Biosensors: a practical approach

No. 268

Biosensors Second edition

A Practical Approach Edited by

Jonathan M. Cooper Department of Electronics, University of Glasgow, Glasgow G12 8LT, UK

Anthony E.G. Cass Department of Biological Sciences, Imperial College London, London SW7 2AZ, UK

OXPORD UNIVERSITY PRESS

OXFORD UNIVERSITY PRESS

Great Clarendon Street, Oxford OX2 6DP It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide in Oxford New York Auckland Bangkok Buenos Aires Cape Town Chennai Dar es Salaam Delhi Hong Kong Istanbul Karachi Kolkata Kuala Lumpur Madrid Melbourne Mexico City Mumbai Nairobi Sao Paulo Shanghai Taipei Tokyo Toronto Oxford is a registered trade mark of Oxford University Press in the UK and in certain other countries Published in the United States by Oxford University Press Inc., New York © Oxford University Press 1990, 2003 The moral rights of the author have been asserted Database right Oxford University Press (maker) First edition published 1990 Second edition published 2004 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, without the prior permission in writing of Oxford University Press, or as expressly permitted by law, or under terms agreed with the appropriate reprographics rights organization. Enquiries concerning reproduction outside the scope of the above should be sent to the Rights Department, Oxford University Press, at the address above You must not circulate this book in any other binding or cover and you must impose this same condition on any acquirer A catalogue record for this title is available from the British Library Library of Congress Cataloging in Publication Data (Data available) ISBN 0 19 963846 2 (Hbk) 0 19 963845 4 (Pbk) 10 9 8 7 6 5 4 3 2 1 Typeset by Newgen Imaging Systems (P) Ltd., Chennai, India Printed in Great Britain on acid-free paper by The Bath Press, Avon

Protocol list

Probe oligonucleotide structure and dynamics Determination of solution concentration of single stranded oligonucleotide and assessment of purity of oligonucleotide 26 Attachment of oligonucleotides 28 CNBr activated coupling to an edge plan graphite electrode 29 Reactive electrophoretic deposition 30 Hybridization conditions Determination of melting temperature 32 Screen-printing technology Preparation of a screen-printed glucose electrode 50 Design of the biosensor Enzyme immobilization on collagen membranes 102 Enzyme immobilization on membranes Immunodyne ABC type from Pall-Gelman 103 Enzyme immobilization on Ultra Bind type membranes 103 Chemical modification of HRP with His-tag 104 Enzyme loading of IDA sepharose beads 104 Preparation of the sensing layer with a photocross-linkable polymer (PVA-SbQ) 104 Fluorescent aryl sulfonamides for zinc(ll) determination Synthesis of ABD-N 115 Removal of zinc from carbonic anhydrase (CA) Preparation of apoprotein 116 Avoidance of metal ion contamination Preparation of solutions free of contaminating metal ions 118 Determination of Zn using a reagent approach Determination of zinc concentration with ABD-N: a reagent approach 322 Calibration of anisotropy Determination of Cu and others by using a reagentless approach 324 Measurement and calibration of anisotropy 128 Single-analyte detection using an enzymatic sensing layer Silanization/functionalization of a fiber tip 335 PAN-enzyme sensors 135 xi

PROTOCOL LIST

Multi-analyte arrays Preparation of acryloylfluorescein (7) 137 Fabrication of an enzyme/pH array 137 Chemical etching of a germania-doped imaging fiber bundle 339 Internal encoding 340 External encoding 340 Enzyme immobilization by physical absorption 343 Enzyme immobilization with glutaraldehyde (15, 18) 342 Amine amplification with PEI (12, 13) 143 Periodate oxidation (19, 20) 143 Preparation of ssDNA probe labeled microspheres (13) 145 Preparation of molecular beacon-modified microspheres (23) 146 Fabrication of a fiber-optic based living array of cells 348 Cell viability assay with BCECF-AM 150 Cell viability test via pH sensitive nanospheres 150 Planar patch clamp Fabrication of silicon chips 357 Dry cleaning and activation of silicon chips 358 Wet cleaning and activation of silicon chips 359 Surface modification of the silicon chips 360 Preparation of giant unilamellar vesicles 363 Formation of PLBs on a silicon chip 162 Fabrication of Sylgard® pads 165 Impedance spectroscopy of tethered lipid membranes Preparation of a Ca2+-sensitive hybrid lipid layer electrode 372 Preparation of an OmpF-containing lipid bilayer tethered on a gold electrode 375 Formation of a proteolipid bilayer tethered by SLIC to a gold electrode 178 Rational protein engineering Protein sequence alignment 388 Creation of three-dimensional models 393 Molecular graphics: display and basic calculations on protein structures 394 Calculation of a map of electrostatic potentials on a protein surface 397 Hot start PCR 207 Agarose gel electrophoresis of DNA 209 Purification of PCR products from agarose gels 233 Megaprimer PCR 213 Restriction digest of DNA 215 Ligation of a DNA insert in a vector 236 Transformation of a plasmid in a bacterial host 217 Isolation of a plasmid from a bacterial host 238 Directed evolution QuikChange® PCR mutagenesis 223 Error prone PCR 225 DNA shuffling 227 High throughput screening for NAD(P)H-dependent activity 233 Other aspects of protein engineering 234 Fluoresence labelling of the S37C mutant of the maltose binding protein 235

xii

Abbreviations

BCA Bicinchoninic acid BEE Benzoin ethyl ether BLM Black lipid membrane bp base pair BSA Bovine serum albumin CVFF Consistent valence forcefield ECL Electrochemiluminescence ESFF Extensible systematic forcefield FIA flow low injection analysis HF Hydrofluoric acid HRP Horseradish peroxidase IPTG Isopropyl-1-thio-BD-galactopyranose ITCHY Iteractive Truncation for the Creation of Hybrid Enzymes LB Luria-Bertani broth NADH Nicotinamide adenine dinucleotide ND Neutral density PAA poly (acrylamide)-co-poly-(N-vinylimidazole) PAN Poly (acrylamide-co-N-acryloxysuccinimide) PBS Phosphate-buffered saline PCR Polymerase chain reaction pdb Protein data bank PEI Polyethyleneimine PLB Planar lipid bilayer PMT Photomultiplier tube PNA Peptide nucleic acids PVI poly(N-vinylimidazole) PVP poly(4-vinylpyridine) RMSD Root mean square deviation SBB Sodium borate buffer SCE saturated calomel electrode SEEP Staggered Extension Protocol SHIPREC Sequence Homology Independent Protein Recombination xiii

ABBREVIATIONS

SNP SPR SSPE TB UV

xiv

single nucleotide polymorphism surface plasmon resonance Saline sodium phosphate EDTA Terrific broth Ultraviolet

Contributors

Phil Bartlett

Michael S. Fleming

School of Chemistry University of Southampton Southampton SO17 1BJ United Kingdom

Department of Chemistry Tufts University Medford, MA 02155 USA

Loïc J. Blum Ldboratoire de Génie Enzymatique et Biomoléculaire Université Claude Bernard Lyon 1 69622 Villeurbanne CEDEX

Daren J. Caruana The Department of Chemistry University College London London WC1H OAJ United Kingdom

Pierre R. Coulet Ldboratoire de Génie Enzymatique et Biomoléculaire Université Claude Bernard Lyon 1 69622 Villeurbanne CEDEX

Michele L. Cramer Department of Biochemistry and Molecular Biology University of Maryland School of Medicine 655 West Baltimore Street Baltimore, Maryland 21201-1559

Gianfranco Gilardi Department of Biological Sciences Imperial College London, Technology and Medicine London SW7 2AY United Kingdom

Laurent Giovangrandi Ecole polytechnique fédérate de Lausanne (EPFL) Ecublens, CH-1015 Lausanne

Adam Heller The University of Texas at Austin Department of Chemical Engineering 1 University Station C0400 Austin, TX 78712-1062

Badri Maliwal Department of Biochemistry and Molecular Biology University of Maryland School of Medicine 655 West Baltimore Street Baltimore, Maryland 21201-1559

Jason R. Epstein Department of Chemistry Tufts University Medford, MA 02155 USA

Michael Mayer Ecole polytechnique fédérale de Lausanne (EPFL) Ecublens, CH-1015 Lausanne

xv

CONTRIBUTORS

Caroline L. Schauer

Chee-Seng Toh

Department of Chemistry Tufts University Medford, MA 02155 USA

Department of Chemistry National University of Singapore Singapore 117543

Shannon E. Stitzel

Ecole polytechnique fédérale de Lausanne (EPFL) Ecublens, CH-1015 Lausanne

Department of Chemistry Tufts University Medford, MA 02155 USA

Thierry Stora Ecole polytechnique fédérale de Lausanne (EPFL) Ecublens, CH-1015 Lausanne

Samuel Terrattaz Ecole polytechnique fédérale de Lausanne (EPFL) EcuUens, CH-1015 Lausanne

Richard B. Thompson Department of Biochemistry and Molecular Biology University Of Maryland School of Medicine 655 West Baltimore Street Baltimore, Maryland 21201-1559

xvi

Horst Vogel

David R. Walt Department of Chemistry Tufts University Medford, MA 02155 USA

Hui Hui Zeng Department of Biochemistry and Molecular Biology University Of Maryland School of Medicine 655 West Baltimore Street Baltimore, Maryland 21201-1559

Xian-En Zhang Wuhan Institute of Virology Chinese Academy of Sciences Wuhan 430071, Hubei, China

Preface

Over the past 20 years, the field of biosensor research have had a significant impact in both laboratory research and the commercial sector. Over that period, biosensors have revolutionized the care and management of diabetes and have had important impacts in several other areas of clinical diagnostics. Europe, North America, and Asia-Pacific have all seen the rise of small- and mediumsized companies seeking technical and application niches in the manufacture or use of biosensors. The current activity in both gene and protein "biochips" can be seen as the latest set of tools that allow users who are not analytical science practitioners to make technically complex and reliable measurements with the minimum of intervention. Similarly, the concern about the dissemination of chemical or biological weapons and the need for their rapid and reliable detection will need to be met by devices that have many characteristics in common with biosensors. When the first edition of this book was published some 13 years ago, the field was dominated by electrochemical devices and the focus was primarily on clinical diagnostics. This dominance was partly a consequence of electrochemical (primarily amperometric) biosensors having been the first examples to be intensively investigated, but also because they formed the basis of the first mass produced and marketed devices (for blood glucose measurement). Whilst electrochemical methods are still major influences in biosensor research, optical methods have come increasingly to the fore and this trend is reflected in the current volume. Other significant developments have included the use of protein engineering methods to design and produce proteins that are better suited for the role as device component rather than constituents of a living cell, and the application of production methods such as screen printing that can manufacture very large number of sensors with a high degree of reproducibility. This latter is a prerequisite as biosensors move from laboratory demonstration to commercial product. The application focus of biosensors has also broadened with time and whilst clinical diagnostics probably remains the single biggest area, roles are also being found in environmental (including food) monitoring, personal security (including warfare), drug discovery, and basic biological research. v

PREFACE

Despite these changes, the field remains one of interdisciplinary challenge and exemplifies the current worldwide trend in engaging physical scientists and engineers in activities at the forefront of the biological sciences. As the latter move toward a more quantitative approach, the need for sophisticated tools for measurement and analysis of biological systems become ever more pressing and many of the ideas that drove and continue to drive biosensor research are directly relevant to this endeavor. The editors of this revised edition exemplify the discipline "hopping" that is characteristic of research in biosensors. One of us (JMC) trained as a biochemist and now holds a chair in an Electronics Department whilst the other (AEGC) trained as a chemist and holds a chair in a Biological Sciences Department. The contributors, who have generously donated their time and experience in writing the chapters that follow, demonstrate not only the progress in biosensors research over the past few years but also the experimental approaches that have enabled this progress. Finally, we would particularly like to acknowledge the contributions to biosensor research made by Professor Pierre Coulet. One of the pioneers of this field, Pierre has recently retired from his chair at the Universite Claude Bernard and we wish him a healthy and prosperous retirement, working on his farm in the Ardeche. JMC AEGC

vi

Contents

Protocol list xi Abbreviations xiii Contributors xv 1

2

Redox hydrogel-based electrochemical biosensors 1 Adam Heller 1 Electron conducting redox polymers in biosensors 1 1.1 Relationship to mediator-based sensors 3 1.2 The electrocatalytic activity of redox hydrogels and the "wiring" of enzymes 2 1.3 Dependence of the diffusivity of electrons on crosslinking and the advantage of composite electrodes 2 1.4 The redox polymers and their electrochemistry 3 1.5 Crosslinkers and crosslinking 4 2

Enzyme electrodes 4 2.1 Electron transfer between enzyme and polymer redox centers 4 2.2 Microscopic homogeneity and salt effects in the redox polymer-enzyme system 6 2.3 Optimal compositions 6 2.4 Special matrices 7

3

Specific sensor examples 7 3.1 Amperometric and potentiometric biosensors of substrates of "wired" redox enzymes 7 3.2 Sensor measuring the turnover rate of hydrolytic and other non-redox enzymes 8 3.3 In vivo glucose sensors 10 3.4 Affinity sensors 30

Hybridization at oligonucleotide sensitive electrodes 19 Daren J. Caruana \ Introduction 19 2

Function of oligonucleotide sensitive electrodes 20

3 Hybridization efficiency and sensitivity 21

vii

CONTENTS

3

4

Probe oligonucleotide structure and dynamics 22 4.1 Surface concentration 22 4.2 Probe length and orientation 23 4.3 Attachment of probe 25

5

Hybridization conditions 30 5.1 Temperature 31 5.2 Ionic strength 32 5.3 Base mismatch 33 5.4 Mass transport 34 5.5 Nonspecific adsorption 34 5.6 Other factors 36

6

Hybridization kinetics

7.

