> 24 hours at 4°C 3 weeks at 4°C in the dark 2 weeks at 4°C in NaCl 45 days in buffer

12 days

Reference

25 days at 4°C, 100% umidity

Kim and Gu 2003 Schreiter et al. 2001 Leth et al. 2002 Nandakumar and Mattiasson 1999 Semenchuck et al. 2000 Philp et al. 2003

40 days

60 days at 4°C in buffer 2 months at room temp 3 months at 4°C in medium 3 months at –20°C 14 days in a desiccator 45 days in buffer solution at 4°C

Rainina et al. 1996 Chan et al. 1999; Jang et al. 2004 Premkumar et al. 2002; Lyngberg et al. 2001 Mulchandani et al. 2001

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Table 8. (Continued). Matrix

Microorganism

Transducer

Sol-gel

Escherichia coli

Luminometer

Latex

Escherichia coli

Luminometer

Carbon paste

Moraxella sp

Electrode

Operational stability

Storage

Reference

3 months at 4°C in medium 3 months at –20°C 14 days in a desiccator 45 days in buffer solution at 4°C

Premkumar et al. 2002; Lyngberg et al. 2001 Mulchandani et al. 2001

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Table 9. Screening-of-properties of attachment and adsorption. Screening of properties shown by microbial biosensors based on attachment and adsorption: + is a positive effect (++ extremely good), - negative effect; (*) indicates that the value depends on the preparation procedure employed for that material; (**) depends on the support employed. The missing values were not available or the property could not be applied to the support Attachment Property Biocompatibility Cell activity

-

Mechanical resistance

-

Adsorption Membrane

Porous carrier

+

+

Chemical resistance Biological resistance

+

-

+

Diffusion

++

++

++

Operational stability

+

+

+

Storage stability

+

+

+

Sterility – no cell leaching

-

-

-

Procedure Simplicity

+

++

++

Cost

+

+

+

Transparency

-

-

-

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Table 9. (Continued). Attachment Property

Bibliographical references

Alkasrawi et al. 1999; Da Silva et al. 2004; Medina 2002; Medina 2004 ; Melo and D'Souza 1999; Mulchandani and Rogers 1998 ; Svitel et al., 1998; Timur et al. 2003 .

Adsorption Membrane Chee et al. 1999 ; Fleschin et al. 1998 ; Håkansson and Mattiasson 2004 . Heim et al. 1999 ; Mulchandani et al. 2002 02; Rastogi et al. 2003a; Rastogi et al. 2003b; Reshetilov et al. 1998a; Reshetilov et al. 1997; Reshetilov et al. 1998b; Reshetilov et al. 2001 ; Rotariu et al. 2002; Rotariu et al. 2004 ; Verma and Singh 2003

Porous carrier

Heim et al. 1999 ; Roach et al. 2003 ; Tkac et al. 2002; Tkac et al. 2003

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Table 10. Screening of properties showed by microbial biosensors based on gel entrapment: + is a positive effect (++ extremely good), - negative effect (--extremely bad); (*) indicates that the value depends on the preparation procedure employed for that material. The missing values were not available or the property could not be applied to the support Entrapment Natural gels Alginate

k-carrageenan

Agarose

Synthetic gels PCS

PVA

Sol-gel

Latex

++

++

++

+

+ (*)

+

+

Mechanical resistance

--

--

+

+

++

+

+

Chemical resistance

-

-

+

+

++

++

+

Biological resistance

--

--

-

+

+

+

+

Diffusion

+

+

+

+

+

Operational stability

+

+

+

+

++

++

++

Storage stability

-

-

-

+

+

+

+

Sterility – no cell leaching

--

--

-

+

+

+

-

Procedure Simplicity

++

++

++

-

+

--

--

Cost

++

++

++

-

+

-

-

Transparency

+

+

++

++

++

+

Property Biocompatibility Cell activity

+

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Table 10. (Continued).

Property

Bibliographical references

Entrapment Natural gels Alginate Cassidy et al. 1997; Corbisier et al. 1999; Davidov et al. 2000; Elasri and Miller 1999; Heitzer et al. 1994 Koler et al. 2000; Semenchuck et al. 2000

k-carrageenan Cassidy et al. 1997; Held et al. 2002; Peter et al. 1996

Agarose

Mulchandani et al. 1998 Semenchuck et al. 2000; Corbisier et al. 1999

Synthetic gels PCS

Chan et al. 1999 Jang et al. 2004 Konig et al. 1998

PVA

Sol-gel

Latex

Horsburgh et al. 2002; Philp et al. 2003; Rainina et al. 1996; Semenchuck et al. 2000; Simonian et al. 1992

Jia et al. 2003; Premkumar et al. 2001b; Premkumar et al. 2002; Ripp et al. 2002; Simpson et al. 2000a

Lyngberg et al. 1999a; Ripp et al. 2002

116

Properties and Choice of Material Used for Microbial Biosensor

Despite the presence of a biocide in the latex solution, luminescence levels attained by immobilized bacteria were similar to the ones due to free cells. Patches could be stored up to 3 months at 20°C and 14 days in a desiccator, retaining their detection activity. Nevertheless, compared to suspended bacteria, immobilized bacteria required a response 10 times slower and a different detection range. This may be due to diffusion limitations and to interactions between the analyte and the matrix; permeability may be enhanced by addition of glycerol or sucrose to latex (Lyngberg et al. 2001).

Carbon Paste Carbon paste is commonly used to prepare working electrodes, though more "convenient" and stable carbon-based electrodes are now developed, including those made from glassy carbon, pyrolytic carbon and porous graphite. As a matter of facts, carbon paste shows lack of mechanical and physical stability under hydrodynamic conditions and poor chemical stability, since they are dissolved by some non-aqueous solvents. Carbon paste electrodes were prepared by manually pressing the carbon paste (90% w/w graphite and 10% w/w paraffin) and the surface is smoothed by a weight paper. Cell suspension is mixed with carbon paste, in order to fix the sensing element directly on the electrode. This method is particularly interesting for recombinant microorganism with surface expressed enzymes, as sensing activity is not inhibited by immobilization procedure. For example, Moraxella sp. was mixed with carbon paste containing graphite powder and mineral oil, then packed firmly into the electrode cavity in order to form the working electrode (Mulchandani et al. 2001), which could be repeatedly used for measurements and stored up to 45 days in buffer solution at 4°C.

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5. CONCLUSIONS Microbial biosensors are promising tools for monitoring and detection in different fields. Cells immobilization is unquestionably essential for further development and commercialisation. The main immobilization methods already employed for microbial biosensors especially in the last decade were herein briefly illustrated, together with their advantages and drawbacks. In order to select the right immobilization method for the biosensor to develop, it is necessary to perform a screening and then to test some of them on the basis of the most important properties required. A systematic characterization is herein proposed and all the immobilization methods previously presented are consequently resumed and evaluated using these criteria. Entrapment methods result to the most reproducible, as cell distribution may be more easily controlled; moreover they provide a real restriction of cell migration, reducing cell leaching or washing out. On the other hand, they may show diffusion limitations, which can be minimized reducing the thickness of the support or enhancing its diffusivity. Among the different matrices, synthetic gels, such as PVA, are the most promising, though they require complex procedures, not always biocompatible. Agarose is the most stable natural hydrogel, showing good mechanical resistance and no swelling. An interesting compromise would be the combination of natural and synthetic gels, such as a natural core with synthetic coat, in order to combine the biocompatibility with a good stability and mechanical resistance. Several matrices have already been used in several biosensors: a summary is given in table 8.

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The immobilization method should always be accurately studied in order to verify its suitability to the biosensor before carrying out the experiments. Though literature data are heterogeneous and difficult to compare, tables 9 and 10 propose a "qualitative" screening of most of the immobilization techniques recently employed for microbial biosensors, evaluated through the criteria previously provided. This would be the base for the further experimental characterization. Some bibliographical references are provided to find further information on each immobilization method.

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Polyak B, Geresh S and Marks RS (2004) Synthesis and Characterization of a Biotin-Alginate Conjugate and Its Application in a Biosensor Construction. Biomacromolecules. 5: 389396 Premkumar JR, Lev O, Marks RS, Polyak B, Rosen R and Belkin S (2001a) Antibody-Based Immobilization of Bioluminescent Bacterial Sensor Cells. Talanta. 55: 1029-1038 Premkumar JR, Lev O, Rose R and Belkin S (2001b) Encapsulation of Luminous Recombinant E.coli in Sol-Gel Silicate Films. Advanced Materials. 13: 1773-1775 Premkumar JR, Rose R, Belkin S and Lev O (2002) Sol-Gel Luminescence Biosensors: Encapsulation of Recombinant E.coli Reporters in Thick Silicate Films. Analytica Chimica Acta. 462: 11-23 Rainina EI, Efremenco EN, Varfolomeyev SD, Simonian AL and Wild JR (1996) The Development of a New Biosensor Based on Recombinant E.coli for the Direct Detection of Organophosphorus Neurotoxins. Biosensors and Bioelectronics. 11: 991-1000 Rastogi S, Kumar A, Mehra NK, Makhijani SD, Manoharan A, Gangal V and Kumar R (2003a) Development and Characterization of a Novel Immobilized Microbial Membrane for Rapid Determination of Biochemical Oxygen Demand Load in Industrial WasteWaters. Biosensors and Bioelectronics. 18: 23-29 Rastogi S, Rathee P, Saxena TK, Mehra NK and Kumar R (2003b) Bod Analysis of Industrial Effluents: 5 Days to 5 Min. Current Applied Physics. 3: 191-194 Rensing C and Maier RM (2003) Issues Underlying Use of Biosensors to Measure Metal Bioavailability. Ecotoxicology and Environmental Safety. 56: 140-147 Reshetilov AN, Iliasov PV, Donova MV, Dovbnya DV, Boronin AM, Leathers TD and Greene RV (1997) Evaluation of a Gluconobacter Oxydans Whole Cell Biosensor for Amperometric Detection of Xylose. Biosensors and Bioelectronics. 12: 241-247 Reshetilov AN, Efremov DA, Iliasov PV, Boronin AM, Kukushskin NI, Greene RV and Leathers TD (1998a) Effects of High Oxygen Concentrations on Microbial Biosensor Signals. Hyperoxygenation by Means of Perfluorodecalin. Biosensors and Bioelectronics. 13: 795-799 Reshetilov AN, Lobanov AV, Morozova NO, Gordon SH, Greene RV and Leathers TD (1998b) Detection of Ethanol in a Two-Component Glucose/Ethanol Mixture Using a Nonselective Microbial Sensor and a Glucose Enzyme Electrode. Biosensors and Bioelectronics. 13: 787-793 Reshetilov AN, Trotsenko JA, Morozova NO, Iliasov PV and Ashin VV (2001) Characteristics of Gluconobacter Oxydans B-1280 and Pichia Methanolica Mn4 Cell Based Biosensors for Detection of Ethanol. Process Biochemistry. 36: 1015-1020 Ripp S, Sayler GS and Sanseverino J (2002) Bioluminescent Methods for Direct Visual Detection of Environmental Compounds. Patent: CA2419481. Roach PCJ, Ramsden DK, Hughes J and Williams P (2003) Development of a Conductimetric Biosensor Using Immobilised Rhodococcus Ruber Whole Cells for the Detection and Quantification of Acrylonitrile. Biosensors and Bioelectronics: 19: 73-78 Roda A, Pasini P, Mirasoli M, Michelini E and Guardigli M (2004) Biotechnological Applications of Bioluminescence and Chemiluminescence. Trends in Biotechnology. 22: 295-303 Rogers KR and Gerlach CL (1999) Update on Environmental Biosensors. Environmental Science and Technology. 33: 500A-506A