Summary 38

36

Screen-printing methods for biosensor production 41 Xian-En Zhang \ Introduction 41 2

Screen-printing technology 42 2.1 Materials and methods 42 2.2 Apparatus 47 2.3 Printing patterns 48 2.4 Printing process 49

3 Applications 51 3.1 Clinical diagnosis 51 3.2 Food analysis bioprocess control 52 3.3 Environmental monitoring 52 3.4 Other approaches 54 4 4

Conclusion 55

Kinetic modeling for biosensors 59 Philip Bartlett and Chee-Seng Toh \ Introduction 59 1.1 The purpose and practice of modeling 59 1.2 Enzyme kinetics 60 1.3 Basic electrochemistry 63 2

Modeling 69 2.1 The flux diagram for the membrane|enzyme|electrode 70 2.2 Simplifying assumptions 70 2.3 The flux equations 71 2.4 Solution of flux equations 73 2.5 Deriving a complete kinetic model 78 2.6 Experimental verification of approximate analytical kinetic models 81 2.7 Numerical simulation methods 82

3 Applications 89 4

viii

Kinetic modeling in other types of biosensors 89 4.1 Potentiometric enzyme electrodes 90 4.2 Optical and photometric biosensors 90 4.3 Immunosensors 91

CONTENTS

5 Conclusions 92 List of symbols 92 5

Bio-, chemi-, and electrochemiluminescence for fiber-optic biosensors 97 Loïc J. Blum and Pierre R. Coulet \ Introduction 97 2 Design of the biosensor 97 2.1 Optical waveguide 97 2.2 Setup 98 2.3 Light-emitting reactions 300 2.4 Preparation of the sensing layer 303 3 Examples of determinations with the luminescence sensors 105 3.1 ATP determination 305 3.2 NADH determination 305 3.3 Extension to other analytes using dehydrogenases as auxiliary enzymes 105 3.4 H2O2 determination 306 3.5 Extension to other analytes involving H2O2 detection 307 4 Concluding remarks 108

6

Determination of metal ions by fluorescence aniostropy: A practical biosensing approach 109 Richard Thompson, Badri Maliwal, Hui Hui Zeng, and Michele Loetz Cramer 1 Introduction and rationale 109 1.1 Why fluorescence anisotropy to determine metal ions? 309 2 Theory of anisotropy-based determination of metal ions 111 2.1 "Reagent" approaches 333 2.2 "Reagentless" approach 332 3 Fluorescent aryl sulfonamides for zinc(II) determination 114 4 Removal of zinc from carbonic anhydrase (CA) 115 5 Avoidance of metal ion contamination 117 6 Determination of Zn using a reagent approach 119 7 Determination of Cu and other ions by using a reagentless approach 123 8 Calibration of anisotropy 124

7

Fluorescence-based fiber-optic biosensors 131 David R. Walt, Caroline L Schauer, Shannon E. Stitzel, Michael S. Fleming, and Jason R. Epstein

\ Introduction 131

1.1 Fiber polishing 333

2 Single-analyte detection using an enzymatic sensing layer 134 2.1 Enzymatic sensing layer 334 2.2 PAN gel immobilization 335

ix

CONTENTS

3 Multi-analyte arrays 136 3.1 Immobilization via polymer photodeposition 136 3.2 Microwell array platform preparation 138 3.3 Live-cell array fabrication 146 4 8

Conclusions 151

Functional analysis of ion channels: Planar patch clamp and impedance spectroscopy of tethered lipid membranes 153 Michael Mayer, Samuel Terrettaz, Laurent Giovangrandi, Thierry Stora, and Horst Vogel \ Introduction 153 2

Planar patch clamp 154 2.1 Concept of patch clamp on a chip 354 2.2 Formation of planar bilayers on a chip 357 2.3 Chip-based planar bilayers: single-channel measurements of alamethicin pores 165

3 Impedance spectroscopy of tethered lipid membranes 168 3.1 Basics of impedance spectroscopy 168 3.2 Measuring technique and electrochemical cell 370 3.3 Hybrid lipid layer 370 3.4 Tethered lipid bilayers 174 3.5 Lipid bilayer tethered via surface-attached proteins 377 3.6 Highly insulating tethered lipid bilayers for single-channel experiments 179 9

Protein engineering for biosensors 185 Gianfranco Gilardi \ Introduction 185 2

Rational protein engineering 187 2.1 Modeling and calculations on protein structures 188 2.2 Site-directed mutagenesis 199

3 Directed evolution 222 3.1 Random mutagenesis: error prone PCR 224 3.2 Recombination: DNA shuffling 226 3.3 Functional screening of the library 230 4

Functional characterization of the mutants 234

5

Other aspects of protein engineering 234

6

Concluding remarks 238

Index

x

241

Chapter 1 Redox hydrogel-based electrochemical biosensors Adam Heller University of Texas at Austin, USA.

1 Electron conducting redox polymers in biosensors 1.1 Relationship to mediator-based sensors Redox hydrogel-based sensors are a subgroup of mediator-based sensors. Unlike their older cousins, the diffusional mediator-based sensors, their electron transport mediating redox centers are polymer-bound. When the resulting redox polymers are crosslinked on electrodes, they become insoluble, but swell in water to form redox hydrogels (1–4). These hydrogels can be as soft as Jell-O, or as tough as leather, depending on the extent of their crosslinking (1, 5). Upon hydration and swelling, the mobility of their segments is increased. This increased segmental mobility translates to an increase in the frequency of electron-transferring collisions between the tethered redox centers, and increases the electronic conductivity of the redox hydrogels. The diffusivity of electrons in the redox hydrogels is typically in the 10~ 6 -10~ 10 cm 2 s" a range (6-9). In addition to conducting electrons, the redox hydrogels, like other hydrogels, also conduct ions (10-18). Because they are permeable to water-soluble species, water-soluble chemicals like nitrite and biochemicals like ascorbate and dopamine can be electrooxidized or electroreduced in their three-dimensional matrices (19-21). When redox enzymes are co-immobilized in the redox hydrogels, their reaction centers can also be electrooxidized or electroreduced. The enzyme-containing redox hydrogels catalyze, therefore, the electrooxidation and the electroreduction of the substrates of the enzymes (22-54). Enzymes with FAD, FMN, PQQ., heme, and copper-containing redox centers have been co-immobilized and their centers are electroactive. Examples of the electrooxidized biochemicals include glucose, fructose, cellobiose, lactate, cholesterol, glycerol-3-phosphate, pyruvate, phenols, primary and secondary alcohols, histamine and other amines, D-amino acids, and glutamate. The electroreduced chemicals include O2 and H2O2. 1

ADAM HELLER

1.2 The electrocatalytic activity of redox hydrogels and the "wiring" of enzymes The electrocatalytic activity of the films formed by crosslinking depends on electron-transferring collisions between redox centers of the co-immobilized enzymes and those of the redox polymer (7-18). When these collisions are frequent enough to assure the efficient collection of the electrons from the enzyme reaction centers by the redox polymer, or the efficient delivery of electrons by the redox polymer to the reaction centers of the enzyme, the enzyme is said to be electrically "wired" and the redox polymer is said to "wire" the enzyme. The crosslinked redox polymers are poor electron conductors when dry: hydration is of essence for the electron transport to be fast and for the current density to be high. When the electron diffusivities reach, after hydration, 10~ 6 -10~ 9 cm 2 s" a and the co-immobilized redox enzyme turns over at a rate of >100 s ~ \ the limiting current density of substrate-electroreduction/oxidation on a semi-infinite planar enzyme electrode is typically of 10~ 3 -10~ 4 Acm~ 2 . Because their electron-transfer mediating centers are not leached, the "wired" enzyme electrodes can be used in microelectrodes (55-59), in experiments in vivo (60-68), and in flow systems (69-79).

1.3 Dependence of the diffusivity of electrons on crosslinking and the advantage of composite electrodes Nanocomposite enzyme electrodes can be made without covalent crosslinking by sequentially adsorbing countercharge films of a redox polymer, usually a polycation and enzymes which is often a polyanion at neutral pH (80-85). In the absence of covalent crosslinking, these films disintegrate in use and their components are slowly leached. Crosslinking by forming covalent bonds prevents the disintegration. While leaching of the redox polymer is prevented by crosslinking, excessive crosslinking reduces the mobility of the segments of the immobilized polymer and thereby the diffusivity of electrons and the current density (5). Excessive crosslinking-related losses are observed not only when the crosslinker forms covalent bonds, but also when ionic bridges are formed. Such bridges are formed in polycationic redox polymer films by large, little-hydrated anions like C1O4~, PO 4 3 ~, and citrate (5, 9,15). Small, well-hydrated anions like Cl~ are less effective crosslinkers. Even though increasing the concentration of NaCl does not reduce the diffusivity of electrons, such an increase can be damaging when it causes the decomposition of electrostatic complexes formed of a polyanionic enzyme and a polycationic redox polymer (86-88). The formation of an electrostatic complex prevents phase-separation of the enzyme and the redox polymer. In the case of polymers formed by the polymerization of weak bases, lowering of the pH increases the fraction of the bases that are protonated and thereby the charge on the polymers. This increases their hydration and thereby the mobility of their segments and the diffusivity of electrons. Plots of the dependence of the electron diffusivity on pH reveal a transition between the less electron-conducting 2

REDOX HYDROGEL-BASED ELECTROCHEMICAL BIOSENSORS

non-protonated and the more electron-conducting protonated forms of the redox polymers (6, 7). The density of positive charge on the crosslinked redox polymer is similarly increased upon increasing the fraction of the transition metal complexed bases (7-9). Even when the electron diffusivity is diminished by excessive crosslinking, high current densities of substrate electrooxidation or electroreduction can be maintained in hydrophilic graphite-based composite electrodes (89). Current densities of >2 mAcm~ 2 of glucose electrooxidation are reached when the composite is made with carbon graphite particles or carbon fibers, when the particles or fibers are of 10 |j,m diameter and are hydrophilic (89). When the particles are hydrophobic or smaller, the current density diminishes because the mass transport in the channels that are narrow or are not wetted by water is poor. This also raises the hydration, the segmental mobility, and the diffusivity of electrons. Nevertheless, when more than 20% of the heterocyclic nitrogens of poly(4-vinylpyridine) (PVP) or poly(N-vinylimidazole) (PVI) are complexed, for example, with [Os(bpy)2Cl]+'2 + , the density of ions and counterions is so high that the polymer becomes rigid and its electronic conductivity decreases.

1.4 The redox polymers and their electrochemistry The redox polymers that were most extensively used in the "wiring" of enzymes were made by attaching redox functions to PVP (1, 8, 9,17,19, 22-25, 90), PVI (32,

91-93), and poly(acrylamide)-co-poly(N-vinylimidazole) (PAA) (34). The redox functions were usually coordinatively pyridine or imidazole-attached complexes of Os 2+/3 + , such as [Os(bpy)2Cl] + /2+ (where bpy = 2,2'-bipyridine). Typically between 20% and 7% of the rings of the polymer were coordinated with the redox polymer, most often about 10%. An example of a redox polymer formed of a PVP is shown in Figure 1. The redox potentials of the polymers depend predictably on their chemistry. The following trends are consistent with the predictions of Lever (94) and are experimentally observed in the redox polymers. The more exoergic the binding of the ligands in the complex, the more positive its redox potential. Because the coordinative binding of osmium to vinyl pyridine is stronger than to N-vinylimidazole, the redox potentials of PVP-based polymers are positive by about 0.1 V relative to the related PVI-based complexes. Substitution of chloride ions in the inner coordination sphere of a complex by a nitrogencontaining heterocyclic ligand such as a pyridine or an alkylimidazole shifts the potential in the oxidizing direction. The redox potentials are more positive for Ru 2+ ' 3 + complexes than they are for Os2+'3+ complexes. The latter are positive relative to the Co2 + '3+ complexes when the ligands are similar. For a particular ligand, such as pyridine, 2,2'-bipyridine or 2,2',6',2"-terpyridine, an electron withdrawing substituent shifts the potential in the positive direction and an electron adding substituent shifts the potential in the negative direction. Thus, the potentials are shifted increasingly in the negative direction in the series of dicarboxylate, unsubstituted, methyl, methoxy and amine 3

ADAM HELLER

Figure 1 An enzyme wiring redox polymer derived of PVP. About one-sixth of the rings of the polymer are complexed with [Os(bpy)CI] +/2+ and about one-sixth are quaternized with bromoethylamine. Electrons are transferred through collisions between the tethered but mobile osmium-based redox centers. Quaternization makes the polymer water soluble and provides reactive primary amines for condensation with aldehydes produced by periodate oxidation of glycoenzymes.

4,4'-disubstituted 2,2'-bipyridines. When the same redox couple is incorporated in different polymers, the redox potential shifts in the positive direction as the polymer is made more polycationic or less polyanionic. Attaching of the redox function to a flexible and long spacer arm increases the volume element in which the couple can collide with a neighboring function and increases the diffusivity of electrons.

1.5 Crosslinkers and crosslinking A particularly convenient way of crosslinking polymers of nitrogen-containing heterocyclics is by reacting these with di- or tri-functional epoxides (1). Polyethylene glycol diglycidyl ether is a convenient crosslinker because of its solubility in water. Its epoxides react at ambient temperature with nitrogens of the heterocyclic rings. The polymers are also crosslinked by oxidizing their Os3+ redox centers to a higher oxidation state osmium compound, for example, by electrooxidation above >0.8 V versus SCE (SCE is the potential of the saturated calomel electrode).

2 Enzyme electrodes 2.1 Electron transfer between enzyme and polymer redox centers The exchange of electrons between a redox polymer and an enzyme is governed by the classical rules of electron transfer. In the multi-step electron transfer 4

REDOX HYDROGEL-BASED ELECTROCHEMICAL BIOSENSORS

process between an electrode and a substrate-reduced or substrate-oxidized enzyme reaction center, the transport of electrons is rapid only if all steps are thermodynamically downhill. Because the electrons cascade to or from the electrode, the redox potential of the polymers must be reducing with respect to the potential of the electrode and oxidizing with respect to the potential of the enzyme when a substrate is electrooxidized. The opposite is true when a substrate is electroreduced (25). The faster the self-exchange between the redox centers of a polymer, the faster the rate of electron transfer to or from an enzyme's redox center. The rate decreases exponentially with the distance between the fixed reaction center of the enzyme and the mobile redox couple tethered to the polymer. At equal distance, the exchange rates are higher when the reorganization energies are smaller. When the octahedral (or other) structure of a complex of a transition metal ion is preserved upon its oxidation and reduction, the reorganization energy is smaller, and electrons are more rapidly transferred when the distance is fixed and transferred to a more remote center when at an equally high rate. This is the case if the coordination is strong and the interatomic distances and angles do not change excessively when the complex is oxidized or reduced. Complexing of the metal ion thus provides for a higher self-exchange rate. However, because complexing also increases the size of the redox couple it can increase the distance of closest approach to an enzyme's deeply buried reaction center. When the reaction centers of the enzyme are deeply buried in its protein or glycoprotein, the redox couple approaches the center mainly through the channel in which the substrate of the enzyme diffuses. Because the rate of electron transfer drops by about a factor of 2.7 for each added angstrom, polymers with less bulky complexes are usually required for effective "wiring" of redox enzymes. By far the most studied enzyme has been glucose oxidase, the structure of which is known. The oxidation of the FADH2 centers of this enzyme is effectively mediated by both positively and negatively charged redox couples. Fe(CN)63~ '4~ is an effective and widely used redox mediator, as is the ferrocene/ferrocinium couple. The osmium complex based redox couples, which are larger, also mediate very effectively the exchange of electrons between the FADH2 centers of glucose oxidase and electrodes. When they are polymer-bound, their effectiveness depends, however, on their size: The bulkier the complexes, the less they approach the reaction centers of the enzyme and the slower the transfer of electrons from these to the redox polymer. Slow electron exchange implies, in the case of FADH2 enzymes, competition by oxygen: The electrons flow not only through the redox polymer to the electrode, but also to O2, the natural co-substrate of the enzyme, reducing it to hydrogen peroxide. Thus, the competition of oxygen for electrons of the redox polymers is a measure of the rate of electron transfer between the enzyme and the redox polymer. It is distinctly slower for very large complexes, even though these have high self-exchange rates. Because the closest distance of approach between a redox center of a polymer and the reaction center of the enzyme is through the narrow channel in which 5

ADAM HELLER

the substrate enters and the product leaves the enzyme, the rate of electron transfer depends on the flexibility of the polymer's backbone. High transfer rates and, therefore, high current densities are observed when the polymers are flexible. The electron-conducting, but rigid polypyrroles and polythiophenes yield high current density enzyme electrodes only when they were modified with pendant flexible hydrophilic or ionic redox functions (95-96).

2.2 Microscopic homogeneity and salt effects in the redox polymer-enzyme system Since the gain in entropy is small when two macromolecules are mixed, polymers are usually not miscible. They become miscible only when a bond is spontaneously formed, in an exoergic reaction, between the two macromolecules. The proteins and glycoproteins of enzymes are usually charged at neutral pH. Their charge is defined by their isoelectric points and on the pH of the solution. Phase separation of the redox polymer and the enzyme is avoided when the charge of the redox polymer is opposite to the charge of the enzyme (86-88). Glucose oxidase and lactate oxidase, which are polyanions at neutral pH, are effectively "wired" by redox polycations, including polymers made by attaching osmium complexes to PVP or PVL When the backbone is a polyanion, as it is in the case of polyacrylic acid, it is more difficult to avoid the separation of the phases and the polyanionic enzymes are less effectively connected to the electrodes by the polyanionic redox polymer. When microscopic homogeneity and effective "wiring" are provided by electrostatic interactions between the redox polymer and the enzyme, the quality of the "wiring" depends on the ionic strength of the solution used. Large salt effects are observed, because at high salt concentrations the charges of the interacting macromolecules are effectively screened by ions. In a concentrated NaCl solution, the dominant anions near the redox polycation are not carboxylate functions of the enzyme protein, but Cl~ anions. Similarly, the dominant cations in the proximity of the polyanionic enzyme are not the cations of the redox polymer, but Na + cations. When the charges of the enzyme and the polymer are balanced and the salt concentration is low, an insoluble enzyme-polymer adduct precipitates. This adduct re-dissolves at high salt concentration where the enzyme is not effectively "wired." (86-88) There is a clear optimum of NaCl concentration for the wiring of enzymes, which happens to coincide for glucose oxidase and its "wires" with the ~0.1 M physiological salt concentration at neutral pH. The loss of effective "wiring" and therefore in current density at high ionic strength can be overcome by crosslinking the enzyme and the polymer when the ionic strength is low. Such crosslinking prevents phase separation by preventing the dissociation of the enzyme and its "wiring" redox polymer.

2.3 Optimal compositions In well-crosslinked electrostatic adducts of enzymes and redox polymers, where phase separation is unlikely, the current densities reach an optimum when 6

REDOX HYDROGEL-BASED ELECTROCHEMICAL BIOSENSORS

enzyme is incorporated in the redox polymer, then decline when the fraction of the enzyme becomes excessive. The optimal enzyme concentration is that concentration where the rate of supply of electrons or holes (electron vacancies) by the substrate to the enzyme equals the rate of their removal from the enzyme to the electrode. When the polymer is in excess, there is not enough enzyme to supply electrons or holes to the wire. When the enzyme is in excess, the electroncurrent carrying capability of matrix is insufficient. For ~100 kDa enzymes turning over at about 1000 times per second the optimal enzyme weight fraction is about 0.3. For slower enzymes it is higher. Comparison of enzymes with different well-bound co-factors suggests that, in general, those with heme and PQQ functions transfer electrons more readily to redox polymers than enzymes with FAD and FMN co-factors. The reason is that the heme and PQQ. functions are usually closer to their periphery. The feasibility of preventing the escape of the weakly bound NADH and NADPH co-factors from the protein through forming specific chemical bonds between the NADH or the NADPH and either the redox polymer or the enzyme has been extensively studied. At this time it does not appear that such systems with immobilized NADH or NADPH can yield high (~10~ 3 Acm 2 ) current densities.

2.4 Special matrices The redox polymers and enzymes can be co-immobilized by the sol-gel method in hydrated silica matrices, in which the enzymes are stabilized (97, 98). These matrices are, however, insulating and hamper, in the absence of carbon or metal additives, the direct electrical communication between the enzymes and the electrodes (99-103). Electrodes can be conveniently made with carbon pastes (104-106), formed of graphite and mineral oil, where stabilization by poly(ethyleneimine) has been reported (105). The hydrated wired enzymes have been used in organic solvents and CO2 (107-109).

3 Specific sensor examples 3.1 Amperometric and potentiometric biosensors of substrates of "wired" redox enzymes When the reaction center of an enzyme is connected through its wire to an electrode, its turnover is observed as an electrical current. Examples of enzymes that were electrically wired include glucose oxidase, PQQ-glucose dehydrogenase, fructose dehydrogenase, cellobiose oxidase, lactate oxidase, cholesterol oxidase, glycerol-3-phosphate oxidase, pyruvate oxidase, tyrosinase, alcohol oxidase, amine oxidase, D-amino acid oxidase, glutamate oxidase, horseradish peroxidase, soybean peroxidase, and laccase. The resulting electrodes measured amperometrically the concentrations of the substrates of their enzyme.

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3.2 Sensor measuring the turnover rate of hydrolytic and other non-redox enzymes When multiple enzymes were incorporated in the redox hydrogel so as to form in situ a precursor the substrate of the electrically connected enzyme, the concentration of the precursor was amperometrically monitored. For example, by co-incorporating cholesterol oxidase and cholesterol esterase in the redox hydrogels, the concentration of the cholesterol esters was monitored. In this case, hydrolysis of the cholesterol esterase, catalyzed by the esterase, yielded cholesterol, which was electrooxidized by the wired cholesterol oxidase. In a second example, co-incorporation of acetylcholine esterase, choline oxidase, and horseradish peroxidase allowed the monitoring of acetylcholine esterase inhibitors. In this case, the acetylcholine esterase hydrolyzes acetyl choline to choline; the choline reacted with oxygen to yield betaine aldehyde and hydrogen peroxide, in the reaction catalyzed by choline oxidase; and the hydrogen peroxide was electroreduced on the electrically connected reaction centers of horseradish peroxidase. The reaction centers of choline oxidase were not wired. If they were connected, the two parallel connections of the choline oxidase and that of the horseradish peroxidase would have produced opposite currents and the measurements would not have been meaningful (41, 42, 110, 111). The amperometric enzyme electrodes formed by connecting the redox centers of enzymes to electrodes to redox hydrogels have, like all other enzyme electrodes, characteristic apparent Michaelis constants and maximal current densities. In the linear part of the current vs concentration diagram, the entire influx of the electroreduced or electrooxidized substrate is reacted. When the influx of substrate exceeds the rate at which it is electroreduced or electrooxidized, the current density no longer increases linearly with its concentration. In this case, the sensitivity is reduced and eventually the electrode "saturates," and the current no longer increases when the concentration of the substrate is increased. In order to extend the linear range of response to higher concentrations, one can overcoat the sensors with a membrane that limits the influx of the substrate. Because the enzyme electrodes have high current densities and it is attractive to miniaturize the electrodes, their overcoating with a membrane causes a particular problem. Pressure sealing of a membrane with an elastomeric O-ring, is obviously irrelevant, because O-rings and pressure seals are not available for very small electrodes. Forming the membrane in situ by casting from a solvent also poses a special problem because it is the residual solvent fraction that determines the pore size and distribution defining the relative rates of nucleation and diffusion, which depend on difficult to control parameters such as temperature gradients, vapor pressure gradients, and the presence or absence of nucleation centers. The most reproducible micromembranes are formed by sequential chemisorption reactions of polymers that react with each other. For example, a membrane of a glucose sensor can be formed by successive chemisorption of a polycation/ polyanion/polycation... sequence (68). The thickness and the mass transport

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REDOX HYDROGEL-BASED ELECTROCHEMICAL BIOSENSORS

characteristics of these layer-by-layer formed membranes are relatively easy to control, allowing precise control of the permeabilities of the substrate, the products, and of all interferants. The layer-by-layer assembled micromembranes allow simultaneous optimization of all biosensor characteristics, including their linear range, their apparent engineering life (the life seen by the user), their drift (the variation of the sensitivity with time), and the sensitivity to interferants (68). Because the typical current densities are of 10~ 4 -10~ 3 AcrrT2 and because currents of 10"9 A can be conveniently measured using inexpensive potentiostats, the typical electrode dimensions are of 10~ 4 -10~ 6 cm2. The diameter of the smallest "wired" enzyme electrodes made thus far was of a few micrometers (55-59). These electrodes had microelectrode characteristics, the diffusion of the electrons to and from the electrode, as well as the substrates, and products being radial (24-26). Biosensors made with polycationic redox polymers have a problem not encountered in sensors made with diffusional mediators. The charge of the cations of the polymers is balanced by the charge of mobile anions in the solution, and the density of these anions in the redox hydrogel greatly exceeds their concentration in the solution. The permeability of the anions in the redox polymers is the product of their diffusivity and their concentration in the film. The high concentrations of anions and the high diffusivity makes the permeability to anions high. This high permeability exacerbates interference by anionic interferants, particularly by urate and ascorbate. While urate interference can be reduced, even eliminated, by poising the electrodes at potentials negative of 100 mV vs Ag/AgCl, interference by ascorbate is severe and can only be eliminated by a membrane in which the solubility of ascorbate is much lower than the solubility of glucose. This is the case when a membrane is formed of multiple layers of polyanions and polycations, the charges being internally balanced. In the absence of an excess of cationic sites, the anions are not particularly soluble in the membrane (68). When the potential at which the electrode is poised exceeds 100 mV (vs Ag/ AgCl), the electrooxidation of urate is particularly damaging. Because the concentration of urate in the redox polymer can be high, urate can electropolymerize and precipitate in the redox polymer film. The polymer formed is much less soluble in water than urate itself. Its presence is seen not only in the reduced current density but also in the reduction of the thickness of the electroactive layer. Transition metal ions, such as Zn2+ and Fe2+ can adversely affect the electrodes by coordinatively crosslinking pyridines and imidazoles of the polymers wiring the enzymes. The crosslinking reduces the diffusivity of electrons, and the thickness of the electroactive film shrinks. This detrimental effect can be avoided by incorporating transition metal ion coordinating centers in the membrane. When the membrane is made of sequential layers of polyacrylic acid and a polycation, then incorporation of polyfvinyl pyridines) in the polycationic layer stops the influx of Fe2 + and Zn2 + and stabilizes the electrode (68).

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3.3 In vivo glucose sensors The tailoring of the characteristics of sensors made by connecting reaction centers of glucose oxidase with redox polymers to electrodes through their overcoating with precisely engineered micromembranes opens the way to implantable biosensors with well-defined characteristics. Specifically, sensors of about 5 x 10 ~ 4 cm2 area have been engineered to simultaneously provide a sensitivity of about 3 nAmM"1; a linear range of 1-30 mM; a drift of < 5% per day; and operational stability sufficient to eliminate the need to recalibrate the sensor for 3 days. When these sensors are overcoated with thin glucose permeable film of poly(ethyleneoxide) and then are heparinized, they accurately measure the subcutaneous and intravenous glycemia in animals. The implanted sensors have no leachable components and are insensitive to changes in partial pressure of oxygen. Because the typical resistance of the skin is about 100 kQ and the currents are of ~10 ~ 8 A, counter-reference electrodes larger than 1 cm2 can be external, that is, placed on the skin. For currents of < 50 nA, the potential drop is about < 1 mV, which is insufficient to perturb the measurement (68).