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Rotariu L, Bala C and Magearu V (2002) Yeast Cells Sucrose Biosensor Based on a Potentiometric Oxygen Electrode. Analytica Chimica Acta. 458: 215-222 Rotariu L, Bala C and Magearu V (2004) New Potentiometric Microbial Biosensor for Ethanol Determination in Alcoholic Beverages. Analytica Chimica Acta. 513: 119-123 Rouillon R, Tocabens M and Carpentier R (1999) A Photoelectrochemical Cell for Detecting Pollutant-Induced Effects on the Activity of Immobilized Cyanobacterium Synechoccus Sp. Pcc 7942. Enzyme and Microbial Technology. 14: 230-235 Schmidt A, Standfuss-Gabisch C and Bilitewski U (1996) Microbial Biosensor for Free Fatty Acids Using an Oxygen Electrode Based on Thick Film Technology. Biosensors and Bioelectronics. 11: 1139-1145 Schreiter PPY, Gillor O, Post A, Belkin S, Schmid RD and Bachmann TT (2001) Monitoring of Phosphorus Bioavailability in Water by an Immobilized Luminescent Cyanobacterial Reporter Strain. Biosensors and Bioelectronics. 16: 811-818 Seki A, Kawakubo K, Iga M and Nomura S (2003) Microbial Assay for Tryptophan Using Silicon-Based Transducer. Sensor and Actuators. B 94: 253-256 Semenchuck IN, Taranova LA, Kalenyuk AA, Il'yasov PV and Rechetilov AN (2000) Effect of Various Methods of Immobilization on the Stability of a Microbial Biosensor for Surfactants Based on Pseudomonas Rathonis T. Applied Biochemistry and Microbiology. 36: 69-72 Simonian AL, Rainina EI, Lozinsky VI, Badalian IE, Khachatrian GE, Tatikian SS, Makhkis TA and Varfolomeyev SD (1992) A Biosensor for Pl-Proline Determination by Use of Immobilized Microbial Cells. Applied Biochemistry and Biotechnology. 36: 199-210 Simpson ML, Sayler GS, Nivens DE, Dionisi HM, Ripp S, Applegate BM, Bolton E, McKnight T and Rochelle J (2000a) Bioluminescent Bioreporter Integrated Circuits (Bbics): Whole-Cell Environmental Monitoring Devices 30th International Conference on Environmental Systems (Society of Automotive Engineers). Simpson ML, Sayler GS and Paulus MJ (2000b) Bioluminescent Bioreporter Integrated Circuit. US Patent US6117643. Smith JJ and McFeters GA (1997) Mechanisms of Int and Ctc Reduction in E.coli K-12. J. Microbiological Methods. 29: 161-175 Stoll A and Duncan JR (1997) Comparison of the Heavy Metal Sorptive Properties of Three Types of Immobilized, Non-Viable Saccharomyces Cerevisiae Biomass. Process Biochemistry. 32: 467-472 Sumino T, Nakamura H, Mori N, Kawaguchi Y and Tada M (1992) Immobilization of Nitrifying Bacteria in Porous Pellets of Urethane Gel for Removal of Ammonium Nitrogen from Wastewater. Applied Microbiology and Biotechnology. 36: 556-560 Svitel J, Curilla O and Tkac J (1998) Microbial Cell-Based Biosensor for Sensing Glucose, Sucrose or Lactose. Biotechnology Applied Biochemistry. 27: 153-158 Svitel J, Vostiar I, Gemeiner P and Danielsson B (1997) Determination of Citrate by Fia Using Immobilized Enterobacter Aerogenes Cells and Enzyme Thermistor/Flow Microcalorimeter Detection. Biotechnology Techniques. 11: 917-919 Tag K, Kwong A, W.K., Lehmann M, Chan C, Renneberg R, Riedel K and Kunze G (2000) Fast Detection of High Molecular Weight Substances in Wastewater Based on an Enzymatic Hydrolysis Combined with the Arxula BOD Sensor System. Journal of Chemical Technology and Biotechnology. 75: 1080-1082

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Taranova L, Semenchuk I, Manolov T, Iliasov P and Reshetilov A (2002) Bacteria-Degraders as the Base of an Amperometric Biosensor for Detection of Anionic Surfactants. Biosensors and Bioelectronics. 17: 635-640 Tercier M-L and Buffle J (1996) Antifouling Membrane-Covered Voltammetric Microsensor for in Situ Measurements in Natural Waters. Analytical Chemistry. 68: 3670-3678 Thouand G, Durand M, Picart P, Massé J and DuBow M (2001) Detection of Bacteria in the Food Industry with Bioluminescent Sensors. Recent Research Developments in Microbiology. 5: 79-93 Timur S, Pazarlioglu N, Pilloton R and Telefoncu A (2003) Detection of Phenolic Compounds by Thick Film Sensors Based on Pseudomonas Putida. Talanta. 61: 87-93 Tkac J, Vostiar I, Gemeiner P and Sturdik E (2002) Monitoring of Ethanol During Fermentation Using a Microbial Biosensor with Enhanced Selectivity. Bioelectrochemistry. 56: 127-129 Tkac J, Vostiar I, Gorton L, Gemeiner P and Sturdik E (2003) Improved Selectivity of Microbial Biosensor Using Membrane Coating. Application to the Analysis of Ethanol During Fermentation. Biosensors and Bioelectronics. 18: 1125-1134 van Dyck TK, DeRose EJ and Gonye GE (2001) Luxarray, a High Density Genomewide Transcription Analysis of Escherichia coli Using Bioluminescent Reporter Strains. Journal of Bacteriology. 183: 5496-5505 Verhaegen K, Simaels J, Driessche WV, Baert K, Sansen W, Puers B, Hermans L and Mertens R (1999) A Biomedical Microphysiometer. Biomedical Microdevices. 2: 93-98 Verma N and Singh M (2003) A Disposable Microbial Based Biosensor for Quality Control in Milk. Biosensors and Bioelectronics. 18: 1219-1224 Vorlop K-D, Muscat A and Beyersdorf J (1992) Entrapment of Microbial Cells within Polyurethane Hydrogel Beads with the Advantage of Low Toxicity. Biotechnology Techniques. 6: 483-488 Walmsley RM and Keenan P (2000) The Eukaryote Alternative: Advantages of Using Yeasts in Place of Bacteria in Microbial Biosensor Development. Biotechnology and Bioprocess Engineering. 5: 387-394 Wang AA, Mulchandani A and Chen W (2001) Whole-Cell Immobilization Using Cell Surface-Exposed Cellulose-Binding Domain. Biotechnology Progress. 17: 407-411 Willke B, Willke T and Vorlop K-D (1994) Poly(Carbamoylsulphonate) as a Matrix for Whole Cell Immobilization - Biological Characterization. Biotechnology Techniques. 8: 623-626 Wu K-Y, A. and Wisecarver KD (1991) Cell Immobilization Using PVA Crosslinked with Boric Acid. Biotechnology and Bioengineering. 39: 447-449 Yamasaki A, Cunha MÂSDA, Oliveira JABP, Duarte AC and Gomes MTSR (2004) Assessment of Copper Toxicity Using an Acoustic Wave Sensor. Biosensors and Bioelectronics. 19: 1203-1208

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In: Biosensors: Properties, Materials and Applications ISBN: 978-1-60741-617-3 Editors: R. Comeaux and P. Novotny © 2009 Nova Science Publishers, Inc.

Chapter 4

NON-CONVENTIONAL STRATEGIES FOR BIOSENSING ELEMENTS IMMOBILIZATION Christophe A. Marquette*, Kévin A. Heyries, Benjamin P. Corgier and Loïc J. Blum Laboratoire de Génie Enzymatique et Biomoléculaire, UMR 5013 EMB2, CNRS -Université Claude Bernard Lyon 1, Bât CPE, 43, bd du 11 novembre 1918, 69622 Villeurbanne, Cedex, France

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1. ABSTRACT The present article draws a general picture of non-conventional methods for biomolecules immobilization. The technologies presented are based either on original solid supports or on innovative immobilization processes. Polydimethylsiloxane elastomer will be presented as a popular immobilization support within the biochip developer community. Electro-addressing of biomolecules at the surface of conducting biochips will appear to be an interesting alternative to immobilization processes based on surface functionalization. Finally, bead-assisted biomolecules immobilization will be presented as an open field of research for biochip developments.

2. INTRODUCTION The development of biochips for the detection of nucleic acids, proteins or enzyme– substrate interactions has benefited from increasing interest over the last decade since those tools were found to provide researchers and diagnostic companies with rapid sample screenings [8, 9]. Nevertheless, one of the main handicaps to the development of these systems appeared to be the lack of generic immobilization procedures of the biologically active compounds, resulting in the obligation to adapt the coating protocols to keep the *

Corresponding author: [email protected]

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immobilized biological molecules active. The early immobilization procedures on silicon and glass supports based on silane technology, still in use for protein and DNA immobilization [912], are now in competition with: (i) the grafting on soft lithography material such as elastomeric polymer, (ii) electrodeposition of biomolecules on conducting surfaces, (iii) micro-contact printing and (iv) beads arraying techniques and particle lithography. The present review will focus on the latest developments and strategies in the field of the biomolecules immobilization for biochips developments. All the methods considered as standard such as photolithography, self assembled monolayers deposition and glass surface functionalization will not be treated in the present paper since numerous reviews were already published on those subjects [13-16]. The presented methods will be the up-to-date and innovative technologies developed for on-chip immobilization of biomolecules.

Figure 1 Schematic representation of the procedure for PDMS biochip surface functionalization with aminobiotin derivatives.

3. BIOMOLECULES IMMOBILISATION ON PDMS BIOCHIPS 3.1. PDMS Properties PDMS (polydimethylsiloxane) is a silicone elastomer widely used for biomedical applications like breast implants, ocular lenses, dentistry [17] and micro- or bio-engineering [18]. Its chemical and physical properties are making the polymer very attractive since it is chemically inert, permeable to gases and few solvents, optically transparent, cheap, not toxic and easy to handle in standard laboratory conditions [19, 20]. Facing the increasing interest in microarrays and microfluidic development [21], PDMS appears more and more to be a powerful material compared to silicon, glass and others immobilization supports [22].

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However, its applications remain limited because of its very hydrophobic behavior (contact angle with water is 117°) [23] and its bio-fouling tendency leading to non specific adsorption of proteins [24]. In order to overcome these problems, several groups currently try to chemically modify PDMS surfaces or turn at their advantage the unique PDMS properties. Based on these surface modification studies, several biochip applications were described. PDMS polymerization is characterized by a hydrosilylation reaction between silane and vinyl residues in the presence of a platinum based catalyst [25] following the reaction : R3SiH + R’3SiCH2=CH2 Î R3SiCH2CH2SiR’3 Once polymerized, PDMS reveals a very hydrophobic surface due to methyl groups beard by the Si atoms of the polymer chain, and this lack of functional groups restrains covalent immobilization of biomolecules. Oxygen plasma treatment is to date the most widespread technique used to convert the methyl groups into hydroxyl ones rendering the surface reactive and hydrophilic [26], but UV-light irradiation (at 184nm and 254nm) [27] or ozone production using a corona discharge have also been used [28]. Nevertheless, this modification is only temporary since un-polymerized buried chain could migrate to the surface, leading to the recovery of the PDMS hydrophobic property [29]. However, a very recent study shown that hydrophobic PDMS recovery could be avoided through washing of the polymerized PDMS with permeable solvents in order to remove the buried unpolymerized chains [30].

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3.2. PDMS Modifications Ozone Treatment for PDMS Surface Functionalization Well established silanisation reaction is commonly used for glass or silicon functionalization [31] and involves Si-OH silanol groups and silanes residues. PDMS oxidation could creates silanol groups and Diaz-Quijada [32] used this property to functionalize PDMS surfaces. They succeeded in oxidizing PDMS with ozone only, without the need of any photolysis. This process allowed a homogeneous surface oxidation avoiding at the same time surface alteration of PDMS. Indeed, it has been shown that UV light, corona discharge and oxygen plasma exposure produce microstructures and crack patterns on PDMS surface. In the Diaz-Quijada study, silanization of the oxidized PDMS with (3aminopropyl)triethoxysilane (APTES) was performed to immobilize single stranded oligonucleotide modified at the 5’-end with an activated carboxylic acid group.

Plasma Oxidation for PDMS Surface Functionalization Jang and co-workers [33] developed an electrochemical immunoassay based on PDMS channels functionalization. In order to activate the inert surface, they exposed PDMS to an air plasma treatment, thus creating silanol groups at the PDMS-air interface. The silanol groups were then reacted with 7-octenyltri(chloro)silane to generate a vinyl residues monolayer (figure 1). An oxidative solution converted the vinyl in carboxylate functions and finally a traditional carbodiimide based coupling chemistry was used to chemically graft an amine containing biotin residue. This procedure allows then the grafting of a far-off biotin on the

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PDMS surface leading to a better recognition by the streptavidin. Finally, avidin was incubated in the channels allowing subsequent grafting of biotinylated mouse type G immunoglobulins (IgG). Eight minutes were necessary to achieve the sampling, reaction with alkaline phosphatase labeled anti-mouse IgG and washing steps, and to achieve a detection limit of 0.02 fmole. However, one has to take in account the number of steps, the time (190 min) and the numerous chemicals needed to achieve such PDMS surface functionalization.