3.4 Affinity sensors By attaching affinity reagents to redox enzymes, it is possible to construct amperometric electrodes tracking affinity reactions. The current increase results in the electrode becoming electrocatalytic when electrical contact is established between the electrode and the reaction centers of the enzyme with which one of the members of the affinity couple is labeled (112-120). Although the affinity reactions can be readily observed with any redox enzyme, it is convenient to use peroxidases, because their heme centers are easy to wire and their turnover rate is high. Their substrate, H2O2, can be generated internally in the affinity sensing layer (117). Hydrogen peroxide is generated by reacting choline with dissolved oxygen to form betaine aldehyde and hydrogen peroxide, a reaction catalyzed by co-immobilized choline oxidase, the reaction centers of which are not wired. The affinity reactions can be carried out on rotating electrodes, so as to prevent the transport of the affinity reagents to the surface of the electrodes from controlling the time required for the detection. The rate of the affinity reaction depends on the transport of the affinity reagents in the redox hydrogels which can be slow for reagents larger than 100 kDa, the detection requiring 10-20 min. In hybridization sensors it is convenient to use soybean peroxidase as the labeling enzyme. Unlike horseradish peroxidase, soybean peroxidase is stable enough to be used for temperatures up to 75 °C (35). Thus, when the enzyme is bound to a DNA segment and if the segment hybridizes to a segment of DNA or RNA immobilized in the redox polymer is targeted, good specificity can be achieved by running the hybridization reactions near the melting temperature of the hybrid. Because the melting temperature of hybrids with defects is lower, one can readily distinguish between perfect and imperfect hybrids. Using the DNA modified redox polymers, one can detect in minutes the presence of about 10

REDOX HYDROGEL-BASED ELECTROCHEMICAL BIOSENSORS 40,000 copies of the enzyme labeled DNAon the electrode. Furthermore, a single mismatch in an 18 base pair sequence can be readily observed (119). Beyond the target hybridization of a redox hydrogel bound DNA sequence with an enzyme labeled sequence, it is also possible to observe the hybridization of a long (~100 base) DNA or RNA sequence with a sequence bound to the redox hydrogel. In this case the long sequence is hybridized to the sequence on the electrode; the presence of the long sequence is then confirmed by hybridizing another part of it with a second complementary sequence of the now immobilized long chain, this one labeled. This scheme is applicable equally to the amperometric detection of DNA and RNA. Because the abundance of RNA organisms is greater, by about 10,000, than the abundance of DNA, the method appears at this time to be suitable for the detection of organisms.

References 1. Gregg, B. A. and Heller, A. (1991) Redox polymer films containing enzymes. 1. A redox-conducting epoxy cement: synthesis, characterization, and electrocatalytic oxidation of hydroquinone, J. Phys. Chem., 95(15), 5970-5. 2. Hillman, A. R., Loveday, D. C., Glidle, A., Vos, J. G., Clarke, A. P., Kelly, D., and Bruckenstein, S. (1993) Electrochemical quartz crystal microbalance studies of redox polymer films, Macromol Symp., 80, 323-35. 3. Calvo, E. J. and Etchenique, R. (1999) Viscoelastic changes in Os-containing poly(allylamine) based redox hydrogels for amperometric enzyme electrodes. ]. Phys. Chem. B, 103, 8944-50. 4. Etchenique, R. A. and Calvo, E. J. (1999) Gravimetric measurement in redox polymer electrodes with the EQCM beyond the Sauerbrey limit, Electrochem, Commun., 1,167-70. 5. Binyamin, G. and Heller, A. (1999) Stabilization of wired glucose oxidase anodes rotating at 1000rpm at 37 °C, J. Electrochem. Soc., 146, 2965-7. 6. Aoki, A. and Heller, A. (1993) Electron diffusion coefficients in hydrogels formed of crosslinked redox polymers, J. Phys. Chem., 97, 11014-19. 7. Aoki, A., Rajagopalan, R., and Heller, A. (1995) Effect of quaternization on electron diffusion coefficients for redox hydrogels based on poly(4-vinylpyridine), J. Phys. Chem., 99, 5102-10. 8. Rajagopalan, R., Aoki, A., and Heller, A. (1996) Effect of quaternization of the glucose oxidase "wiring" redox polymer on the maximum current densities of glucose electrodes,/. Phys. Chem., 100, 3719-29. 9. Rajagopalan, R. and Heller, A. (1997) Electrical "wiring" of glucose oxidase in electron conducting hydrogels, Ch. 7. In Molecular electronics (ed. J. Jortner and M. Ratner), pp. 241-54, Blackwell Science Publishers. 10. Pickup, P. G., Kutner, W., Leidner, C. R., and Murray, R. W. (1984) Redox conduction in single and bilayer films of redox polymer, J. Am. Chem. Soc., 106, 1991-8. 11. Oyama, N. and Hatozaki, O. (1988) Redox active membranes and electron transfer, Kagaku (Kyoto), 43, 280-1. 12. Forster, R. J., Vos, J. G., and Lyons, M. E. G. (1991) Controlling processes in the rate of charge transport through osmium 2,2'-bipyridine authors poly(4-vinylpiperidine) complex [Os(bipy)2(PVP)nCl]Cl redox polymer-modified electrodes, J. Chem. Soc., Faraday Trans., 87, 3761-7. 13. Mathias, M. F. and Haas, O. (1992) An alternating current impedance model including migration and redox-site interactions at polymer-modified electrodes,/. Phys. Chem., 96, 3174-82.

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ADAM HELLER 14. Surridge, N. A., Zvanu, M. E., Keene, F. R., Sosnoff, C. S., Silver, M., and Murray, R. W. (1992) Effects of mixed-valent composition and bathing environment on solid-state electron self-exchanges in osmium bipyridine redox polymer films, /. Phys. Chem., 96, 962-70. 15. Mathias, M. F. and Haas, O. (1993) Effect of counterion type on charge transport at redox polymer-modified electrodes, /. Phys. Chem., 97, 921-5. 16. Kelly, D. M. and Vos, J. G. (1994) Osmium and ruthenium poly(pyridyl) redox polymers as electrode coatings, Ekctroact. Polym. Ekctrochem., 2, 173-232. 17. Sharp, M. and Larsson, H. (1995) Studies of the temperature dependence of charge propagation rates in quaternized poly(4-vinylpyridine) polymers containing electrostatically bound and coordinated redox sites, Electroanal. Chem., 386, 189-95. 18. Sun, L, Jiao, K., and Weber, S. G. (1998) Charge transport through osmium-containing redox polymers in nitrophenyl-based solvents: effect of solvent size, /. Phys. Chem. B, 102, 1945-50. 19. Doherty, A. P. and Vos, J. G. (1992) Electrocatalytic reduction of nitrite at an [Os(bipy)2(PVP)10Cl]Cl-modified electrode, /. Chem. Soc., Faraday Trans., 88, 2903-7. 20. Doherty, A. P., Stanley, M. A., and Vos, J. G. (1995) Electrocatalytic oxidation of ascorbic acid at [osmium(2,2'-bipyridyl)2-(poly-4-vinylpyridine)10Cl]Cl modified electrodes: implications for the development of biosensors based on osmium-containing redox relays, Analyst (Cambridge, UK), 120, 2371-6. 21. Ju, H., Ni, J., Gong, Y., Chen, H., and Leech, D. (1999) Electrocatalytical oxidation and determination of dopamine at redox polymer/Nafion modified electrodes, Anal. Lett, 32, 2951-64. 22. Gregg, B. A. and Heller, A. (1990) Cross-linked redox gels containing glucose oxidase for amperometric biosensor applications, Anal. Chem., 62, 258. 23. Heller, A. (1990) Electrical wiring of redox enzymes, Ace. Chem. Res., 23, 128. 24. Gregg. B. A. and Heller, A. (1991) Redox polymer films containing enzymes. 2. Glucose oxidase containing enzyme electrodes,/. Phys. Chem., 95(15), 5976-80. 25. Heller, A. (1992) Electrical connection of enzyme redox centers to electrodes, /. Phys. Chem., 96, 3579-87. 26. Katakis, I. and Heller, A. (1992) L-a-glycerophosphate and L-lactate electrodes based on the electrochemical "wiring" of oxidases, Anal. Chem., 64, 1008-13. 27. Elmgren, M., Lindquist, S.-E., and Henriksson, G. (1992) Cellobiose oxidase crosslinked in a redox polymer matrix at an electrode Surface—a new biosensor,/. Electroanal. Chem., 341, 257-73. 28. Wang, D. L. and Heller, A. (1993) Miniaturized flexible amperometric lactate probe, Anal. Chem., 65, 1069-73. 29. Ye, L., Hammerle, M., Olsthoorn, A. J. J., Schuhmann, W., Schmidt, H.-L, Duine, J. A., and Heller, A. (1993) High current density "wired" quinoprotein glucose dehydrogenase, Anal. Chem., 65, 238-41. 30. Vreeke, M., Maidan, R., and Heller, A. (1992) Hydrogen peroxide and (3-nicotinamide adenine dinucleotide sensing amperometric electrodes based on electrical connection of horseradish peroxidase redox centers to electrodes through a three-dimensional electron relaying polymer network, Anal. Chem., 64 3084-90. 31. Elmgren, M., Lindquist, S.-E., and Sharp, M. (1993) Charge propagation through a redox polymer film containing enzymes—effects of enzyme loading, pH and supporting electrolyte,/. Electroanal. Chem., 362, 227-35. 32. Ohara, T. J., Rajagopalan, R., and Heller, A. (1994) "Wired" enzyme electrodes for amperometric determination of glucose or lactate in the presence of interfering substances, Anal. Chem., 66, 2451-7. 33. Prayda, M., Jungar, C. M., Iwuoha, E. L, Smyth, M. R., Vytras, K., and Ivaska, A. (1995) Evaluation of amperometric glucose biosensors based on co-immobilization of glucose

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34. 35. 36. 37. 38.

39. 40. 41.

42. 43. 44. 45. 46. 47. 48. 49. 50. 51.

oxidase with an osmium redox polymer in electrochemically generated polyphenol films, Anal. Chim. Ada, 304, 127-38. De Lumley-Woodyear, T., Rocca, P., Lindsay, J., Dror, Y., Freeman, A., and Heller, A. (1995) Polyacrylamide based redox polymer for connecting redox centers of enzymes to electrodes, Anal. Chem., 67, 1332-8. Vreeke, M. S., Yong, K. T., and Heller, A. (1995) A Thermostable biosensor of hydrogen peroxide, Anal. Chem., 67, 4247-9. Robinson, G., Leech, D., and Smyth, M. R. (1995) Electrically "wired" tyrosinase enzyme inhibition electrode for the detection of respiratory poisons, Electroanalysis, 7, 952-7. Vijayakumar, A. R., Csoregi, E., Heller, A., and Gorton, L. (1996) Alcohol biosensors based on coupled oxidase-peroxidase systems, Analytica Cliimica Acta, 327, 223-34. Ruzgas, T., Csoregi, E., Katakis, I., Kenausis, G., and Gorton, L. (1996) Preliminary investigations of an amperometric oligosaccharide dehydrogenase-based electrode for the detection of glucose and some other low molecular weight saccharides, J. Mol. Recognit., 9, 480-4. Hedenmo, M., Narvaez, A., Dominguez, E., and Katakis, I. (1996) Reagentless amperometric glucose dehydrogenase biosensor based on electrocatalytic oxidation of NADH by osmium phenanthrolinedione mediator, Analyst (Cambridge, UK), 121,1891-5. Larsson, T., Elmgren, M., Lindquist, S.-E., Tessema, M., Gorton, L, and Henriksson, G. (1996) Electron transfer between cellobiose dehydrogenase and graphite electrodes, Anal. Chim. Acta, 331, 207-15. Katakis, I. and Heller, A. (1997) Electron transfer via redox hydrogels between electrodes and enzymes, In Frontiers in biosensorics, I, Fundamental aspects (ed. F. W. Scheller, F. Schubert, and I. Fedowitz), pp. 229-41, Birkhauser Verlag, Basel, Switzerland. Kenausis, G., Chen, Q., and Heller, A. (1997) Electrochemical glucose and lactate sensors based on "wired" thermostable soybean peroxidase operating continuously and stably at 37 °C, Anal. Chem., 69, 1054-60. Tessema, M., Csoregi, E., Ruzgas, T., Kenausis, G., Solomon, T., and Gorton, L. (1997) Oligosaccharide dehydrogenase-modified graphite electrodes for the amperometric determination of sugars in a flow injection System, Anal. Chem., 69, 4039-44. Paredes, P. A., Parellada, J., Fernandez, V. M., Katakis, L, and Dominguez, E. (1997) Amperometric mediated carbon paste biosensor based on D-fructose dehydrogenase for the determination of fructose in food analysis, Biosens. Bioekctron., 12, 1233-43 Daigle, F. and Leech, D. (1997) Reagentless tyrosinase enzyme electrodes: effects of enzyme loading, electrolyte pH, ionic strength, and temperature, Anal. Chem., 69, 4108-12. Trudeau, F., Daigle, F., and Leech, D. (1997) Reagentless mediated laccase electrode for the detection of enzyme modulators, Anal. Chem., 69, 882-6. Stigter, E. C. A., De Jong, G. A. H., Jongejan, J. A., Duine, H. A., Van DerLugt, J. P., and Somers, W. A. C. (1997) Electron transfer and stability of a quinohemoprotein alcohol dehydrogenase electrode,/. Chem. Technol. Biotechnol, 68, 110-16. Daigle, F., Trudeau, F., Robinson, G., Smyth, M. R., and Leech, D. (1998) Mediated reagentless enzyme inhibition electrodes, Biosens. Bioelectron., 13, 417-25. Somers, W. A. C., Stigter, E. C. A., Van Hartingsveldt, W., and Van Der Lugt, J. P. (1999) Enantioselective oxidation of secondary alcohols at a quinohemoprotein alcohol dehydrogenase electrode, Appl. Biochem. Biotechnol, 75, 151-61. Alkasrawi, M., Popescu, I. C., Mattiasson, B., Csoregi, E., and Laurinavicius, V. (1999) A redox hydrogel integrated PQ_Q;glucose dehydrogenase based glucose electrode, Anal. Common., 36, 395-8. Belay, A., Collins, A., Ruzgas, T., Kissinger, P. T., Gorton, L, and Csoregi, E. (1999) Redox hydrogel based bioenzyme electrode for L-glutamate monitoring, J. Pharm. Biomed. Anal, 19, 93-105.

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ADAM HELLER 52. Gajovic, N., Binyamin, G., Warsinke, A., Scheller, F. W., and Heller, A. (2002) Operation of a miniature redox hydrogel-based pyruvate sensor in undiluted deoxygenated calf serum, Anal. Chem., 72, 2963-68. 53. Niculescu, M., Frebort, I., Pec, P., Galuszka, P., Mattiasson, B., and Csoregi, E. (2000) Amine oxidase based amperometric biosensors for histamine detection, Electroanalysis, 12, 369-75. 54. Niculescu, M., Nistor, C, Frebort, I., Pec, P., Mattiasson, B., and Csoregi, E., (2000) Redox hydrogel-based amperometric bienzyme electrodes for fish freshness monitoring, Anal. Chem., 72, 1591-7. 55. Pishko, M. V., Michael, A. C., and Heller, A. (1991) Amperometric glucose microelectrodes prepared through immobilization of glucose oxidase in redox hydrogels, Anal. Chem., 63, 2268-72. 56. Rohde, E., Dempsey, E., Smyth, M. R., Vos, J. G., and Emmons, H. (1993) Development of a flow-through electrochemical detector for glucose based on a glucose oxidasemodifled microelectrode incorporating redox and conducting polymer materials, Anal. Chim. Ada, 278, 5-16. 57. Horrocks, B. R., Schmidtke, D., Heller, A. and Bard, A. J. (1993) SECM 24. Enzyme microelectrodes for the measurement of hydrogen peroxide at surfaces, Anal. Chem., 65, 3605-14. 58. Sakai, H., Baba, R., Hashimoto, K, Fujishima, A., and Heller, A. (1995) Local detection of photoelectrochemically produced H2O2 with a "Wired" horseradish peroxidase microsensor, J. Phys. Chem., 99, 11896-900. 59. Niwa, O., Kurita, R., Liu, Z., Horiuchi, T., and Torimitsu, K. (2000) Subnanoliter volume wall-jet cells combined with interdigitated microarray electrode and enzyme modified planar microelectrode, Anal. Chem., 72, 949-55. 60. Csoregi, E., Quinn, C. P., Schmidtke, D.W., Lindquist, S.-E., Pishko, M.V.,Ye, L, Katakis, I., Hubbell, J. A., and Heller, A. (1994) Design, characterization, and one-point in vivo calibration of a subcutaneously implanted glucose electrode, Anal. Chem., 66, 3131-8. 61. Csoregi, E., Schmidtke, D. W., and Heller, A. (1995) Design and optimization of a selective subcutaneously implantable glucose electrode based on "wired" glucose oxidase, Anal. Chem., 67, 1240-44. 62. Quinn, C. P., Pishko, M. V., Schmidtke, D. W., Ishikawa, M., Wagner, J. G., Raskin, P., Hubbell, J. A., and Heller, A. (1995) Kinetics of glucose delivery to subcutaneous tissue in rats measured with 0.3 mm amperometric microsensors, Am. ]. Physio!., 269 (Endocrinol. Metab. 32), E155-E161. 63. Schmidtke, D., Pishko, M. V., Quinn, C. P., and Heller, A. (1996) Statistics for critical clinical decision making based on readings of pairs of implanted sensors, Anal. Chem., 68, 2845-49. 64. Schmidtke, D. W. and Heller, A. (1998) Accuracy of the one-point in vivo calibration of "wired" glucose oxidase electrodes implanted in jugular veins of rats in periods of rapid rise and decline of the glucose concentration, Anal. Chem., 70, 2149-55. 65. Wagner, J. G., Schmidtke, D. W., Quinn, C. P., Fleming, T. F., Bernacky, B., and Heller, A. (1998) Continuous amperometric monitoring of glucose in a brittle diabetic chimpanzee with a miniature subcutaneous electrode, Proc. NatlAcad. Set, 95, 6379-82. 66. Schmidtke, D. W., Freeland, A. C., Heller, A., and Bonnecaze, R. T. (1998) Measurement and modeling of the transient difference between blood and subcutaneous glucose concentrations in the rat after injection of insulin, Proc. NatlAcad. Set, 95, 294-9. 67. Heller, A. (1999) Implanted electrochemical glucose sensors for the management of diabetes, Ann. Rev. Biomed. Eng., 1, 153-75. 68. Chen, T., Friedman, Keith A., Lei, I., and Heller. A. (2000) In situ assembled mass transport controlling micromembranes and their application in implanted amperometric glucose sensors, Anal. Chem., 72, 3757-63.

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REDOX HYDROGEL-BASED ELECTROCHEMICAL BIOSENSORS 69. Csoregi, E., Laurell, T., Katakis, I., Heller, A., and Gorton, L. (1995) On-line glucose monitoring using microdialysis sampling and amperometric detection based on "wired" glucose oxidase in carbon paste, Microchimica Acta, 121, 31-40. 70. Huang, T., Yang, L., Gitzen, J., Kissinger, P. T., Vreeke, M., and Heller, A. (1995) Detection of basal acetylene line in rat brain microdialysate, J. ChromatographyB, Biomed. Appl., 670, 323-27. 71. Yang, L., Janle, E., Huang, T., Gitzen, J., Kissinger, P. T., Vreeke, M., and Heller, A. (1995) Application of "Wired" peroxidase electrodes for peroxide determination in liquid chromatography coupled to oxidase immobilized enzyme reactors, Anal. Chem., 67, 1326-31. 72. Kato, T., Liu, J. K., Yamamoto, K., Osborne, P. G., and Niwa, O. (1996) Detection of basal acetylcholine release in the microdialysis of rat frontal cortex by high performance liquid chromatography using a horseradish peroxidase-osmium redox polymer electrode with pre-enzyme reactor, J. Chromatogr., B: Biomed. Appl., 682, 162-6. 73. Yang, L. and Kissinger, P. T. (1996) Determination of oxidase enzyme substrates using cross-flow thin-layer amperometry, Electroanalysis, 8, 116-21. 74. Tessema, M., Larsson, T., Buttler, T., Csoregi, E., Ruzgas, T., Nordling, M., Lindquist, S.-E., Pettersson, G., and Gorton, L. (1997) Simultaneous amperometric determination of some mono-, di-, and oligosaccharides in flow injection and liquid chromatography using two working enzyme electrodes with different selectivity, Anal. Chim. Acta, 349, 179-88. 75. Tessema, M., Csoregi, E., Ruzgas, T., Kenausis, G., Heller, A., Solomon, T., and Gorton, L. (1997) Oligosaccharide dehydrogenase modified graphite electrodes for the amperometric determination of sugars in flow injection system, Anal. Chem., 69, 4039-44. 76. Niwa, O., Horiuchi, T., Kurita, R., and Torimitsu, K. (1998) Online electrochemical sensor for selective continuous measurement of acetylcholine in cultured brain tissue, Anal. Chem., 70, 1126-32. 77. Osborne, P. G., Niwa, O., and Yamamoto, K. (1998) Plastic film carbon electrodes: enzymic modification for online, continuous, and simultaneous measurement of lactate and glucose using microdialysis sampling, Anal. Chem., 70, 1701-6. 78. Larsson, N., Ruzgas, T., Gorton, L, Kokaia, M., Kissinger, P., and Csoregi, E. (1998) Design and development of an amperometric biosensor for acetylcholine determination in brain microdialyzates, Electrochim. Acta, 43, 3541-54. 79. Yao, T. and Ogawa, H. (2000) Highly sensitive and selective detection of pyruvate by amperometric flow-injection analysis based on enzymatic substrate recycling and sensitive detection of hydrogen peroxide, J. How Injection Anal., 17, 37-42. 80. Pishko, M. V., Katakis, L, Lindquist, S.-E., Heller, A., and Degani, Y. (1990) Electrical communication between graphite electrodes and glucose oxidase/redox polymer complexes, Mo!. Cryst. Liq. Cryst., 190, 221. 81. Pishko, M. V., Katakis, L, Lindquist, S.-E., Ye, L., Gregg, B. A., and Heller, A. (1990) Direct electrical communication between graphite electrodes and surface adsorbed glucose oxidase/redox polymer complexes, Angew. Chem. Intl. Ed., 29, 1, 82. 82. Sirkar, K., Revzin, A., and Pishko, M. V. (2000) Glucose and lactate biosensors based on redox polymer/oxidoreductase nanocomposite thin films, Anal. Chem., 72, 2930-36. 83. Narvaez, A., Suarez, G., Popescu, I. C., Katakis, L, and Dominguez, E. (2000) Reagentless biosensors based on self-deposited redox polyelectrolyte-oxidoreductases architectures, Biosens. Bioelectron., 15, 43-52. 84. Cheng, L., Liu, J., and Don, S. (2000) Layer-by-layer assembly of multilayer films consisting of silicotungstate and a cationic redox polymer on 4-aminobenzoic acid modified glassy carbon electrode and their electrocatalytic effects, Anal. Chim. Acta, 417, 133-42.

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ADAM HELLER 85. Li, W., Wang, Z., Sun, C, Xian, M., and Zhao, M. (2000) Fabrication of multilayer films containing horseradish peroxidase and polycation-bearing Os complex by means of electrostatic layer-by-layer adsorption and its application as a hydrogen peroxide sensors, Anal. Chim. Ada, 418, 225-32. 86. Katakis, I., Ye, L, and Heller, A. (1994) Electrostatic control of the electron transfer enabling binding of recombinant glucose oxidase and redox polyelectrolytes, J. Am. Chem. Soc., 116, 3617-18. 87. Katakis, I., Ye, L., Kenausis, G., and Heller, A. (1994) Design of redox polyelectrolyte "wires" for glucose electrodes, Polym. Mater. Sci. Eng., 71, 592-3. 88. Katakis, L, Vreeke, M., Ye, L., Aoki, A., and Heller, A. (1996) Electron conducting adducts of water soluble redox polyelectrolytes and enzymes. In Advances in molecular & cell biology, Volume 15B, (ed. E. Edward Bittar, B. Danielsson, and L. Billow), pp. 391-409, JAI Press Inc. 89. Gary Binyamin, Jason Cole, and Adam Heller (2000) Mechanical and electrochemical characteristics of composites of wired glucose oxidase and hydrophilic graphite. J. Electrochem. Soc., 147, 2780-3. 90. Kenausis, G., Taylor, C., and Heller, A. (1996) Wiring of glucose oxidase and lactate oxidase within a hydrogel made with poly(vinyl pyridine) complexed with [Os(4,4'dimethoxy-2,2'-bipyridine)2 Cl] +/2 + , J. Chem. Soc., Faraday Transactions, 92, 4131-6. 91. Taylor, C., Kenausis, G., Katakis, L, and Heller, A. (1995) "Wiring" of glucose oxidase within a hydrogel made with polyvinyl imidazole complexed with [(Os-4,4'-dimethoxy 2,2'-bipyridine)Cl]+/2 + , J. Electroanal. Chem., 396, 511-15. 92. Ohara, T. J., Rajagopalan, R., and Heller, A. (1993) Glucose electrodes based on crosslinked [Os(bpy)2Cl]+ '2+ complexed poly (1-Vinyl-imidazole) films, Anal. Chem., 65, 3512-17. 93. Kenausis, G., Taylor, C., Rajagopalan, R., and Heller, A. (1996) "Wiring" of lactate oxidase within a low redox potential electron conducting hydrogel, J. Mol. Recog., 9, 626-31. 94. Lever, A. B. P. (1990) Electrochemical parametrization of metal complex redox potentials, using the ruthenium(III)/ruthenium(II) couple to generate a ligand electrochemical series, Inorg. Chem., 29, 1271-85. 95. Schuhmann, W., Kranz, C., Huber, J., and Wohlschlaeger, H. (1993) Conducting polymer-based amperometric enzyme electrodes. Towards the development of miniaturized reagentless biosensors, Synth. Met, 61, 31-5. 96. Gajovic, N., Habermuller, K., Warsinke, A., Schuhmann, W., and Scheller, R W. (1999) A pyruvate oxidase electrode based on an electrochemically deposited redox polymer, Electroanalysis, 11, 1377-83. 97. Heller, J. and Heller, A. (1998) Loss of activity or gain in stability of oxidases upon their immobilization in hydrated silica: Significance of the electrostatic interactions of surface arginine residues at the entrances of the reaction channels, J. Am. Chem. Soc., 120, 4586-90. 98. Chen, Q., Kenausis, G. L, and Heller, A. (1998) Stability of oxidases immobilized in silica gels, J. Am. Chem. Soc., 120, 4582-85. 99. Park, T.-M., Iwuoha, E. L, Smyth, M. R., and MacCraith, B. D. (1996) Sol-gel-based amperometric glucose biosensor incorporating an osmium redox polymer as mediator, Anal. Commun., 33, 271-3. 100. Park, T.-M., Iwuoha, E. L, and Smyth, M. R. (1997) Development of a sol-gel enzyme inhibition-based amperometric biosensor for cyanide, Electroanalysis, 9, 1120-3. 101. Park, T.-M., Iwuoha, E. L, Smyth, M. R., Freaney, R., and McShane, A. J. (1997) Sol-gel based amperometric biosensor incorporating an osmium redox polymer as mediator for detection of L-lactate, Talanta, 44, 973-8.