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Figure 2. PDMS activation with thiophosgen for RGD peptide, DNA and PSCA protein immobilization.

PDMS Oxidation by Hydrogen Peroxide In a different approach, Sui and co-workers [34] developed an improved and convenient way to oxidize PDMS using an acidic hydrogen peroxide solution trough microfluidic channels. Following the oxidizing step, PDMS was silanized either by a PEG-silane derivative for non-specific protein adsorption or by an amino-silane derivative. The amino group was then activated by thiophosgen to obtain isothiocyanate-grafted PDMS microchannels (figure 2), which were subjected to attachment reactions with different amino containing biomolecules such as tripeptide RGD, single stranded DNA and PSCA (Protein Sequence Comparative Analysis) proteins. The PDMS modification was shown to be stable in time with no hydrophobic recovery and was successfully used for immunoassays, DNA hybridization and cell immobilization. This method has the particularity of avoiding radiation exposure or energetic beam commonly used to modify PDMS surface.

Lipid Bilayer Formation Yang and co-workers [35] developed a heterogeneous microfluidic immunoassay based on solid supported bilayer containing dinitrophenyl (DNP)-conjugated lipids. The lipid bilayer was deposited on previously oxidized PDMS surface using oxygen plasma treatment and the lipid bilayer was created by vesicle fusion method. A linear array of channels was

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deposited and fluorescently labeled antibodies directed against DNP were flowed, giving a binding constant of 1.8µM for the antibody toward the immobilized antigen. This approach, taking advantage of the lateral diffusion properties of lipids, allows movements of the immobilized probe using the lipid bilayers properties. This method is particularly interesting for proteins which could suffer of unfolding when deposited using adsorption or covalent bonding on solid support.

Ester Terminated Silicones Surfaces Recently, a generic route has been proposed by Chen and co-workers [36] to functionalize PDMS containing hydrosilane residues (figure 3). First, hydrosilane residues were created on polymerized PDMS surfaces using strong acidic conditions (triflic acid) in the presence of (MeOSiO)n. In parallel, α-Allyl-ω-N-succinimidyl carbonate-poly(ethylene glycol) molecules were synthesized and reacted with the hydrosilane PDMS in the presence of little amount of Karstedt’s Pt catalyst. Thus, covalent grafting was created between PDMS and PEG modified molecule which contained a free N-hydroxy-succinimide (NHS) group. The procedure allowed then the immobilization of PEG chains acting as a brush with its NHS groups pointing to the solution. Various biomolecules such as peptides for cell culture or proteins (mucin, heparin, lysozyme, human serum albumin and epidermal growth factor) were successfully immobilized on this PEG modified PDMS material.

ester reactive nucleophilics T1OH

PDMS polymer

PDMS polymer

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Pt catalyst

N1N1-disuccinimidyl carbonate poly(ethylene glycol) monoallyl ether

PDMS polymer Allyl--N-succinimidyl Carbonate Poly(ethylene glycol)

PEG modified surfaces

Figure 3. Overview of the strategy for PDMS biochip surface functionalization with PEG derivatives.

Chemical Vapour Deposition Enable Lahann and co-workers [37, 38] used chemical vapor deposition (CVD) to deposit thin layers (90nm to 150nm) of poly(p-xylylene carboxylic acid pentafluorophenolester-co-pxylylene) (PPX-PPF) on PDMS surfaces (figure 4). First, PPF was sublimated at 180°C at reduce pressure and was then pyrolyzed at 600°C leading, with customized conditions, to the formation of p-quinodimethane. Afterwards, these reactive species were brought in a cold chamber (15°C) to polymerize homogeneously on PDMS surfaces. This technique has the advantage of being performed at room temperature without the need of additional catalyst, solvent or initiator, and to lead to a nanometer size PDMS modified layer. Furthermore, the polymer layer is insoluble in aqueous solutions or organic solvents. Using this strategy, the authors were able to coat PDMS-(PPX-PPF) with an amino-

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biotin derivative via an amide bond formation, followed by an incubation step with a streptavidin containing solution. As streptavidin can bind four biotins residues, two were believed to be linked with the functionalized surface, leaving two binding sites for biomolecules assembly. The authors validated their approach through cells adhesion on immobilized integrins for the development of on-chip cell-based bioassays.

Figure 4. Chemical vapour deposition (CVP) of poly(p-xylylene carboxylic acid pentafluorophenolester-co-p-xylylene) on PDMS biochip surface.

Photo-Linking Brooks and co-workers [39] reported the use of a carbene generating photolinker to pattern biotin derivatives on various surfaces, including PDMS. A solution containing a biotin derivative bearing a (trifluoromethyl)diazarine function was deposited on a flat PDMS

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surface and carbene formation was generated using UV light exposure (254nm). A particular feature of this experiment is the use of a dynamic confocal apparatus to create customized pattern of biotin by simply moving and focusing the UV light beam on un-activated PDMS surfaces. The authors didn’t investigate the chemical modification involved in the biotin grafting onto PDMS substrate but proposed that the carbene function was inserted in C-H and C=C bonds of the hydrophobic chains of the polymer. This procedure created a homogeneous layer of biotin (6nm thick) subsequently incubated with streptavidin. The system was efficient for the detection of biotinylated antibodies or fluorescent probes.

Photo-Grafting Using a photografting procedure, Wang and co-workers [40] investigated an efficient procedure to functionalize PDMS surfaces. Benzophenone used as a photoinitiator, was adsorbed and diffused in PDMS. These benzophenone groups were then used to graft polyacrylic acid (PAA) under UV-light exposition. The use of an exposure mask led to the achievement of localized grafting. After 25 minutes of UV-light illumination, the polymeric layer created (about 150nm above the PDMS surface) allowed conventional carbodiimide amide bond formation with biomolecules containing free amino groups. As biochip applications, the authors performed on chip cells culture on immobilized ethylenediamine layers and immunoassays using anti-GFP antibodies covalently attached on PAA modified PDMS. Interestingly, no clean-room facilities were required for the achievement of the present PDMS surface patterning and the formation of the interpenetrating polymeric layer leads to a spatial resolution of 5µm.

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3.3. Immobilisation of Biomolecules on Un-Modified PDMS Surfaces Considering the highly hydrophobic properties of PDMS, some biomolecules strongly adsorb on it by their hydrophobic domains. Taking advantages of this property, Etheshola and co-workers [41] have built the first heterogeneous immunoassay using bare PDMS surfaces coated with anti-sheep type M immunoglobulins (IgM) antibodies. Non-specific adsorption problem following the immobilization step was overcome by using an appropriate solution containing BSA (Bovine serum albumin), casein and Tween20. The sensitivity of the assay reached 425 fmole of IgM in the PDMS modified channels. Using a similar approach, Linder and Verpoorte [42] used biotin-conjugated protein coating as the first layer for PDMS microchannels surface treatment. Neutravidin solution was then flowed through the system and reacted with the immobilized biotin to create a uniform layer which was functionalized with a second biotinylated antibody, thus creating a three layer sandwich surface. However, when an antigen solution was added to the system, some unwanted binding with the first antibody was detected, leading to interferences between the analyte and the coating. More recently, Cesaro-Tadic [43] used a microfluidic based immunoassay for the detection of tumor necrosis factor α (TNF-α) by simply coating the capture antibodies on a PDMS surface. The target protein was injected into the chip followed by fluorescently labeled antibodies and the detection limit reached was 0.02 fmole.

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Figure 5. The four main steps for micro-contact printing preparation of biochips.

Microcontact Printing (µCP) Considering the particular properties of PDMS, Whitesides group’s introduced in the early 1990’s an original way to pattern biomolecules, using PDMS as a soft stamp [44, 45]. This soft lithographical technique, called micro-contact printing (µCP), is simple, inexpensive and offers multiple possibilities to transfer monomolecular layers onto surfaces. Indeed, PDMS is the most widely used polymer for this application because it can be easily molded on a 3D master in classical laboratory conditions. Once peeled off, an “ink” containing the desired molecules is applied and incubated to the elastomeric stamp. The inked stamp is then subsequently brought into contact (i.e. printed) with a flat surface, enabling the transfer of the biomolecules to the biochip surface [46] (figure 5). One of the main advantages of this technique is to pattern homogeneously large or small surface areas with biomolecules, particularly proteins, with very good spatial resolution [46]. Unfortunately, µCP is also known to lead to proteins drying, and one have to consider that only proteins remaining active in a dry state, such as some antibodies [46] or resistant proteins, can be used with this method.

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In the field of the well established DNA biochip, Thibault and co-workers [47] used this technology to adsorb oligonucleotides on a hydrophobic PDMS surfaces using µCP. They demonstrated the efficient DNA transfer from a PDMS stamp to flat substrate leading in an increase of the detection signal when compared to traditional pin spotted slides. µCP seems then to be a very promising procedure to address biomolecules but some limitations exist. Inking process tends to create protein diffusion at few micro-meter or nanometer scale and one has to take in account some stamp deformation during the process using low scale 3D features [47]. Another phenomenon pointed out is the siloxane contamination [48, 49] of the surface during the printing step. Indeed, it is well known that PDMS exhibits a dynamic behavior related to un-polymerized buried PDMS chain migration from the bulk to the surface [26] and, very recently, Asberg and co-workers [49] were able to assess the conformational states of different printed biomolecules (a synthetic peptide, calmodulin and the horseradish peroxidase).

Affinity Contact Printing (αCP) An original way to use µCP is to functionalize the PDMS stamps with antibodies or other affinity proteins. Thus, immersing the stamp in a complex solution enables the selective extraction of antigens and their patterning on a dedicated surface. Bernard and co-workers [50] were the first to develop this technique by chemically grafting proteins on a previously activated PDMS stamp with an hetero-bifunctional crosslinker (oxygen plasma oxidation followed by silanization). In this particular case, stamps modified with NgCAM (a protein responsible for neural cell adhesion) specific antibodies were used to extract the protein from a culture medium and print it to generate a cell growth pattern. More recently, Renault and co-workers [51] demonstrated the possibility to use αCP to create proteins microarray with resolution of the immobilized protein patterns of few micrometers. The authors also introduced the concept of microfluidics networks (µFN), exhibiting high resolution pattern of 3 x 3 µm2. This technique allows an easy reproduction of the target array from the initial α-stamp which represents the critical step. Patterning Functionalized Lipid-Bilayer Membranes Supported lipid bilayers are useful to study dynamic processes between proteins and lipids and Hovis and co-workers [52] introduced two methods for patterning fluid lipid bilayers based on µCP. In the first one, named blotting, a lipid bilayer is created on a flat surface by the vesicle fusion method and used to ink a PDMS stamp. The second one, called stamping, implies lipid bilayer formation directly on the hydrophobic surface of the PDMS stamp. The modified stamp is then bring into contact with a glass slide, creating a lipid bilayer pattern. Kung and co-workers [53] created complex surfaces containing both lipid bilayers and protein layers. Using µCP of proteins with a particular pattern on a glass slide, the authors were able to fill the areas leaved unmodified with lipid bilayers. The resolution of the patterned features between proteins and lipids can be scaled down to one micrometer depending of the stamp pattern resolution but one of the main advantages of this technique is to restrict the natural tendency of lipids to diffuse, through the use of protein layers as barriers. With the same approach, Philips and Cheng [54] used phosphatidylcholine membranes containing receptors specific of cholera toxin to create a heterogeneous immunoassay on

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oxidized PDMS surface. Vesicles carrying the receptor were fused on the polymer surface and the so designed biochip succeeded in the detection of 210 pM of cholera toxin. These results shown how lipid deposition can be an efficient way to functionalize PDMS surfaces since proteins with appropriate domains can be inserted inside the bilayer and used for biochip fabrication [55].