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REDOX HYDROGEL-BASED ELECTROCHEMICAL BIOSENSORS 102. Kane, S. A., Iwuoha, E., and Smyth, M. R. (1998) Development of a sol-gel based amperometric biosensor for the determination of phenolics, Analyst (Cambridge, UK), 123, 2001-6. 103. Park, T.-M. (1999) Amperometric determination of hydrogen peroxide by utilizing a sol-gel-derived biosensor incorporating an osmium redox polymer as mediator, Anal. Lett., 32, 287-98. 104. Pravda, M., Adeyoju, O., Iwuoha, E. I., Vos, J. G., Smyth, M. R., and Vytras, K. (1995) Amperometric glucose biosensors based on an osmium (2+/S + ) redox polymermediated electron transfer at carbon paste electrodes, Electroanalysis, 7, 619-25. 105. Jezkova, J., Iwuoha, E. I., Smyth, M. R., and Vytras, K. (1997) Stabilization of an osmium bis-bipyridyl polymer-modified carbon paste amperometric glucose biosensor using polyethyleneimine, Ekctroanalysis, 9, 978-84. 106. Parellada, J., Narvaez, A., Dominguez, E., and Katakis, I. (1997) A new type of hydrophilic carbon paste electrodes for biosensor manufacturing binder paste electrodes, Biosens. Bioelectron., 12, 267-75. 107. Iwuoha, E., Smyth, M. R., andVos.J. G. (1994) Amperometric glucose sensor containing nondiffusional osmium redox centers: analysis of organic-phase response, Electroanalysis, 6, 982-9. 108. Iwuoha, E. I. and Smyth, M. R. (1996) Organic phase enzyme electrodes: kinetics and analytical applications, Biosens. Bioelectron., 12, 53-75. 109. Dressman, S. F., Garguilo, M. G., Sullenberger, E. V., and Michael, A. C. (1993) Characterization of the electroenzymic reduction of hydrogen peroxide in carbon dioxide by voltammetry with a chemically modified microelectrode, J. Am. Chem. Soc., 115, 7541-2. 110. Heller, A., Maidan, R., and Wang, D. L. (1993) Amperometric biosensors based on 3-dimensional hydrogel-forming epoxy networks, Sensors and Actuators, 13, 180-3. 111. Ohara, T. J., Vreeke, M. S., Battaglini, F., and Heller, A. (1993) Bienzyme sensors based on "electrically wired" peroxidase, Electroanalysis, W. Simon Memorial Issue, 5, 825-31. 112. Vreeke, M., Rocca, P., and Heller, A. (1995) Direct electrical detection of dissolved biotinylated horseradish peroxidase, biotin, and avidin, Anal. Chem., 67, 303-6. 113. Lu, B., Iwuoha, E. I., Smyth, M. R., and O'Kennedy, R. (1997) Development of an "electrically wired" amperometric immunosensor for the determination of biotin based on a non-diffusional redox osmium polymer film containing an antibody to the enzyme label horseradish peroxidase, Anal Chim. Acta, 345, 59-66. 114. Lu, B., Iwuoha, E. I., Smyth, M. R., and O'Kennedy, R. (1997) Development of an amperometric immunosensor for horseradish peroxidase (HRP) involving a nondiffusional osmium redox polymer co-immobilized with anti-HRP antibody, Anal. Commun., 34, 21-4. 115. de Lumley-Woodyear, T., Campbell, C. N., and Heller, A. (1996) Direct enzymeamplified electrical recognition of a 30-base model oligonucleotide, J. Am. Chem. Soc., 118, 5504-5. 116. de Lumley-Woodyear, T., Campbell, C. N., Friedman, E., Friedman A., Georgiou, G., and Heller, A. (1999) Rapid amperometric verification of PCR amplification of DNA, Anal. Chem., 71, 535-8. 117. Campbell, C. N., de Lumley-Woodyear, T., and Heller, A. (1999) Towards Immunoassay in whole blood: separationless sandwich-type electrochemical immunoassay based on in-situ generation of the substrate of the labeling enzyme, Fresenius]. Anal Chem., 364, 165-9. 118. Danilowicz, C. and Manrique, J. M. (1999) A new self-assembled modified electrode for competitive immunoassay, Ekctrochem. Commun., 1, 22-5.

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ADAM HELLER 119. Caruana, D. J. and Heller, A. (1999) Enzyme-amplified amperometric detection of hybridization and of a single base pair mutation in an 18-base oligonucleotide on a 7|xm diameter microelectrode, J. Am. Chem. Soc., 121, 769-74. 120. de-Lumley-Woodyear, T., Caruana, D. J., Campbell, C. N., and Heller, A. (1999) Reactive electrophoretic activation of a microelectrode for enzyme-amplified recognition and for melting-temperature determination of a simple oligonucleotide, Anal. Chem., 71, 394-8.

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Chapter 2 Hybridization at oligonucleotide sensitive electrodes Daren J. Caruana Department of Chemistry, University College, London, 20 Gordon St, London WC1H OAJ, UK.

1 Introduction DNA hybridization on solid surfaces was developed on membranes as a routine biotechnological technique in the 1970s (1). With the invention and refinement of new surface analytical techniques, oligonucleotide detection by surface confined hybridization is the fundamental principle behind the function of most oligonucleotide sensitive devices. These devices are capable of high throughput DNA analysis for applications such as sequencing, genetic diagnosis, drug phenotyping, computing, and gene expression analysis. Regardless of application, a common aspect of biosensor devices is the conversion of a physiochemical process confined at a surface into an interpretable signal. In the case of oligonucleotide sensitive electrodes, the transducer element of the device differentiates between single and double stranded oligonucleotides at a solid interface. The challenge is to define the optimum conditions for maximum hybridization efficiency, to obtain a high degree of sensitivity. The aim here is to discuss the important aspects of oligonucleotide attachment to conducting surfaces and the process of hybridization to the complementary strand for the fabrication of oligonucleotide sensitive electrochemical sensors. Conceptually, hybridization at a surface may appear to be the same as the equivalent process in bulk solution; however, a unique set of conditions needs to be defined. The emphasis of this chapter will be to provide practical information to aid in the determination of optimum hybridization conditions. Different applications or electrode models will require different set of conditions for optimum hybridization and detection sensitivity. Therefore, it is unconstructive to provide a set of experimental conditions, which will not always apply for every situation.

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DAREN J. CARUANA These variables will be divided into two groups: (a) Characterization of the surface confined probe; defining the environment of the probe, attachment method and surface structure. (b) Hybridization conditions defining the conditions under which association of a solution-phase target oligonucleotide with a surface tethered probe oligonucleotide. Many of these experimental variables are interrelated and all must be carefully considered to determine optimum conditions for hybridization. Discussion will be confined to oligonucleotides of 14-50 nucleotides in length.

2 Function of oligonucleotide sensitive electrodes Random collision between two complementary strands is the fundamental requirement for hybridization to take place on a solid surface. The probability or frequency for collision will dictate the kinetics of this process. Once collision has occurred, the annealing must then take place to produce a hybrid. If the resulting hybrid is completely complementary, the hybrid will be stable. Hybridization will still take place if the sequences are not completely complementary, but the hybrid will be less stable than the completely complementary hybrid. Differentiation between interactions resulting in complementary hybrids and other interactions leading to less stable non-complementary hybrids may be carried out by carefully defining the experimental conditions. This is the key to the function of oligonucleotide sensitive electrodes that rely on hybridization, and is the subject of this chapter. Although the discussion here will be confined to DNA, which is readily available by custom synthesis, there are a number of other nucleic acid analogs that may also be used. RNA may be used but is experimentally difficult to manipulate due to its susceptibility to degradation from RNAses that are more abundant than DNAses. Uncharged analogs such as peptide nucleic acids (PNA), and other non-phosphorous containing analogs are potentially suitable for use as probes in oligonucleotide sensitive electrodes (2). The structure of the nucleotide duplex is an intertwined structure of two antiparallel molecules. The three-dimensional shape is maintained by an array of individually weak interactions which are strong collectively, and which stabilize the molecules in a rigid familiar double helical structure. The interactions are mainly hydrogen bonds, hydrophobic, electrostatic, van der Waals, and dipole interactions. Hybridization involves the formation of these interactions to produce a close packed helix. The formation of these forces holding the two strands together, most notably the hydrogen bonding between the individual bases and ionic interactions (Figure 1) can be translated into a range of physiochemical experimental parameters. These parameters in some way contribute to the formation or disruption of the individual weak interactions and determine which hybrids remain on the surface and which are removed. 20

OLIGONUCLEOTIDE SENSITIVE ELECTRODES

Figure 1 Showing the Watson-Crick hydrogen bonding between (a) A-T, (b) G-C base pairs, and (c) the phosphate deoxyribose backbone (3).

3 Hybridization efficiency and sensitivity In bulk solution, the process of hybridization of two complementary oligonucleotide strands is a specific interaction between two molecules and occurs homogeneously in solution. In the case of surface hybridization, the process may be looked upon as a specific adsorption process. Essentially the same interactions are taking place but may be characterized differently. The function of these oligonucleotide devices may be characterized by the hybridization efficiency and hybridization rate, which are not normally issues when describing hybridization process in the bulk solution. Hybridization efficiency is a measure of the proportion of probe oligonucleotides that successfully hybridize to the complementary strand under optimum hybridization conditions at the surface. The hybridization rate describes the flux of target strand to the surface to facilitate hybridization. The equilibrium constant for the hybridization process for two complementary single stranded oligonucleotides: Probe + Target -> Hybrid is given by:

where K is the equilibrium constant. Experimental hybridization data may also be expressed as a ratio, v, of hybridized to single stranded probe oligonucleotide:

21

DAREN J. CARUANA The quantitative determination of the proportion of hybridized oligonucleotides on a surface is not trivial, and requires the use of sensitive quantitative analytical techniques such as radiolabeling or fluorescence. If indirect methods of detection are used, then only relative amounts of hybridization may be obtained. Ultimately the extent of hybridization may be determined by the stringency of the hybridization conditions and surface concentration of the tethered oligonucleotide.

4 Probe oligonucleotide structure and dynamics Although the structure of double stranded DNA is well studied, single stranded DNA is much less defined especially at a solid surface. It is tempting to picture a solid surface decorated with probe single stranded oligonucleotides standing up straight pointing toward the solution ready to hybridize with the complementary target single stranded oligonucleotide to form the duplex. In reality the probe structure and surface density are difficult to characterize due to the minute amount present on the surface. The probe architecture will substantially affect the hybridization efficiency and, therefore, the sensitivity of the oligonucleotide sensitive electrode. The probe structure at the surface will be dependent on a number of parameters including the surface charge, electrical potential, hydrophobicity, probe attachment method, and also solution physicochemical or chemical conditions. These variables, specifically for probe structure, will be discussed in relation to how these properties affect hybridization efficiency. The discussion will enable the experimentalist to identify a unique set of conditions for a unique surface and application. The probe concentration, length, spacer group, and surface orientation will be discussed in some detail.

4.1 Surface concentration Steric hindrance to hybridization as a consequence of high surface packing of oligonucleotides on the surface has a direct impact on the hybridization efficiency (4). There are methods of oligonucleotide surface modifications that lead to a highly dense layer of single stranded oligonucleotide which may lead to hybridization efficiencies of less than 5%. Therefore, strict control of surface concentration is enormously important to conserve a reasonable hybridization efficiency and detection limit. There is a relationship between probe length in base pairs, and surface distribution based only on nearest neighbor interactions contributing to steric interactions for monolayer coverage. As a rough "rule of thumb" a relationship may be formulated between the number of molecules that may be attached to the surface, assuming the probe oligonucleotide exists as a stretched linear molecule and the oligonucleotide is attached at only one point. The equations given in Table 1 provide an estimate of the theoretical optimum surface concentration that will minimize steric interactions between probe oligonucleotides. Another consideration is the difference in length of the target 22

OLIGONUCLEOTIDE SENSITIVE ELECTRODES Table 1 Relating length of probe oligonucleotide in base pairs with maximum close packed (^/3) surface coverage to minimize nearest neighbor interactions. Roughness is r= (actual surface area)/ (geometric area) Attachment point

Equation

Variables

Terminus

b is length in bases and r is roughness factor

Terminus via spacer

b is length in bases, r is roughness factor, and s is spacer length in A

Midpoint

b is total length in bases and r is roughness factor

Midpoint via spacer

b is length in bases, r is roughness factor, and s is spacer length in A

strands that can influence near neighbor hybridization. When the target strand is smaller than the probe, the hybridization rate increases roughly proportionately to the difference in length. When the target strand is longer, the hybridization rate is reduced. Accurate quantitative measurement of surface concentration of single stranded oligonucleotides may be carried out by surface sensitive techniques in combination with radioisotope (32P) or fluorescence labeling methods (5).

4.2 Probe length and orientation The availability of the probe strand to the solution has a direct affect on both the rate and efficiency of hybridization. The lengths of the probe and the spacer group determine the secondary structure that the molecule can exhibit, and the orientation of the probe molecule is largely dependent on the surface properties of the support, as shown in Figure 2. Attachment of the oligonucleotide through a spacer group reduces nearest neighbor steric effects. The length and properties (charge, hydrophobicity, flexibility) of the spacer are important and influence the structure of the probe layer. The longest aliphatic spacers available commercially are already attached to oligonucleotides at 12C in length. Longer aliphatic chains become less soluble and may behave as surfactants. Glycols and amino-based spacers are more water soluble and are available with different lengths and terminal reactive groups. As yet there has been no comprehensive study on the surface properties and spacer properties on the hybridization efficiency; however, it is clear that attachment via a spacer improves the hybridization efficiency, although an optimum length has not been determined. The simplest spacer is to use a poly(dT) which extends the oligonucleotide and places the important sequence further from the surface. However, this does not overcome problems of secondary structure of probe molecules. 23

DAREN J. CARUANA

Figure 2 Schematic of single stranded oligonucleotides attached to the surface (a) stretched out to the solution, (b) with the charged phosphate backbone interacting with the surface, (c) with the bases interaction with the surface, and (d) via spacer groups.

Figure 3 Schematic representation of a probe oligonucleotide with a hairpin structure.

Single stranded oligonucleotide molecules probably behave as a polyelectrolyte with either no fixed or random secondary structure. For single stranded oligonucleotides with lengths less than 8-10 bases, the molecules will exhibit rather little secondary structure. An oligonucleotide longer than 11 nucleotides may fold back on itself and form a hairpin, if the sequence supports it, as shown in Figure 3. The occurrence of such hairpin structures dramatically reduces the availability of probe to the solution and reduces hybridization efficiency. Hairpin

24

OLIGONUCLEOTIDE SENSITIVE ELECTRODES Table 2 Surface sensitive techniques suitable for the determination of structure of probe oligonucleotides Technique

Description/requirement

Neutron reflection

Requires Synchrotron Radiation Provides high resolution surface and involved data analysis density and thickness in the axis perpendicular to the surface plane

Scanning probe microscopy

Atomic Force or scanning tunneling microscope

References

Comments

9

X, Y, and Z spatial surface structure

6, 10, 11

X-ray photoelectron Scanning electron microscope spectroscopy with XPS capability

Thickness of surface layers and elemental density

4, 12

Fluorescence

Inter- and intramolecular interactions. An indirect method requiring chemical labeling

13

High sensitivity photon counting mode equipment

structures are easily destabilized by heating (the melting temperature may be calculated using the formula in Section 5.1) or with the addition of denaturing agents such as formaldehyde or glyoxal (ethanedial). Hairpin structures are frequently associated with RNA molecules. The physicochemical nature of the surface (i.e. hydrophobicity, charge, electrical potential) will affect the structure of the probe in various ways. Electrostatic forces between the surface and the negatively charged backbone of oligonucleotides do influence the structure of the molecule. Intuitively, negatively charged surfaces repel the oligonucleotides away from the surface, whilst positively charged surfaces cause the molecule to lie down with the phosphate groups in contact with the surface. Interestingly, studies using positively charged surfaces decorated with oligonucleotides of 24 bases appear to form non-helical structures not found free in solution (6). The influence of an electrochemical potential applied to the surface will have a similar effect to chemical surface charges. The effect of electrochemical potential on a surface decorated with a dense covering of short double stranded oligonucleotide has been observed (7), however, the effect on single stranded oligonucleotides has not been investigated in detail. Hydrophobic surfaces tend to interact with the bases making the oligonucleotide unavailable for hybridization, as shown in Figure 2 (8). The lack of information on the fine structure of submonolayer single stranded oligonucleotide is mainly due to technical difficulty with observing small changes in structure in situ. Such studies require refinement of sophisticated surface sensitive techniques and heavy investment of personnel and money. A list of techniques that have been applied to these studies with the information that they provide is given in Table 2.

4.3 Attachment of probe The immobilization of probe onto a solid support has been used as an analytical tool in molecular biology after it was first described by Gillespie and 25

DAREN J. CARUANA Speigelman (14). Nitrocellulose, nylon, or polystyrene supports were used to bind fragments of DNA and RNA with lengths greater than 500 nucleotides. The attachment to these membranes was purely physisorption and behaved well at low temperatures. There are currently several commercially available membranes designed to covalently bind nucleic acids either by UV radiation or by chemical treatment of activated membranes. For electrochemical-based detection of hybridization, the methods for attachment have been adapted for attachment onto solid conducting surfaces. Regardless of how DNA or hybridization is detected, the method chosen for the attachment of single stranded oligonucleotides onto the appropriate surface depends on the nature of the surface. Several approaches have been developed for surface attachment and may be broadly divided into two: The first involves the base-by-base synthesis of oligonucleotides on the surface using highly efficient chemical reactions (15,16). The second involves direct attachment of ready synthesized single stranded sequences onto the surface. Both approaches have been extensively developed; however, the former involves a larger degree of investment in specialist equipment and is an exceedingly attractive method for DNA array fabrication due to the light addressable nature of the synthesis. Certainly, the attachment of the ready synthesized oligonucleotides on the surface is the most convenient, and will be discussed on a practical level here. The choice of method is frequently dependent on the nature of surface, whether a hard or soft (polymeric coated solid) conducting surface is used. For surface modification, the concentration of probe in solution and the purity of the DNA may be measured as described in Protocol 1. There are many sources of custom sequence oligonucleotides for different lengths, linker groups and functional groups at 5', 3' and internal positions. The product purity may be specified; either ethanol or phenol precipitated which is minimum purity or HPLC, PAGE and ion exchange purified for higher purity. For oligonucleotide sensitive electrode preparation precipitated purity is probably sufficient.

Protocol 1

Determination of solution concentration of single stranded oligonucleotide and assessment of purity of oligonucleotide Equipment and materials UV spectrophotometer Quartz cuvette of path length 1 cm Buffer containing: 0.1M Trizma® base buffer (Sigma Aldrich chemical company), 10 mM EDTA (ethylenediaminetetraacetic acid), 2 mM NaCl, adjusted to pH 7.4 26

All pipette tips and glassware should be sterilized or thoroughly cleaned to remove nuclease contamination.

OLIGONUCLEOTIDE SENSITIVE ELECTRODES Protocol 1 continued

Method 1 Add 1 ml of buffer to a cuvette and zero (blank) the spectrometer. 2 Place the sample in a similar cuvette and take absorbance reading at 260 and 280 nm, and 230 and 325 nm. 3 The concentration is given by the following equation:

where N is the number of bases indicated by the subscript.

4 If the ratios A26o/A28o and A230lA325 are between 1.8 and 1.9, and 1.9 and 2.0, respectively, the sample is highly purified. Lower ratios may contain protein or phenol (phenol is used to precipitate the DNA) contamination. This procedure works for both DNA and RNA either single or double stranded.

4.3.1 Attachment to gold by chemisorption Attachment via a thiol onto gold is a very popular method due to the availability of thiol terminated single stranded oligonucleotides (5). There are no complex chemical steps or chemistry involved and is relatively easy to control in terms of oligonucleotide surface concentration. Protocol 2 describes the method used for the deposition of oligonucleotides onto clean gold surfaces. The surface concentration of oligonucleotides may be controlled by using a mixture of different thiol bearing molecules in the deposition mixture. The gold pre-treatment is extremely important. It is recommended that thorough cleaning of the surface should be carried out by repetitive voltammetric cycling in a weak acid (H2SO4 0.05M) solution. This is a controlled electrochemical technique, which ensures that the surface is prepared reproducibly. Alternative methods of producing a clean surface would be to sputter gold on the surface of a flat substrate (if using glass as the substrate, the surface needs to be coated with Cr to aid adhesion). The surface of sputtered gold using a lowpressure gold sputterer in an argon atmosphere is reported to be predominantly Au [111] crystal face exposed to the liquid (although this does not mean that the surface is atomically flat). The gold electrodes produced in this way are convenient because the surface is relatively clean as long as care is taken to reduce exposure to polluting environments.

4.3.2 Non-covalent or physisorption This method was developed for the hybridization of DNA on nitrocellulose membranes for techniques such as Southern blots. The adsorption of oligonucleotides normally longer than 500 bases works well for high area surfaces where surface concentration and hybridization efficiency are not of concern. This method is difficult to control and relies on the adsorption of the unmodified 27

DAREN J. CARUANA oligonucleotide on a clean surface. There are methods developed for electrochemical hybridization detection at Indium doped tin oxide surfaces with oligonucleotides in the order of 400-1500 bases, which are strongly adsorbed (6,17).

Protocol 2

Attachment of oligonucleotides Materials Oligonucleotide modified with a thiol terminated 3' or 5' purchased from Genosys or other custom synthesis source. Sterile pH 7.2 HEPES (0.01M) buffer

Cysteamine as the diluent thiol All pipette tips and glassware should be sterilized or thoroughly cleaned to remove nuclease contamination.

Method 1 Make the appropriate mixture of thiol modified oligonucleotide and diluent thiol cystamine in HEPES buffer. 2 Place the clean electrode surface into an aqueous solution containing the thiol modified oligonucleotide and leave at room temperature for 24 h. 3 Remove and wash the electrode thoroughly with deionized water. 4 The electrode should be modified immediately prior to use.

4.3.3 Chemical attachment A variety of chemical attachment methods are available. All effectively produce covalently attached oligonucleotide on a range of conducting surfaces. The first step is to introduce reactive chemical groups on the chosen surface. This is not always straightforward and frequently determines the electrode material. There are several methods, which are specific for different surfaces or pre-treatment of surfaces (18). The surface of conducting silicon after silanization or other chemical treatments to introduce reactive anchor groups are very numerous. Characterization of the efficiency of each chemical step and the number density of sites introduced by each method is difficult and requires sensitive surface analysis. Electrochemical methods of surface modification of edge plane graphite with oxygen containing reactive groups may be used for oligonucleotide attachment through CNBr coupling (see Figure 4(a) and Protocol 3).

4.3.4 Deposition onto a polymer coated electrode Using a polymeric support on a conducting surface is a convenient way of increasing the hybridization signal by deposition of more probe oligonucleotides due to the higher active area. Optimization of this method is difficult, as hybridization within the polymer matrix will depend on the microenvironment of the polymer such as density, charge, hydrophobicity, thickness, etc. 28

OLIGONUCLEOTIDE SENSITIVE ELECTRODES

Figure 4 Reaction schemes for the (a) CNBr activated coupling and the (b) reactive electrophoretic deposition on electrodes baring reactive amino groups, respectively.

Protocol 3

CNBr activated coupling to an edge plan graphite electrode Materials Cyanogen bromide, sodium hydroxide Amino terminated single stranded oligonucleotide

Sterile 0.1M sodium bicarbonate buffer pH8.5 Ice and magnetic stirrer.

Method 1 To 25 ml of deionized water in a fume hood add 5 g of CNBr and adjust the pH to 11.0 with 5% NaOH and place the oxidized edge plane graphite electrode in the solution and stir for 20 min. 2 Remove the electrode and place in 20 ml ice-cold 0.1M sodium bicarbonate buffer pH 8.5 and add amino terminated single stranded oligonucleotide (ca. 300-500 |j,g) and leave stirring for 48 h at 4 °C. 3 Wash electrode in potassium phosphate buffer and use immediately. The DNA solution can be used only once. For control of surface concentration, use an amine (such as CH3CH2NH2) added to the DNA solution to dilute the surface concentration.

Attachment of oligonucleotides to the polymer support may be done in a variety of ways, before, during, or after deposition of the polymer matrix. Electropolymerization of pyrrole monomer units modified with oligonucleotide at an electrode surface is an example of codeposition (19). Protocol 4 outlines an 29

DAREN J. CARUANA electrophoretic method of deposition of oligonucleotide with a 5' chemically reactive group, which forms a covalent bond to an amine on the polymer backbone (20, 21). These techniques are very attractive where spatial segregation of the oligonucleotides is important. The drawback is that characterization is not easy and the determination of the optimum hybridization efficiency is difficult.

Protocol 4

Reactive electrophoretic deposition Equipment and reagents Bench top centrifuge CH instruments potentiostat with chronoamperometric capabilities Electrode of diameter between 10 and 25 |im pre-coated with polymeric matrix with reactive amino groups

l-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDC) (Aldrich. Cat. No. 16,146-2) Single stranded oligonucleotide (15-35 bp) from Genosys Microamplicon tube (Amicon) with a 3000 Da cutoff membrane.

Method 1 450-550 |j,g of probe oligonucleotide is dissolved in 50 |j,l of pH 7.0 methylimidazole (0.02 M) buffer. 2 To the oligonucleotide solution was added 50 |j,l of EDC (0.2M) and was stored at 4 °C for 16 h. 3 450 |jl of deionized water was added to the mixture and placed into a microamplicon tube to reduce the volume to ca. 50 |jl by spinning at 50,000 rpm (approx. 15 min). This procedure was repeated twice to remove excess buffer and EDC. The final concentration of EDC and methylimidazole will be ca. 2 x 10~ 4 M and 2 x 10 ~ 5 M, respectively. 4 The 50 |jl of oligonucleotide was then transferred to an electrophoretic cell with 100 |jl of deionized water. 5 The microelectrode was placed in the cell containing the oligonucleotide approx. 1 mm from the counter electrode. A constant potential of 0.9 V for 5 min was applied to the working electrode.

Hybridization conditions In practical terms, the hybridization is dependent on the thermodynamic stability of the duplex formed and the collision frequency; both are interrelated and difficult to define independently. However, both are controlled by the physicochemical conditions under which hybridization takes place (22). The kinetics of this reaction in solution is well studied and, for the simple case it is described 30

OLIGONUCLEOTIDE SENSITIVE ELECTRODES

by a second order rate equation. Hybridization kinetics at a solid surface is experimentally more difficult to monitor in real time, and is comparatively less well understood compared to the equivalent process in solution (23). Nevertheless, the physical and chemical parameters that dictate the stability of the surviving hybrid need to be considered for optimizing hybridization efficiency. There is no single protocol that is appropriate for all applications of oligonucleotide sensitive electrodes. There are many variables to be considered when designing the hybridization conditions that will either affect the stability of the hybrids, the rate at which the hybrids form, or both. The discussion here will focus on the effect of temperature, number of base pair mismatches, ionic strength, formamide concentration, nonspecific adsorption, and mass transport on the hybridization stability and hybridization efficiency.