4. BIOMOLECULES IMMOBILISATION THROUGH ON-BIOCHIP ELECTROCHEMISTRY

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4.1. Conductive Electro-Polymers The electro-addressing of biomolecules is a modern objective to create new kinds of immobilization strategy for biochips. This wish responds to the ever-increasing demand of new efficient technologies for biochip development. The electro-chemical addressing of biomolecules intends to take advantage of direct, specific and spatially controlled way of immobilization, thus reducing time and cost of biochip production. Compared to other classical immobilization methods which necessitate a chain of chemical reactions and/or further consecutive technical steps, the electro-addressing of biomolecules can be considered as a direct method. Fundamentally, the method requires the application of a difference of potential, using a basic electrochemical setup, to achieve the localized fixation of the biomolecules, i.e. the "addressing". The technical advantage seek is to avoid the use of mechanical apparatus such as solution deposition systems. Nevertheless, the biochips useful for electro-addressing will have to be composed of either a matrix of electrodes or at least a conductive material. Historically, biomolecules immobilization via electro-addressing is considered as a recent technology which emerged with the conductive electro-polymer researches. Innovations in the field started in 1979 [56], with the demonstration and the study of the electropolymerization of pyrrole at a platinum electrode surface. Polypyrrole films prepared under controlled electrochemical environment were shown to be interesting materials with improved conductivity, strong adhesion to the metal surface and good stability under electrochemical conditions. The conducting property of these polymers is due to the strong electron mobility related to the regular conjugation of C=C bonds inside the polymer structure (figure 6).

Figure 6. Polymerization of pyrrole through electro-oxidation process.

Years after years, numerous electrochemical techniques such as cyclic voltammetry, potential step experiments and coulometry were used to investigate the electrodeposition of polypyrrole films from acidic, neutral and basic aqueous or organic solutions [57]. Biosensors: Properties, Materials and Applications : Properties, Materials and Applications, Nova Science Publishers, Incorporated, 2009. ProQuest

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The first immobilization procedure experimented for biosensing was realized through the electro-polymerization of pyrrole, simply used to entrapped biomolecules during the polymerization process (figure 7-a). The technique used the ability of pyrrole and/or its derivatives to form insoluble films attached on the electrode surface. Thus, Umana and Waller [58] reported a protein-modified glassy carbon electrode based biosensor, using glucose oxidase entrapped in a polypyrrole film available for electrochemical detection of glucose. At this point two major classes of electro-addressing immobilization using electropolymers have to be distinguished: (i) the entrapment of biomolecules during the electro-polymerization process which was shown to be efficient with high molecular weight molecules, (ii) the co-polymerization of single unit monomers together with monomer units functionalized biomolecules, which seems to be more efficient with small biomolecules such as oligonucleotides and peptides (figure 7b).

Figure 7. a) Immobilization via entrapment of biomolecules during the electro-polymerization process at the electrode surface. b) Immobilization via co-electro-polymerization of monomer units and monomer-functionalized biomolecules at the electrode surface.

On-Chip Electro-Entrapment of Biomolecules On the model of the entrapment, oxidase enzymes were immobilized by electropolymerization into conductive polymers films of polyaniline, polyindole, polypyrrole and poly(o-phenyldiamine) [59]. The kinetic and the behavior of the entrapped enzyme toward temperature, solvent and pH were studied. All the bio-films tested evidenced a clear decrease of the current generated in the presence of electro-active interfering species. This behavior was attributed to the permselective effect of the polymer. Other enzyme types were successfully immobilized using this approach, extending the usefulness of the technology to a wide variety of molecules. For example, Bekir Yildiz and co-workers [60] have shown that the electro-addressing of invertase through the electropolymerization of poly(pyrrole)/PMMA-co-PMMT (polymethyl methacrylate-co-polymethyl thienyl methacrylate) matrices was possible. Results shown that the enzyme was kept active, and that the biosensor offered great measurement stability over time (40 measures in 1 day). In a similar way, Vedrine and coworkers [61] immobilized a tyrosynase in an electrogenerated polythiophene film at the

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surface of a glassy carbon electrode. It was then shown that the electro-polymerized film provided an optimal response of the biosensor. The amperometric enzyme sensor generated was used to detect toxic mono- and di-phenols but also herbicides through the inhibition of the tyrosinase activity. In addition to immobilized enzyme, the electro-polymerization entrapment technique was described to generate immunosensors. A one-step entrapment of type G immunoglobulins (IgG) during the polymerization of pyrrole was shown to generate a sensitive immunosensor at the surface of a glassy carbon electrode biochip [62]. Following the immobilization, impedance measurements were used to provide a labelless method to detect antibodies/antigens interactions. Recently, Shi and coworkers [63] also entrapped antibodies in polypyrrole films during electro-polymerization, leading to a specific electro-addressing on interdigitated micro-electrodes array. Antigens in sera samples were detected through cyclic-voltammetry experiments in Fe(CN)63-/4-. This chip based immunoassay offered high sensitivity and good specificity against antigens for testing in human sera samples.

On-Chip Co-Polymerization of Biomolecules The second class of electro-polymer immobilization is based on the use of biomolecules functionalized with monomer and co-electro-polymerized with free monomer (figure 7b). This co-polymerization procedure was mainly used to immobilize small molecular-weigh biomolecules. In this way, Garnier and co-workers [64] have built a peptide functionalized conductive polymer layer. Monomers were prepared on a pyrrole basis, allowing the co-electropolymerization. The sensing layers formed permitted the selective recognition of the immobilized peptides by the carboxypeptidase A. Using the same co-polymerization addressing approach, biotin derivative was electroaddressed in polydicarbazole film at the surface of a glassy carbon electrode [65]. A complex of avidin-polyphenol oxidase was used to immobilize the enzyme which enabled the detection of L- and D-norepinephrines. Microarrays composed of 50µm gold electrodes were used as support for oligonucleotides/pyrrole electro-addressing, via the co-electro-polymerization of single pyrrole molecules together with oligonucleotides-5'-pyrryole modified. The oligonucleotides biochips realized were successfully tested for the genotyping of hepatitis C virus in blood samples [66]. The immobilization via direct electro-addressing on electrode microarray was also demonstrated for the achievement of a nucleic acid biochip. The system was used to detect nucleic acid gene mutation [67]. Peptide biochips were developed based on this pyrrole co-polymerization technology. Immobilized peptides on micro-electrode arrays were then available for the immunodetection of specific antibodies [68]. Interesting applications of the pyrrole co-polymerization procedure were developed on surface plasmon resonance imaging gold substrates. The co-polymerization of biomoleculepyrrole adducts and pyrrole units was used to generate spots through an electro-spotting procedure (figure 8)[69]. This strategy was shown to be successful for the direct monitoring of DNA interactions through SPRi measurement [70].

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The same method was extended to the co-polymerization of pyrrole and pyrrole-modified proteins in order to address antibodies at surface of the gold chip. This approach enabled the real time observation of interactions between antibody and antigens [71].

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Figure 8. Arraying using the electro-spotting method onto gold plated SPRi biochip.

Further sophisticated immobilization strategies were studied for protein electroaddressing on screen-printed microarray, involving different reactive conjugates. Therefore, HIV-1 P24 capsid protein modified with pyrrole monomers was immobilized through copolymerization [72]. Various pyrrole-protein complexes were tested for electro-addressing, notably protein fixed on (maleic-anhydride-alt-methyl vinyl ether). This latter was shown to provide the most accessible immobilized protein. Electro-polymerized films sensing layers were then evidenced as really versatile and powerful tools which could be used for numerous analytical systems such as optical or electrochemical, immuno- and enzyme-based sensors. However their involvement for biochip mass arraying and detection are not yet fully exploited.

4.2. Diazonium Salt Based Electro-Addressing The biomolecules electro-addressing based on the diazonium electro-reduction is a recent technology, taking advantage of the electrochemical grafting possibilities of aryl-diazonium residues. Figure 9 presents the reaction sequence involved: (i) an aryl-amine residue is diazotated in presence of NaNO2 and HCl giving an aryl-diazonium, (ii) this latter is electroreduced to an aryl-radical which (iii) reacts with the electrode material surface. The radical generated attacks the surface and forms a C-X bond; where X is the electrode material, namely Au, Cu, C (carbon or diamond) or Si [73-81].

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Figure 9. Reaction sequence for the electro-grafting of aryl-diazonium molecules.

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The use of this reactions was first described on carbon material surface [82, 83], and was applied to the indirect covalent immobilization of an enzyme. A glassy carbon electrode surface was functionalized by the electro-reduction of 4-acetic-phenyl-diazonium, leading to a 4-phenyl-acetic layer. Then a chemical covalent grafting of the glucose oxidase was performed to generate a glucose sensitive electrode. Diazotation reaction on an aniline derivative was also used to create a biotin layer, electro-addressed at a screen-printed carbon electrode surface (figure 10) [84]. An anilinebiotin residue was diazotated and subsequently electro-grafted on the electrode surface, providing a covalent anchoring point for streptavidin.

Figure 10. Strategy for creating biotin layer covalently attached to an electrode surface.

Recently, our team has developed a real one-step electro-addressing immobilization strategy using aryl-diazonium modified biomolecules [85]. The overview of this electroaddressing strategy is presented in figure 11. The initial model developed was an immunobiochip. Immunoglobulins were first functionalized with an aniline derivative through carbodiimide activation (figure 11-i) leading to immunoglobulin-anilines adducts. The aryl-amine present on the biomolecule could then be transform in diazonium via a diazotation in the presence of HCl and NaNO2 (figure 11-ii). Afterwards, the diazonium bearing immunoglobulins were shown to be successfully electroaddressed on specific electrodes of a screen-printed carbon microarray. The immuno-biochip obtained using this novel approach enabled the specific detection of anti-rabbit IgG with a detection limit of 50 fmole of protein. The immobilization strategy via

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diazonium-protein electro-addressing was also shown to provide an excellent spatial specificity of the immobilization on the screen-printed carbon microarrays. This method was successfully extended to the detection of rheumatoid factor (RF, which is a group of antibodies) in human sera samples [86]. A capture type immunoassay was then realized (figure 12-a) where rabbit IgG were electro-addressed on the carbon electrodes of a screen-printed microarray and were recognized by RF antibodies present in the serum samples. It was also demonstrated that eletro-grafted immunoglobulins could be used as recognition element and were then keeping their fully binding ability (figure 12-b). The functionalization protocol and the electro-addressing technique were pushed forward in order to perform oligonucleotides grafting. A 20mer sequence from a “hot spot” of the exon 8 of the p53 tumor suppressor gene was functionalized with 4-aminobenzylamine, electroaddressed and used as stationary phase probe sequence for hybridization testing of biotinylated target sequence (figure 12-c). As for the protein biochips, the system was shown to provide a strong specific signal, only due the specific recognition of immobilized biomolecules. This biomolecule electro-addressing technology appears to be promising, taking advantage of simple, direct and covalent immobilization of biomolecules. Up to now, this technology was demonstrated on carbon material surfaces, but may be extended to a wide variety of conducting materials, such as iron [74, 78], platinum, cobalt, nickel, zinc, copper [77], gold [87, 88], indium thin oxide [89] and silicon [75].

Figure 11. Biomolecules electro-addressing strategy onto screen-printed electrochemical biochips.

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Figure 12. Schematic representations of different biochip models using the electro-addressed immobilization of (a) an immunoglobulin antigen, (b) an active anti-human antibody and (c) a probe DNA sequence

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5. BIOMOLECULES IMMOBILIZATION THROUGH BEADS Micro- and nano-beads have become a major tool in analytical chemistry sciences. Indeed, micrometer size beads were developed with a very large range of properties: magnetic and/or fluorescent beads, having different surface functional groups for coupling chemistry or having different physicochemical properties. Biomolecule immobilization on beads, when compared to a flat surface, presents the advantage of generating higher specific surfaces. Those textured surfaces obtained were shown to improve the sensitivity of the developed assays [90]. Three different systems could be distinguished according to the organization of the beads: bead arrays, composed of highly organized beads; bead biochips, using beads as immobilization support without precise positioning of each bead; and the beads in microfluidic networks, characterized by a possible displacement of the beads.

5.1. Bead Arrays

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Arraying beads in a highly ordered manner requires the development of a bead localization system. This technical difficulty has been overcome by different groups through the formation of physical traps as illustrated in figure 13. The beads are in those cases of micrometer size (2-5 µm).

Figure 13. Scanning electron microscopy (SEM) images showing a: multiple well pits used to confine the sensor beads (from [3]). b: etched fiber optic end filled with 3 µm beads (from [4]). c: etched array housing 2 µm bead (from Illumina®).

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Such traps were obtained by chemical etching (hydrofluoric acid) of a silicon wafer [3], or imaging fiber optic bundles [4, 91, 92]. A precise control of the basin size enabled a perfect matching of the beads in their traps. Moreover, as presented in figure 14-a, such traps could have a pyramidal shape and be open at both ends, leaving the solutions flowing from one side of the chip to the other, and then bringing the beads in a constant reagent flow. This last type of chip is therefore an interesting approach to obtain systems with high mass transfer and a high hybridization rate. DNA and other biomolecules immobilization chemistries on the beads used in these systems are resumed in figure 14. Two main techniques were used, a direct immobilization through primary amino groups (figure 14-a), or an affinity procedure using avidin modified beads and biotinylated biomolecules (figure 14-b). In the second case, immobilization chemistry is required to graft avidin on the beads. When using agarose beads, the microspheres were purchased as terminated with aldehyde groups to which proteins could be linked via reductive amination (figure 14-b). Another possibility is to use commercially available neutravidin coated beads [93]. This affinity immobilization system enables, in the case of oligonucleotide immobilization, the achievement of perfectly orientated probes since the biotin is introduced, during the nucleic acid synthesis, only at one end of the sequence. One major handicap of these bead arraying systems is that usually no physical addressing of the beads in a particular trap can be achieved; these ones are then randomly self organized in the traps. Currently, two methods co-exist to determine the position of the probe grafted beads on a self-assembled bead arrays. The first method requires fluorescent dye encoding of the microspheres, where each microsphere is labeled with a unique ratio of dyes in order to identify the attached probe sequence [94] The second method is based on the use of addressing oligonucleotide sequences coimmobilized on each bead with the sequence of interest. The microsphere positions were then determined by hybridization to a series of fluorescent complements [91, 95]. Both approaches present limitations, since on one hand the number of unique, distinguishable optical signatures that can be prepared with a fluorescent dye are limited, and on the other hand, decoding the array with the hybridization method could be time consuming and dependent on the addressed sequence quality. A solution to cope with this problem is to use beads with a larger diameter (230 µm) in order to permit their manipulation with a micro-manipulator [96]. Each particular bead could then be placed at a precise location of the etched array. Nevertheless, at the present time, only low density bead arrays (4*3 array) could be physically addressed simultaneously, since the procedure is found to be time consuming. A last addressing system named LEAPS (for Light-controlled Electrokinetic Assembly of Particles near Surface), has been described by Seul and co-workers [93, 97] and appears to be more powerful. It is based on the concomitant use of (i) an in situ generated electric field, (ii) a computer generated illumination pattern and (iii) interfacial patterning. An electric field is generated at a silicon substrate surface patterned with etched traps. Beads present in this field will assemble into a cluster and are subjected to be retained in traps. The illumination of the silicon wafer enables the local modulation of the electrical properties of the semiconductor, leading to a possible addressing of the beads in particular traps.

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Figure 14. Probe immobilization onto a: amino modified beads, b: after the immobilization of avidin via reductive amination onto Sepharose beads (3 µm).

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All those systems, based on beads arraying and using optical detection are characterized by a very low nucleic acid target detection limit with a lowest value at 1 zmole [92]. The bead arrays are therefore interesting tools in terms of integration and miniaturization. Indeed, having sensing elements localized in a micron size sphere could lead to the achievement of highly integrated systems. Nevertheless, the integration degree appeared at the present time to be proportional to the technical difficulty degree.

5.2. Bead Biochips

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Bead biochips are characterized by a non-ordered relative position of each bead. The microspheres are in this case used to increase the specific surface of the support and to facilitate either the immobilization chemistry or the handling of the biomolecules. Two main categories of bead biochips could be distinguish associated to the use of the beads in an immobilized state or in a homogeneous phase.

Immobilized Beads Systems using immobilized beads bearing nucleic acid sequences are suffering from a lack of technical possibility to immobilize the beads at the surface of a solid support. Indeed, only a few examples are found in the literature which propose such analytical systems. The electron microscopy images presented in figure 15-a and 15-c illustrate two possibilities of arraying populations of identical beads at the surface of a biochip [2, 6, 98]. Figure 15-c shows the organization of immobilized 2.8 µm sized beads at a biotin modified surface. The surface was previously modified by micro-contact printing with a biotinylated protein that reacted subsequently with streptavidine grafted beads to generate a self assembled and self sorted array. In a similar way, modifying the surface by microcontact printing with particular chemical functions (anhydride) immobilization of hydroxy- or aminofunctionalized beads was enabled [6]. Therefore, using beads bearing both the immobilization function (biotin or chemical) and the biomolecule of interest, led to the grafting of a high surface density of probes, generated by the surface enhancement obtained. Nevertheless, since these methods are using a chemical modification of the flat surface, the problem is then resumed to the classical addressed modification of a flat and homogeneous surface. The use of bead assisted biomolecule immobilization is in that case only useful to increase the specific immobilization area. Another interesting bead immobilization technique is based on the entrapment of 1-100 µm sized microspheres at an elastomer (PDMS: polydimethyl siloxane)/air interface. Such a method enabled the immobilization of biomolecules bearing beads in an addressed manner (spotted) and with a high microsphere density, as could be seen in figure 15-b. Compared to the methods presented above, this system enables the use the bead assisted immobilization with a large scale of bead coverage, since the bead surface chemistry is not used during bead immobilization. Numerous analytical applications are described using this technique in particular for the study of point mutation in the codon 273 of the gene of the anti-cancer p53 protein [2] and the detection of anti-HIV capsid protein antibodies. In this example, 5'-amino-C6 probe sequences were immobilized via carbodiimide (dicyclohexylcarbodiimide) reaction onto carboxylate modified latex beads (1µm), prior to their immobilization (figure 16-a).

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Figure 15. Scanning electron microscopy (SEM) images of a: a 150 µm diameter latex beads spot, b: a closer view of the latex beads arrangement within a spot (from [2]) c: immobilized streptavidine-coated beads (2.8 µm) on a biotin modified surface (from [6]), d: 3D representation of the SEM image of a Sepharose bead trapped at the PDMS / air interface (from [7]).

In another study, 5'-amino-C6 modified nucleic acid was immobilized onto cyanogen bromide activated Sepharose beads (figure 16-b) [7]. The 100 µm diameter beads were then subsequently transferred at the PDMS/air interface (figure 15-d). Such porous polymeric beads have enabled a high enhancement of the specific surface since targets could be hybridized with the probes immobilized outside but also inside the Sepharose beads. Target detections (20mer sequences) with such systems were in the 0.1 pmole to 0.1 fmole range. Immobilized bead biochips are furthermore an interesting alternative to the dramatically complex bead arrays. Indeed, different populations of biomolecule-grafted beads could be easily immobilized at the surface of a solid support, leading to the achievement of easy to prepare, adjustable nucleic acid biochips.

Homogeneous Phase Bead Biochips: Suspension Array Technology Biomolecules immobilized beads could be used in a non-heterogeneous phase to perform hybridization assays. The probe molecules are in this case grafted onto non-immobilized beads.

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Figure 16. Biomolecule immobilization on a: carboxylate modified latex beads (1 µm) via carbodiimide reaction and b: cyanogen bromide (CNBr) activated Sepharose beads (100 µm). (φ: cyclic structure, e. g. dicyclohexylcarbodiimide).

Such systems present the advantage of being heterogeneous regarding the immobilized probes and to enable therefore the separation between hybridized and non-hybridized target. Moreover, bead suspensions could be approximated to homogeneous solutions, which could be turned into heterogeneous through the application of physical forces (gravity, magnetism). Numerous studies on bead biochips were therefore based on magnetic [99, 100], glass or silica [101-103], and polystyrene beads [104]. The biomolecule immobilization chemistry on those beads could be very different, from the classical avidin/biotin affinity reaction (figure 14-b) to the disulfide bridging onto thiol modified silica [102] (figure 17-a), the thiocyanate reaction onto amino terminated latex beads [103] (figure 17-b), and finally the hybridization based immobilization of poly(A) tagged probes onto poly(T) bearing magnetic beads [99] (figure 17-c). A last possibility to obtain probes grafted beads is to directly synthesize oligonucleotide probes onto glass beads via phosphoramidite reaction [101].

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Figure 17. Biomolecule immobilization onto a: silica 60 nm particles, b: amino terminated 0.31 µm latex beads and c: poly(T) modified Dynabeads®. MPTS: 3-mercaptopropyltrimethoxysilane.

Hybridization onto those different homogeneous systems could be detected through a large range of detection methods such as fluorescence [102, 104, 105], fluorescence quenching [101] or stripping voltammetry [100]. Biosensors: Properties, Materials and Applications : Properties, Materials and Applications, Nova Science Publishers, Incorporated, 2009. ProQuest

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Interesting studies were also performed based on a suspension of beads in conjunction with flow cytometry measurements [106, 107]. Flow cytometry, which was the standard methodology for cell population study during the last 20 years, has now begun to serve for in vitro microspheres analysis [108]. Such systems were described as multiplex microsphere bead assays and were used to detect different nucleic acid sequences hybridized on beads having different properties (size, fluorescent label). Discriminating on one hand the bead type and on the other hand the hybridized sequence leads to a sensitive and high throughput technique with detection limits in the 10 fmole range [107]. Finally, as a prospective issue, flow cytometry is planned to have a potential throughput of nearly 300 thousand analyses per day, and to play an important role in genomics and proteomics [108].

5.3. Beads in Micro-Fluidic Systems

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Beads in microfluidic systems are an interesting evolution of the homogeneous phase beads biochips. Indeed, examples of microfluidic platforms in which biospecific molecules are immobilized on an internal channel surface are still rare [109]. Bead based material is therefore a clearly viable alternative to introduce immobilized biological compounds in micro-channels.

Figure18. a: Beads packing device working in a flow system, taking advantage of a "leaky" wall (from [1]), b: A scanning electron microscopy image of a flow through reactor composed of pillar made walls used to pack microspheres (from [5]).

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Figure 19. Classical optical image of the bead arrays capillary (a) and fluorescent microscope images (b-k) of the bead arrays after hybridization (from [112]).

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Microfluidic structures used to retain beads might then fulfill the dual purpose of holding back particles while allowing samples and reagents to be delivered. A first type of device is presented in figure 18 in which microspheres are confined either in a microchamber bordered by a "leaky" wall (figure 18-a) [1] or by a pillar made wall (figure 18-b) [5]. Such systems were used to perform single nucleotide polymorphism (SNP) analysis of the codon 72 of the gene of the anti-cancer p53 protein. In a similar way, magnetic beads supporting nucleic acid sequences could be retained in a microfluidic system by a magnet and act as supporting phase [110, 111]. A good example is the study presented by Fan and co-workers [111] in which magnetic beads bearing different nucleic acid probes (up to eight) were magnetically packed in an eight channels microfluidic biochip. Hybridization was then taking place in a well adapted flow through format. More organized systems were also described based on the alignment of beads bearing biomolecules in capillaries [112, 113]. Such systems enabled the achievement of ordered beads as presented in figure 19-a. Different beads bearing different DNA probes could then be aligned in a capillary. In a typical experiment performed by Kambara and co-workers [113], 103 µm sized glass beads modified with DNA were arrayed in a capillary with an internal diameter of 150 µm. This bead handling required the development of bead alignment devices such as microchamber rotating cylinders or microvacuum tweezers, in order to manipulate and introduce the beads in the capillary in an ordered manner. The hybridization of the different probes immobilized on the different beads led to the achievement of the images presented in figure 19-b,k. Such systems therefore have a real potential in analytical development since beads with particular bio-specificity could be addressed and arrayed in a fluidic system which could be used to carry the different reagents and which could be read out optically

6. CONCLUSION The present review has proposed a selection of non-conventional methods to obtain biomolecules immobilization on biochip. As a matter of fact, each technology – i.e. PDMS, electrochemical and bead based immobilization – appeared to be linked to very special biochip designs. Indeed, these extremely particular methods, which were demonstrated as generic, were also highly dependant of (i) the use of a special polymer for the first one, (ii) the conductivity of the biochip support for the electro-immobilization and (iii) the possibility to handle bead for the last system. Thus, the current multiplication of coexisting powerful methods for biochip development leads to an interesting situation where one developed system could match one analytical need of the biomedical, safety, research or diagnostic field.

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[107] Chandlera, D.P. and A.E. Jarrellb, Enhanced nucleic acid capture and flow cytometry detection with peptide nucleic acid probes and tunable-surface microparticles. Analytical Biochemistry, 2003. 312(2): p. 182-190. [108] Nolan, J.P. and L.A. Sklar, Suspension array technology: evolution of the flat-array paradigm. Trends in Biotechnology, 2002. 20(1): p. 9-12. [109] Verpoorte, E., Beads and chips: new recipes for analysis. Lab on a Chip, 2003. 3(4): p. 60N-68N. [110] Jiang, G. and D.J. Harrison, mRNA isolation in a microfluidic device for eventual integration of cDNA library construction. The Analyst, 2000. 125(12): p. 2176-2179. [111] Fan, Z.H., et al., Dynamic DNA Hybridization on a Chip Using Paramagnetic Beads. Analytical Chemistry, 1999. 71(21): p. 4851-4859. [112] Kohara, Y., Hybridization Reaction Kinetics of DNA Probes on Beads Arrayed in a Capillary Enhanced by Turbulent Flow. Analytical Chemistry, 2003. 75(12): p. 30793085. [113] Noda, H., et al., Automated Bead Alignment Apparatus Using a Single Bead Capturing Technique for Fabrication of a Miniaturized Bead-Based DNA Probe Array. Analytical Chemistry, 2003. 75(13): p. 3250-3255.

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Chapter 5

ELECTROCHEMILUMINESCENT SENSORS: FABRICATIONS AND APPLICATIONS Hui Wei and Erkang Wang* State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, People’s Republic of China, and Graduate School of the Chinese Academy of Sciences, Beijing 100039, People’s Republic of China

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ABSTRACT Over the past decades, electrochemiluminescence (ECL) of tris(2,2’bipyridyl)ruthenium [Ru(bpy)32+] has received considerable attention from many researchers and been widely used to detect a variety of analytes that range from metal ions and small molecules to DNA, peptides, and proteins. Among all the areas of Ru(bpy)32+ ECL, the Ru(bpy)32+ based ECL sensors are of great active. In this chapter we will review the state of the art of Ru(bpy)32+ ECL sensors. After a brief introduction of Ru(bpy)32+ ECL and its mechanisms, the fabrications and applications of the Ru(bpy)32+ ECL sensors are discussed in details. It also indicates the future outlook in this field.

Keywords: electrochemiluminescence; ECL; sensors; tris(2,2’-bipyridyl) ruthenium; fabrications; applications; bioassay.

1. INTRODUCTION Electrochemiluminescence (also called electrogenerated chemiluminescence, abbreviated as ECL) is a means of converting electrochemical energy into radiative energy at the surface of an electrode via an applied potential.[1] Luminescent signals could be obtained from the excited states of an ECL active species generated at electrode surfaces during the electrochemical reaction. Among many organic and inorganic ECL systems studied, ECL of *

Tel: +86 431 85262003; Fax: +86 431 85689711; Email: [email protected] (E. K. Wang)

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tris(2,2'-bipyridyl) ruthenium(II) [Ru(bpy)32+] has played an important role in the development of ECL and its applications, as manifested in, for example, the growing interest in clinical tests and biomolecule detection.[1-8] Ru(bpy)32+ ECL offers several distinct advantages over other detection systems such as strong luminescence, good solubility in a variety of aqueous and nonaqueous solvents, inherent sensitivity, good spatial and temporal resolution, and wide linear range in the utility in different analytical areas. Since the first detailed studies of Ru(bpy)32+ ECL was realized in solution by Tokel and Bard in 1972,[9] the Ru(bpy)32+ ECL has been extensively studied, from reaction mechanisms to clinical applications, synthesis of ECL-active compounds, solid-state ECL systems, and ECL instrumentation.[1-8, 10-20] Up to date, more than 2000 scientific literatures (including papers, patents and book chapters) on various topics of ECL have been published and a considerable number of excellent reviews have also appeared.[1-6, 16, 21-28] Thus, we will present the state of the art of ECL sensors, including their fabrications and applications, in this chapter and mainly focus publications appeared after 2000. The interested readers are encouraged to consult several outstanding reviews for further and deep discussions on certain specific topics.[1-3]

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2. MECHANISMS OF RU(BPY)32+ ECL The mechanisms of Ru(bpy)32+ ECL have been extensively studied and have been well summarized.[1-3, 10] According to how the excited Ru(bpy)32+* was produced, the Ru(bpy)32+ ECL can be generally classified into two types: ion annihilation ECL and coreactant ECL. Most ECL sensors developed so far are based on coreactant ECL technology. Though several kinds of coreactants, including oxalate, peroxydisulfate, and amine, have been investigated to study the mechanisms of Ru(bpy)32+ ECL, the majority of study has been focused on the coreactant tri-n-propylamine (TPrA) due to its highest ECL efficiency and its importance to immunoassay and DNA analysis. Therefore, the Ru(bpy)32+/tri-n-propylamine (TPrA) system is taken as an example to elucidate the ECL mechanisms here. As shown in figure 1, the excited Ru(bpy)32+* can be generated via three different routes: (1) Ru(bpy)33+ reduction by TPrA● free radicals (figure 1A); (2) the Ru(bpy)33+ and Ru(bpy)3+ annihilation reaction (figure 1B); and (3) Ru(bpy)3+ oxidation by TPrA●+ cation radicals (figure 1C). However, from an experimental viewpoint, the mechanisms can be divided into two types according to the amount of Ru(bpy)32+ used. In relatively high concentrations of Ru(bpy)32+ (~millimolar) solutions, an ECL wave in 1.0-1.4 V (vs. Ag/AgCl) will be observed (Type I); while in dilute Ru(bpy)32+ solutions (less than approximately micromolar) containing ~0.1 M TPrA, another ECL wave in 0.7-1.0 V (vs. Ag/AgCl) will be observed (Type II). Type I ECL wave can be explained by the mechanisms in figure 1A and figure 1B; while Type II ECL wave can be explained by the mechanism in figure 1C, respectively.

3. FABRICATIONS OF RU(BPY)32+ ECL SENSORS As shown in figure 1, the ECL signal intensity is positively related to the concentration of Ru(bpy)32+ or the coreactant, so ECL can be used to detect for both. According to the two types ECL mechanisms discussed above (Type I and Type II), two corresponding kinds of

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ECL sensors could be designed. For Type I ECL sensor, Ru(bpy)32+ or its derivatives is usually immobilized onto an electrode surface, so the local concentration of Ru(bpy)32+ is high and constant, thus the concentration of coreactants and their analogues (i.e. analytes themselves are used as coreactants) could be determined by measuring the ECL emission intensity.

Figure 1. Proposed mechanisms of Ru(bpy)32+/TPrA ECL system. (A) Electrogenerated Ru(bpy)33+ reacts with TPrA as well as by direct reaction of TPrA at the electrode; (B) Ru(bpy)32+ reacts with TPrA● to form Ru(bpy)3+, which can then interact with Ru(bpy)33+ to form ECL via annihilation; (C) generation of ECL within the potential range before the oxidation of Ru(bpy)32+ at the electrode, involving formation of excited state Ru(bpy)32+* on reaction of TPrA●+ with Ru(bpy)3+ [formed by reaction of Ru(bpy)32+ with TPrA● free radical].[2-3, 10].

3. FABRICATIONS OF RU(BPY)32+ ECL SENSORS As shown in figure 1, the ECL signal intensity is positively related to the concentration of Ru(bpy)32+ or the coreactant, so ECL can be used to detect for both. According to the two types ECL mechanisms discussed above (Type I and Type II), two corresponding kinds of ECL sensors could be designed. For Type I ECL sensor, Ru(bpy)32+ or its derivatives is usually immobilized onto an electrode surface, so the local concentration of Ru(bpy)32+ is high and constant, thus the concentration of coreactants and their analogues (i.e. analytes themselves are used as coreactants) could be determined by measuring the ECL emission intensity. However, not all the analytes are ECL active, especially some bio-related analytes

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(such as tumor markers and antigenes). In this case, Type II ECL sensors are usually fabricated and Ru(bpy)32+ derivatives are employed to label one of the species involved in an affinity binding reaction. The concentration of an analyte can be determined by measuring the emission of the Ru(bpy)32+ derivative labels in the presence of an excess and constant concentration of coreactant (usually TPrA). We will discuss the fabrications and applications of these two types of ECL sensors in detailed in the following sections.

3.1. Fabrications of Type I ECL Sensors To fabricate Type I ECL sensors, Ru(bpy)32+ or its derivatives should be immobilized on an electrode surface. Up to date, a large number of different methods and matrix materials have been explored for Ru(bpy)32+ immobilization.

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3.1.1. Cation Ion-Exchange Approach Nafion, a cation ion-exchange polymer, can be used as matrix to immobilize positively charged Ru(bpy)32+ via cation ion-exchange approach. Since Rubinstein and Bard’s seminal report of immobilization of Ru(bpy)32+ on an electrode surface using Nafion,[29-31] quite a lot of extended work have been done to make robust and sensitive ECL sensors. New materials, such as carbon nanotube (CNT), titania (TiO2), silica and V2O5, have been incorporated into Nafion films to accelerate the charge transfer and enhance its long-term stability.[32-42]

Figure 2. The schematic approach for the fabrication of Ru(bpy)32+ ECL sensor on a glassy carbon (GC) electrode using Nafion and CNT.[32].

As shown in figure 2, by employing the composite film of Nafion and CNT, Dong’s group has developed an ECL sensor on a glassy carbon (GC) electrode with improved sensitivity, reactivity, and long-term stability.[32] Nafion was used as the ion exchanger for Ru(bpy)32+, the solvent of CNT and the membrane material, while the CNT incorporated greatly enhanced the electronic conductivity of Nafion film and played an important role in adsorbing Ru(bpy)32+. By combining the merits of the CNT and Nafion, their ECL sensor exhibited a more than 3 order of magnitude higher ECL signals than the pure Nafion films modified electrode towards TPrA detection towards TPrA determination. Other ECL sensors based on Nafion have also been fabricated and studied by many researchers. For example, Zhang and co-workers have reported the simple and sensitive ECL

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detection of an antihistamine chlorphenamine maleate (CPM) by using TiO2/Nafion composite film modified GC electrode.[34] Lee’s group has developed a Ru(bpy)32+ ECL sensor based on sol-gel-derived V2O5/Nafion composite films and their sensor exhibited greatly enhanced ECL responses towards analytes compared to other types of sol-gel ceramic/Nafion composite films such as SiO2/Nafion and TiO2/Nafion.[39] Although Nafion has been the one most studied to construct Ru(bpy)32+ ECL sensor, other ion-exchange polymers have also been investigated.[43-49] For example, Eastman AQ55D polymers (AQ), an alternative to Nafion, have been used to make a silica/AQ/Ru(bpy)32+ ECL sensor for the determination of a commonly used depressant chlorpromazine (CPZ).[43] AQ is a kind of poly(ester sulfonic acid) cation exchange polymer similar to Nafion, but has some advantages over Nafion due to its more hydrophilic characteristic, lower cost, more rapid response and anti-fouling properties. As shown in Figure 3, our group has demonstrated a simple and effective method to construct an ultrasensitive ECL sensor by using novel platinum nanoparticles(PtNPs)/AQ/Ru(bpy)32+ colloidal materials.[44] The sensor was fabricated via a two-step approach: first, the colloidal materials of PtNPs/AQ/Ru(bpy)32+ were synthesized through a wet-chemical routine; and then the colloidal materials were cast onto a GC electrode surface to produce an ECL sensor. In our sensor, AQ was used not only to immobilize Ru(bpy)32+ but also as the dispersant of PtNPs. Due to the electronic conductivity and electroactivity of PtNPs in composite film, this ECL sensor exhibited enhanced ECL responses and especially a very low limit of detection (1 fM) of TPrA.

Figure 3. The schematic approach for the preparation of PtNPs/AQ/Ru(bpy)32+ composite film.[44].

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Poly(sodium 4-styrenesulfonate) (PSS), another ion exchange polymer, has also been used to improve the ECL sensor sensitivity.[46-49] Recently, PSS was extended to partial sulfonation of polystyrene (PSP) to develop a solid-state ECL sensor.[49] The PSP was used as the matrix to immobilize the ECL reagent Ru(bpy)32+ due to the electrostatic interactions between the sulfonic acid groups of PSP and Ru(bpy)32+ cations. Such a sensor has been used for the sensitive determination of 2-(dibutylamino)ethanol (DBAE) (figure 4).[49]

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Figure 4. ECL intensity-time curve of the PSP/CNT based Ru(bpy)32+ ECL sensor in 0.25 mM DBAE solution under continuous cyclic voltammetry (CVs) for 8 cycles at the scan rate of 50 mV/s.[49].

3.1.2. Covalent Approach Covalent approach can also be employed to immobilize Ru(bpy)32+ and therefore to prepare Ru(bpy)32+ ECL sensors. As shown in figure 5, Dennany and co-workers have synthesized Ru(bpy)32+ contained metallopolymer by covalently incorporating Ru(bpy)32+ into poly(vinylpyridine) (PVP).[50-51] Then they used the metallopolymer to prepare catalytic oxidation DNA ECL sensors via the electrostatic layer-by-layer assembly of [Ru(bpy)2(PVP)10]2+ and DNA.

Figure 5. The chemical structure of [Ru(bpy)2(PVP)10]2+.[50].

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Lu and Whang have used the metallopolymer to perform detailed studies on the ECL and electrochemical behaviors of [Ru(bpy)2(PVP)10]2+/oxalate system on an indium tin oxide (ITO) electrode.[52] Besides metallopolymer, Ru(bpy)32+ can also be covalently attached into silica polymers.[53-55] Through hydrolysis these silica polymers could be easily converted into sol-gel films which could be readily coated on an electrode surface. We have synthesized silica hybridized ruthenium bipyridyl complex through amidation reaction by covalent attachment of bis(bipyridyl)-4,4'-dicarboxy-2,2'-bipyridyl-ruthenium to (3-aminopropyl)triethoxysilane (APTS) (see figure 6).[53] The hybrid complex then was coated onto an ITO electrode through acid catalytic hydrolysis and spin-coating techniques to fabricate an ECL sensor. As prepared ECL sensor possessed good stability therein with excellent ECL behavior.

Figure 6. Synthetic procedure for formation of bis(bipyridyl)-4,4'-dicarboxy- 2,2'-bipyridyl- ruthenium-silica hybrid materials and their predicted structure.[53].

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3.1.3. Electrostatic Approach Since Ru(bpy)32+ is positively charged, it can also be immobilized through electrostatic interactions.[56-65] Dong and co-workers have developed an ECL sensor based on the multilayers of silica nanoparticles/Ru(bpy)32+ (figure 7).[56] Since the ITO substrate was negatively charged, positively charged poly (diallyldimethylammonium chloride) (PDDA) could be adsorbed onto the ITO surface to provide a preassembled layer. Then negatively charged silica nanoparticles and positively charged Ru(bpy)32+ could be alternately assembled to fabricate the sensor. The assembly process was characterized by cyclic voltammograms and absorption spectroscopy. Their sensor showed high sensitivity and long-term stability due to the high surface area and special structure of the silica nanoparticles. The similar approach could also be extended to the clay/Ru(bpy)32+ and decatungstate/Ru(bpy)32+ systems where clay and decatungstate both were negatively charged.[57].

Figure 7. The schematic approach for the preparation of silica nanoparticles/Ru(bpy)32+ ECL sensor by electrostatic layerby-layer assembly.[56].

Other negatively charged materials, especially the emerging nanomaterials, have also been explored to develop new ECL sensors.[58-65] For example, Wang and coworkers have developed a novel method to construct a stable ECL sensor (figure 8).[62] The sensor was fabricated via a two-step approach: first, Ru(bpy)32+-Au nanoparticles (Ru-AuNPs) aggregates were obtained via the electrostatic interactions between Ru(bpy)32+ and citratecapped AuNPs; then the Ru-AuNPs aggregates were assembled on a mercaptopropyl triethoxysilane (MPTES) modified ITO electrode surface via Au-S interaction. The same group has also used platinum nanopartilces (PtNPs) to fabricate solid-state ECL sensors.[63] Even inorganic anions, such as PtCl62- and Fe(CN)63-, have been used to develop new Ru(bpy)32+ ECL sensors via the electrostatic interactions between Ru(bpy)32+ and anions approach.[64-65]

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Figure 8. Scheme illustrating (A) the formation of Ru(bpy)32+-AuNPs (Ru-AuNPs) in aqueous medium due to electrostatic interactions between Ru(bpy)32+ and citrate-capped AuNPs and (B) the immobilization of Ru-AuNPs on a sulfhydrylderivated ITO electrode surface.[62].

3.1.4. Physical Entrapment Approach Physical entrapment approach can also be employed to construct type I ECL sensors. In most cases silica materials are used for physical entrapment of Ru(bpy)32+ owing to their porous properties, and ease to prepare, to modify and to dope with various reagents.[66-76] Pastore and coworkers have produced an ECL sensor on K-glass conducing substrates by embedding Ru(bpy)32+ inside silica glass thin films.[66] Their sensor exhibited a diffusioncontrolled redox behavior of the Ru(bpy)32+/Ru(bpy)33+ system. They found that the thermal treatment (i.e. annealing) could improve the sensor’s performance. Collinson et al. have investigated the ECL of Ru(bpy)32+ and its co-reactants entrapped within sol-gel-derived silica monoliths using an immobilized ultramicroelectrode.[67] Dong’s group has developed a novel ECL sensor based on Ru(bpy)32+-doped silica (RuDS) nanoparticles conjugated with a biopolymer chitosan membrane.[68] The RuDS nanoparticles were prepared by the water/oil microemulsion method. Their sensor showed good sensitivity and reproducibility for TPrA detection. As shown in figure 9, we have synthesized the similar RuDS nanoparticles.[69] Then the ECL behavior of the RuSi nanoparticles was investigated after deposition with biomolecules through LBL self-assembly. Our results indicated that the biopolymer coatings could improve its stability though the sensor’s ECL signals were inhibited. The ECL sensor could also be fabricated via Stöber method.[74-76] For example, Yang’s group has prepared a solid-state ECL sensor with good reproducibility and stability through Stöber method.[74]

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Besides all the approaches and materials mentioned above, many other approaches and materials have also been investigated to develop Type I ECL sensors. Very recently, a review has well summarized the recent developments in fabrication of solid-state ECL sensors and their analytical applications.[16] Interested readers are referred to it for the further information.

Figure 9. (A) The preparation of Ru(bpy)32+-doped silica (RuSi) nanoparticles via a water/oil microemulsion method and (B) TEM image of the as-prepared RuSi nanoparticles.[69].

3.2. Fabrications of Type II ECL Sensors As for the Type I ECL sensors described in section 3.1, the analytes of interest must be used as the ECL coreactants. Usually, the presence of an amine group on the analytes, such as alkylamines, antibiotics, antihistamines, opiates, and nicotinamide, is required. However, most of biologically and clinically important targets, such as DNA, peptides, and proteins, are not the ECL coreactants. In this case, Type II ECL sensors are usually fabricated and Ru(bpy)32+ derivatives are employed as ECL labels that bind the analytes. In this section we will first discuss the Ru(bpy)32+ derivatives explored as ECL labels, and then address the assay formats for Type II ECL sensors in details.

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3.2.1. ECL Labels for Type II ECL Sensors Ru(bpy)32+ derivatives conjugated with suitable groups, such as N-hydroxysuccinimide (NHS) ester and phosphoramidite conjugates, have been synthesized and used as ECL labels in Type II ECL sensors. Shown in figure 10 are the chemical structures of some ECL labels mostly used. In 1991, Blackburn et al. reported the first Ru(bpy)32+ NHS ester, namely [4-(Nsuccinamidyloxy -carbonylpropyl)-4’-methyl-2,2’-bipyridine] bis(2,2’-bipyridine) ruthenium (II) dihexa -fluorophosphates (see figure 10 for the chemical structure, Ru-Label 1).[8] RuLabel 1 has been successfully commercialized and has been widely used to label targets with amine groups in biological assay and clinical tests. Ru-Label 1 can be used to label both proteins and DNA (RNA). Some analogues of Ru-Label 1, such as Ru-Label 2 and Ru-Label 3 in figure 10, have also been developed by researchers.

Figure 10. Chemical structures of Ru(bpy)32+ derivatives as ECL labels.[8, 15, 77-79].

Though Ru-Label 1 has been demonstrated to label DNA and RNA, the target DNA and RNA must be modified with amine groups. In 1993, DiCesare et al. have developed a highsensitivity ECL-based detection system for automated PCR product quantitation by using the ECL label Ru-Label 4 conjugated with a phosphoramidite group (figure 10).[77] Using RuLabel 4, unmodified DNA and RNA can be directly labeled. Other kinds of ELC labels have also been developed for specific analytical applications. For example, an ECL sensor for the determination of Pb2+, Hg2+, Cu2+, and K+ in solution has been developed using Ru(bpy)2(AZA-bpy) (AZA-bpy =4-(N-aza-18-crown-6-methyl-2,2’bipyridine).[78] The structure of Ru(bpy)2(AZA -bpy) was given as Ru-Label 5 in figure 10. As for Na+ ECL detection in both aqueous and nonaqueous media, Ru-Label 6 in figure 10 was used as an ECL probe.[79] Recognition of Na+ by the crown ether moiety in Ru-Label 6 resulted in a significant increase in the ECL emission intensity of the complex.

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Figure 11. Chemical structures of dendritic Ru(bpy)32+ derivatives as ECL labels.

With the goal of realizing the ultrasensitive ECL bio assays, multiple labels at multiple accessible sites of a target molecule has been investigated. However, such multilabeling at multiple sites could affect the bioreactivity of biomolecules and could even cause the precipitation of target proteins. Therefore, multilabeling at a single site strategy by using dendritic ECL labels has been studied. As shown in figure 11, using a dendritic prototype label with three Ru(bpy)32+ linked to a NHS group, Zhou et al. have demonstrated multilabeling a model protein at a single NH2 position.[80] Other amplification methods have also been developed to enhance the sensitivities of Type II ECL sensors. By entrapping Ru(bpy)32+ into silica particles, polystyrene beads or liposomes, or loading Ru(bpy)32+ onto CNT, enhanced ECL labels for Type II ECL sensors could be prepared (figure 12).[68-69, 81-84] Since thousands of Ru(bpy)32+ molecules entrapped in a single enhanced ECL label, the detection sensitivity for the biological recognition event can be amplified using these kinds of new labels. When the surfaces of these enhanced ECL labels are conjugated with suitable groups, such as NH2, COOH and biotin conjugates, they can be readily used to label the analytes of interest via proper bioconjugation reactions.

Figure 12. Enhanced ECL labels for Type II ECL sensors prepared by entrapping Ru(bpy)32+ into silica particles (A) or liposomes (B), or by loading Ru(bpy)32+ onto CNT (C).[68-69, 81-84].

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Recently, we have studied the ECL properties of Ru(bpy)32+-doped silica nanoparticles within layer-by-layer (LBL) biomolecular coatings and investigated their possible usage as ECL label materials in details.[69] We found that the biomolecular coatings could improve their biocompatibility and prevent the leaking of the Ru(bpy)32+ ions, the Ru(bpy)32+-doped silica nanoparticles within the LBL biomolecular coatings could be readily used as stable and efficient ECL tag materials.

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3.2.2. Assay Formats for Type II ECL Sensors In their seminal work, Blackburn and coworkers have demonstrated that Type II ECL sensors were suitable for immunoassays and DNA probe assays in clinical diagnostics.[8] In a recently review, Miao has well summarized the assay formats for Type II ECL sensors.[3] As shown in figure 13, eight typical assay formats are given where most of biologically important analytes, such as DNA, antibody-antigen, and peptide-related, could be detected.

Figure 13. Eight examples of ECL assay formats: (A) DNA hybridization assay based on an immobilized ssDNA hybridizes with a labeled target ssDNA; (B) sandwich type DNA biosensor; (C) assay used for integrase activity test with immobilized and free labeled dsDNA; (D) sandwich type immunoassay; (E) direct immunoassay; (F) competitive assay in which analyte competes with labeled analyte for antibody binding sites on immobilized antibody; (G) protease activity assay in which cleavage of the immobilized peptide results in the decrease in ECL emission due to the removal of the ECL label; (H) kinase activity assay using a labeled antibody to recognize the phosphorylated product.[3].

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sensor based on the method depicted in figure 13D.[85] For this sandwich type immunoassay, biotinylated anti-CRP species were first immobilized onto the Au (111) substrate premodified with an avidin layer; then CRP and anti-CRP tagged with Ru(bpy)32+ labels were conjugated to the surface layer. Finally, the ECL signals were obtained from the sandwich sensing electrode by immersing them in a TPrA containing electrolyte solution.

Figure 14. The schematic diagram of direct ECL quantification of BSA by using Ru-Label 3.[86].

Recently, we have reported a quantitative ECL detection of bovine serum albumin (BSA) via biotin-avidin interaction using an avidin-based sensor and Ru-Label 3 as ECL labels and TPrA as coreactant (figure 14).[86] To detect BSA, first, an avidin layer was first immobilized onto a glassy carbon electrode pretreated with CNT; then, biotinylated BSA tagged with Ru-Label 3 was attached to the as-prepared avidin surface; finally, ECL response was generated when the self-assembled modified electrode was immersed in a TPrAcontaining electrolyte solution. Cao et al. developed an interesting approach for quantitative DNA detection via quenching Ru(bpy)32+ ECL by ferrocene.[87] As shown in figure 15, the ssDNA with a Ru(bpy)32+ label gave intense ECL signal; however, when it was hybridized with its complementary DNA sequence labeled with ferrocene, an intramolecular ECL quenching occurred and the ECL signal was inhibited. This quenching sensor provided a promising approach for application to sequence specific DNA detection.

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Ferrocene Ru(bpy)32+

Ru(bpy)32+

Quenched

ECL

An Electrode

An Electrode

Figure 15. Quantitative DNA detection via quenching Ru(bpy)32+ ECL by ferrocene.[87].

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Beyond the traditional immunoassay, novel recognition elements, especially aptamers, have also been involved to construct Type II ECL sensors. Aptamers are in vitro selected DNA or RNA sequences that are capable of binding to specific targets.[88-89] As far back as in 1999, Bruno and Kiel have reported the in vitro selection of DNA aptamers to anthrax spores with ECL detection.[90] Recently, a lot of aptamer-based ECL sensors have been developed to detect small molecules and proteins.[91-94] We have designed an aptamerbased biosensor combined with gold nanoparticles amplification for the determination of lysozyme with ECL method (figure 16).[94] The lysozyme detection was realized with a competition assay format. In this format, unlabeled lysozyme in the test sample could replace the labeled lysozyme from the labeled lysozyme/aptamer complexes due to its higher affinity to the aptamer than the labeled lysozyme. This article is not included in your organization's subscription. However, you may be able to access this article under your organization's agreement with Elsevier.

Figure 16. Schematic diagrams for the fabrication of an aptamer-based ECL sensor for lysozyme detection.[94].

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4. APPLICATIONS OF RU(BPY)32+ ECL SENSORS Due to their intrinsic advantages mentioned above, Ru(bpy)32+ ECL sensors have shown great promise in analytical application. With the Ru(bpy)32+ ECL sensors developed, quite a lot of analytes including amines, oxalate, amino acids, drugs, alcohol, phenols, glucose and biomolecules, etc. have been detected in varieties of matrix, ranging from assay buffer to urine, serum, plasma, blood, and raw water samples.

4.1. Applications of Type I ECL Sensors

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For Type I ECL Sensors, the concentration of Ru(bpy)32+ immobilized on an electrode surface is constant, thus the ECL signals can be used to detect the concentration of coreactants and their analogues. Both non-biological and biological analytes could be detected with Type I ECL Sensors.

4.1.1. The ECL Assay of Non-Biomolecules Since the Ru(bpy)32+/TPrA system has been most extensively studied and exhibits the highest ECL efficiency, TPrA is usually used as a typical coreactant to validate the Type I Ru(bpy)32+ ECL sensors. For most sensors, the detection limit for TPrA is from 1 μM to 1 nM. However, by using novel Pt nanoparticles/AQ as matrix to construct the Ru(bpy)32+ ECL sensor, as low as 1 fM detection limit for TPrA has been finished by Wang and coworkers.[44] Xu et al. have developed a regenerable ECL sensor by incorporating Ru(bpy)32+ in ceramic carbon electrode.[95] They then combined the ECL detection with a solid-phase extraction strategy to detect dioxopromethazine in urine sample. Their sensor exhibited good selectivity for dioxopromethazine over other 17 interference species (such as phenylalanine and tryptophan). Also, the sensor improved the sensitivity for the determination of dioxopromethazine by ~3 orders of magnitude. By immobilizing Ru(bpy)32+ and alcohol dehydrogenase in the same sol-gel hybrid film, Dong’s group has developed an ethanol biosensor based on ECL detection.[96] Their sensor showed a linear response to ethanol from 2.5×10-5 M to 5.0×10-2 M with a detection limit of 1×10-5 M. The ECL detection of other analytes (including positively charged, neutral and negatively charged analytes) has also been finished by using Type I Ru(bpy)32+ ECL sensors.[55] More interesting, gas samples could also be analyzed with Type I ECL sensor. Egashira et al. have reported a trimethylamine gas ECL sensor based on Ru(bpy)32+/Nafion gel.[97] The sensor was successfully applied to monitor the seafood as practical samples. The ECL response for a squid was almost consistent with a profile curve obtained from the classic K value measurement. 4.1.2. The ECL Assay of Biomolecules Many biomolecules could also be analyzed using Type I ECL sensors. An ECL sensor for practically important polyamines, spermidine and spermine, has been fabricated by using bifunctional nanopartices of magnetic core luminescent shell.[75] The sensor showed an

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improved performance with the linear ranges of spermidine from 4×10-6 M to 5×10-3 M (R=0.988) and of spermine from 5.5×10-7 M to 10-4 M (R=0.999), respectively. The sensor reported gave 2 orders of more sensitive detection than the ultraviolet method. Other biologically important molecules, such as glucose, coenzyme NADH, and neurotransmitter dopamime, have also been analyzed using the Type I ECL sensors developed.[33, 40, 98-99] Guanine (and adenine sometimes) bases in DNA can be catalytically oxidized by Ru(bpy)32+. Thus direct ECL detection of DNA could be finished by using Ru(bpy)32+ immobilized onto an electrode surface. We reported one-electron catalytic oxidation of guanine bases in DNA using a Ru(bpy)32+-doped silica nanoparticles modified ITO electrode.[76] In the following work, we have distinctly discriminated native salmon testes DNA (ST-DNA) and its thermally denatured counterpart via label free ECL protocol.[100] As shown in figure 17, though both electrochemical and ECL signals could distinct native ds-STDNA from its thermally denatured counterpart, ECL signals gave a more efficient discrimination than electrochemical signals did. Moreover importantly, sensitive single-base mismatch of p53 gene segment has been realized with 39.3 nM.

Figure 17. CVs (a) and corresponding ECL intensity-potential curves (b) of the Ru(bpy)32+ modified electrode in the absence (line 1) and presence of 3.04×10-8 mol/L ST-DNA (ds) (line 2) and 3.04×10-8 mol/L thermally denatured ST-DNA (ds) (line 3) in 10 mM acetate buffer containing 50 mM NaCl (pH=5.50).[100].

Rusling’s group has employed ECL sensors to study the DNA damage caused by chemical or biological routes.[50, 101-104] In their design, the ultrathin films of [Ru(bpy)2(PVP)10]2+, DNA and enzyme were assembled onto an electrode surface (figure 18A). These ultrafilms can mimic a major toxicity pathway in the human liver, and thus are being used to develop toxicity sensor arrays (figure 18B). In a recently report, they have demonstrated ECL arrays for high throughput in vitro genotoxicity screening.[104] The ECL sensors were constructed by assembling various human cytochrome P450 enzymes, DNA and [Ru(bpy)2(PVP)10]2+ onto the electrode surface as shown in figure 18. The sensors were then exposed to H2O2 to activate the enzymes. Using benzo[α]pyrene as a test substrate, enzyme activity for producing DNA damage was tested and was found in the same order as their metabolic activity.

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Figure 18. (A) Idealized representations of film configurations used in ECL sensors for detection of DNA damage; and (B) conceptual diagram of an ECL array instrumentation.[101, 104].

The Type I ECL sensors developed usually bear a relatively poor selectivity. By coupling with separation techniques (such as high performance liquid chromatography, capillary electrophoresis and microchip capillary electrophoresis), the selectivity of the Ru(bpy)32+ ECL sensors could be greatly improved. However, these discussions are out of the scope of this chapter. So interested readers are referred to several reviews for further information.[26, 28]

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4.2. Applications of Type II ECL Sensors Most of Type II ECL sensors are developed for immunoassays and biomolecular assays. In these assays, targets (such as proteins, DNAs, and peptides) having no “coreactant functionalities” are tagged with ECL labels, then ECL signals associated with the target concentration are produced in the presence of a high and constant concentration of TPrA solution. Until now, biologically important targets from tumor markers to cardiac markers, vascular markers, growth factors, fertility markers, Alzheimer’s disease markers and HIV related genes, have been successfully studied using various assay formats for Type II ECL sensors (figure 13).[3] Miao and Bard have reported the ECL determination of immobilized DNA on Au(111) electrodes using Ru(bpy)32+ labels (figure 19).[85] An amino-modified 23-mer ssDNA derived from the Bacillus anthracis as probe was first attached to the Au(111) substrate, then the target ssDNA (complementary or noncomplementary ssDNA) tagged with Ru(bpy)32+ labels was hybridized to form the duplex. When a potential is applied to the electrode in TPrA solution, an ECL signal was generated and detected. As shown in figure 19B, the complementary DNA hybrid gave a much higher ECL response with respect to that for the noncomplementary DNA. Note that the CV response could not distinct the complementary DNA from the noncomplementary one due to the CV signals were mainly originated from TPrA oxidation. To further improve the sensitivity for DNA detection, polymerase chain reaction (PCR) amplification could be coupled.[8, 105-110] For example, Blackburn et al. have coupled ECL with PCR amplification and lowered the detection limit of HIV 1 gag DNA to PbSO4. The application of bone meal as an ameliorant had a significant (pplant height>pod dry weight>flower pod number. This was closely related to Kapustka et al. (1995) who proposed that the sensitivity of vegetative response follows the order: root length>root mass>shoot length>total mass (root+shoot)>shoot mass>germination. The vegetative responses (root length (r=0.86), root dry weight (r=0.83), shoot dry weight (r=0.85), plant height (r=0.82), pod dry weight (r=0.63) and flower number (r=0.76) had a positive correlation to the bioluminescence. This observation demonstrated that an increase in arsenic toxicity (through irrigation water) resulted to a decrease of all the measured vegetative and reproductive responses of lentil plant. The bioassay results from studies carried out by Sadeque, 2005 further indicated that the effect of filtration was probably attributed to physically held (e.g. arsenic complexes) but not chemically bond arsenic compounds (e.g. arsenite are more soluble in aqueous media) in the soil matrices (figure 6). This observation is related to the behaviour of clay which are negatively charged silicate minerals and surfaces of Fe-, Al- and Mn- oxides or hydroxides which have net negative charges, therefore, they preferably adsorb negatively charged ions, not oxyanions of arsenic. However the sorption of arsenic oxyanions from soil leachate occurs on clay and other colloidal surfaces (Sadiq, 1997). Sopper (1992) further reported that binding of metals to organic materials and clay minerals, precipitation, complexation and

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An Overview of Selected Lux-Marked Biosensors and Its Application…

ionic interactions significantly reduces their inhibitory influences on microbial activity, so that toxicity is substantially less in a soil system compared to pure culture systems. Clay soils are also considered to play a major role in the immobilisation of heavy metals by sesquioxides, which are involved, in two main steps.

% Bioluminescence of the control

125 100

M=NS As