5.1 Temperature By far the most important variable is temperature. Hybridization temperature is always discussed relative to the melting temperature, Tm, for a given oligonucleotide at which an equilibrium is reached where 50% of single stranded oligonucleotides are hybridized. The melting temperature, Tm, of an oligonucleotide is dependent on the length in base pairs base composition and salt concentration, which are related according to the following relationship (24):

where (%G + C) is the content of G and C in the oligonucleotide and I is the length in base pairs. There are many different versions of melting temperature equations and different equations apply at slightly different conditions. There is no equation which will estimate the melting temperature for all lengths of oligonucleotides. The equation given above holds for 14 to ca. 72 nucleotides for sequences in solution at pH between 5 and 9. As the length in base pairs increases, the melting temperature becomes more independent of the length. Theoretical equations also give Tm values based on nearest neighbor and thermodynamic changes in entropy and enthalpy when hybridization occurs (25, 26). There have been few studies to assess the effect of surface confinement of the hybridization process. Krull et al. (13) have shown that when a single base pair mismatch in the middle of a model 20 nucleotide changed the melting temperature 6-10 °C rather than 3.8-6.1 °C change in solution. The melting temperature for oligonucleotides in solution may be determined experimentally by measuring the absorbance at 260 nm using a spectrophotometer whilst increasing the temperature linearly, as outlined in Protocol 5.

31

DAREN J. CARUANA

Protocol 5

Determination of melting temperature Equipment and reagents UV spectrophotometer with a programmable temperature controlled micro-cuvette holder Quartz cuvette of path length 1 cm, small volume (200 pi) Buffer containing: 0.1M Trizma® base buffer (Sigma-Aldrich chemical company), 1 mM EDTA (Ethylenediaminetetraacetic acid), 2 mM NaCl, adjusted to pH 7.4

Two complementary oligonucleotide samples All pipette tips and glassware should be sterilized or thoroughly cleaned to remove nuclease contamination.

Method 1 Zero the spectrophotometer with buffer at 260 nm. 2 Place 200 |j,l single stranded oligonucleotide dissolved in buffer in the cuvette to obtain an absorbance between 0.05 and 0.1. 3 Mix equimolar amounts of the two complementary single stranded oligonucleotides in buffer and place the solution in the cuvette and thermostat at ca. 30 °C below the calculated melting temperature for 30 min. 4 Increase the temperature of the cuvette at 1 °C min"1 and monitor the absorbance at 260 nm. 5 At the point of melting, the absorbance will increase over a range of 15 °C. The Tm is the temperature when the absorbance is halfway between starting and the finishing values.

Ionic strength Ionic strength affects the stability of hybrids, rate of hybridization, and interactions between probe molecules attached to the surface. High ionic strength improves the stability of hybrids and increases the rate of hybridization when hybridization is not limited by mass transport. Sodium chloride is commonly used in hybridisation buffers, but the effects are more pronounced with divalent cations such as Mg2 + . Quantitative effects of salt concentration on the rate of hybridization and hybrid stability have not been determined in detail for oligonucleotides at surfaces, but probably reflect the trend observed in solution. 1M sodium chloride is frequently used, as higher ionic strength will tend to stabilize mismatched hybrids and slow down the hybridization rate (for filter hybridization above 0.1M NaCl, the hybridization rate is not markedly affected) (27). The high salt concentration is important to reduce the electrostatic interactions between single and double stranded molecules attached to the surface. When using peptide nucleic acids, the ionic strength is less important. 32

OLIGONUCLEOTIDE SENSITIVE ELECTRODES

In the presence of quaternary ammonium salts such as tetraethylammonium or tetramethyl ammonium salts, the hybridization is independent of base composition, only length is important (28). This is very useful when an array of probes with different sequences (%G & C) is spatially localized on the surface. In the presence of TEA+ or TMA + , the Tm is dependent only on the number of base pair mismatches and length of probe (28). Typical concentration ranges of TMAC1 used in hybridization buffers are between 2M and 3M which can lower the melting temperature by 10-15 °C for a 20 base pair oligonucleotide.

5.3 Base mismatch The reassociation requires that all the bases meet up and form hydrogen bonds when the sequences are complementary. Hybridization will also occur when base sequences are not completely complementary leading to base pair mismatch. The degree of association strongly depends on the number and nature of the mismatch. The stability is dependent on the following factors: (a) A mismatch occurring on the center section of the molecule is less stable than molecules with mismatches occurring at the periphery. (b) The number of mismatches as a percentage of the length. If multiple mismatches are dispersed, the stability of the hybrid will be less than if they were clustered. (c) The identity of the mismatched nucleotide can yield different stability due to greater steric disruption to the duplex. The order of stability for a single position from most stable is T-A, A-T> G-T, G-A, > A-A, T-T, C-T, C-A (28). Very frequently differentiating between single base mismatch and complementary duplexes is required for many oligonucleotide sensitive electrodes. As a rule, the shorter the probe, the easier it is to differentiate between them by melting temperature differences. In an analytical sense, mismatched hybrids may be considered as nonspecific interactions, and their occurrence needs to be reduced. As discussed above, there are several degrees of mismatching and there are no quantitative rules to characterize their stability in terms of melting temperature. As a general guide, with every 1% mismatch bases in a DNA duplex, the Tm is reduced by 1±0.5 °C. For an estimate of the hybridization temperature for the differentiation of complementary and mismatched sequences, the following equation maybe used:

where M is the minimum number of mismatches and L is the total number of base pairs. A target strand of 20 base pairs with one mismatch will have a melting temperature ca. 5 °C less than the Tm of the fully complementary duplex.

33

DAREN J. CARUANA

5.4 Mass transport The probability for a target strand to collide with the complementary strand tethered to a surface is increased by physically increasing the flux of the solution containing the target to the surface. The rate of mass transport may be improved by engineering of the hybridization cell, shaking, stirring, or affecting migration of the target. Reducing the volume and/or increasing the probe-modified surface to volume ratio will improve the diffusional mass transport to the surface. The hybridization process involves not only planar diffusion toward the surface, but two-dimensional diffusion across the surface of the probe modified interface (8). The architecture of the oligonucleotide decorated surface is important. If the surface is porous or covered by a polymeric matrix, then diffusion will be heavily impeded thus reducing the collision frequency. Optimum surface area without impeding the diffusion will require nanoscale surface design to increase the solution availability of probe. Migration in an applied electric field has been studied in the context of improving the mass transport to the surface. However, there have been reports of other effects associated with the electric field at the surface where hybridization takes place. The physical basis of these are not clear but may be a direct or indirect effect of the electric field on the hybridization.

5.5 Nonspecific adsorption From a detection point of view, lowering nonspecific adsorption with the surface is important to maintain a measurable signal-to-noise ratio. The surface of the oligonucleotide sensitive layer maybe viewed as having two adsorption sites: the probe, a specific binding site, and the area around the probe which is nonspecific. The aim would be to restrict the binding to the probe to form a duplex. The importance of nonspecific adsorption is dependent on the method of detection. When detection relies on labeled target (fluorescent or enzyme) brought to close proximity to the electrode, then nonspecific adsorption is important and will lower sensitivity. Other complications may arise regarding the label used for hybridization indication, such as the nonspecific adsorption of the label itself to sites on the surface. If the detection is specific to duplex formation, then nonspecific adsorption is less important. The extent of adsorption of oligonucleotide molecules is dependent on the nature of the surface surrounding the probe oligonucleotides, the pH, ionic strength, and temperature (29). There are strategies that have been developed for filter hybridization to reduce nonspecific adsorption, which are effective for high molecular weight immobilized probes. These strategies include buffers (such as Denhardt's buffer) containing high ionic strength buffer salts, surfactants, and large molecular weight molecules such as Ficoll (a synthetic non-ionic sucrose polymer) polyvinylpyrrolidone, BSA, and also nonhomogeneous heat denatured sheared DNA (Salmon sperm DNA or yeast tRNA). For low molecular weight probe molecules on the 34

OLIGONUCLEOTIDE SENSITIVE ELECTRODES

surface, such strategies are not so effective and would mask much of the surface and reduce hybridization efficiency. Strategies that are effective in reducing nonspecific adsorption: (a) Introducing a wash step after hybridization has taken place to remove nonspecific adsorption. This approach is restricted to post-hybridization detection, not real time. (b) Maintaining the surface, predominantly, negative charged by surface modification, or addition of surfactant such as SDS. (c) Using polymeric coated surfaces with low surface Gibbs free energies of adsorption such as poly(acrylamide). Figure 5 shows the response from an acrylamide-based redox polymer decorated with 18 nucleotide probe. Addition of enzyme (Soyabean Peroxidase, SBP) labeled noncomplementary target at 550 s gives a low signal indicating small amount of nonspecific adsorption. When enzyme labeled complementary stand is added to the hybridization solution at 1450 s, a large amperometric response results from the in situ hybridization (30).

Figure 5 Current-time plot of the catalytic current of a microelectrode coated with the probe-bearing redox polymer, (a) 10 jil of 40 nM SBP-labeled target with four mismatched bases were introduced at 550 s, followed by (b) 10 jil of 40 nM SBP-labeled perfectly matching target at 1450 s. Stirred 1 ml pH 7 HEPES buffer, 1M NaCI with 1.0 mM H202; -0.06 V (Ag/AgCI) thermostated at 45 °C stirred. (Reproduced with permission from ref. 21.) 35

DAREN J. CARUANA

5.6 Other factors Other factors that may affect the stability of the hybrids are pH and formamide concentration. Within the pH range 5-9, there is no effect in the melting temperature. However, pH effect on surface hybridization has not been studied extensively, it is likely to affect the ionizable groups on the electrode surface rather than a direct effect on the hybridization. This is of importance when using polymer-modified surfaces with an increased surface area. Formamide decreases the stability of oligonucleotide hybrids by lowering the Tm. This is a useful property when temperature is an issue for detection or if attachment of probe to the electrode surface is non-covalent. In solution 30% formamide reduces the melting temperature by ca. 20 °C. When formamide is present, the term — 0.72 (%formamide) is added to the equation on page 33.

6 Hybridization kinetics The kinetics of the hybridization of a single strand target in solution is dependent on the mass transport to the surface bearing the probe oligonucleotides. If the sequences are completely complementary, the hybridization may be described by a Langmuir-type isotherm for all sites being equal. The diffusion limited first-order Langmuir model:

where t is the time, r and T0 are the coverage and the total coverage at infinite time, and kLD is the rate constant. This equation appears to fit the hybridization transient well when mass transport is diffusion limited. There are two cases that may be limiting; either the rate of mass transport or the rate of surface confined hybridization. If the concentration of the target is low relative to the probe concentration, the limiting factor would be the mass transport; if however the target concentration is high, then hybridization rate would be limiting. The rate of hybridization is dependent on the hybridization conditions chosen and the application for the oligonucleotide sensitive electrode. Manipulation of hybridization rate is important when concentration of target is required. The dynamic range can be achieved when the hybridization rate is mass transport limited. This is a simplified picture and the rate is ultimately dictated by the stringency of the conditions chosen for hybridization: (a) Temperature. A maximum rate of hybridization occurs at 20-25 °C below the Tm in solution. As the temperature approaches Tm, the hybrids tend to dissociate leading to a low overall rate of hybridization. The temperature dependence on the rate of surface-confined hybridization has not been studied in detail but it is likely that the trends would be similar. (b) Concentration of target. As the single stranded target concentration increases, the hybridization rate will increase. However, high concentration will lead to nonspecific adsorption/binding which will depress the signal-to-noise ratio. 36

Table 3 Summary of effect of experimental variables on the hybridization and stringency of conditions for sequence discrimination Variable

Hybrid stability

Hybridization rate

Attached probe

High stringency

Low stringency

Temperature

10 °C Below 7m hybrid is stable

Maximum rate is ca. 25 °C below 7m Closer to the 7m the rate is slower

No effect

Temperature close to the 7m above the 7m of the single base pair mismatch

Temperature approx. 20 °C lower than 7m

Ionic strength

High ionic strength high stability

Rate increases x 4 up to 0.4M. No significant change up to 1M

High ionic strength leads to screening of anionic charges on adjacent probes

1M NaCI

Above 0.4M NaCI

Formamide

Reduce 7m by 0.72 (%Formamide)

No effect

Unknown

Lowering of 7m destabilizes complementary and mismatched hybrids equally

Not required

DAREN J. CARUANA (c) Target. If the target is double stranded in the test solution, melting before hybridization occurs at the surface of the electrode. This will reduce the rate of hybridization due to a competing hybridization process in the solution. (d) Formamide. From filter studies, formamide does not appear to affect the hybridization rate but shifts the optimum temperature by the same amount as the melting temperature. Formamide at 30-50% in the hybridization conditions reduces the stability of hybrid 30-42 °C depending on the length of the molecule. (e) Ionic strength. At high ionic strength (1M NaCl) hybridization rate is fast due to stabilization of duplex once hybridization has taken place. At low ionic strength, < 0.1M Na+ , a twofold increase in concentration increases the rate by 5-10-fold. (f) Dextran sulphate. 10% dextran sulfate gives a 10-fold increase in reassociation rate in solution. The effect is believed to be associated with volume exclusion raising the effective concentration. For short sequences hybridization accelerators are probably not effective.

7 Summary In summary the process of hybridization is a complex interaction dependent on an array of physicochemical and chemical variables, see Table 3. The topics covered in this chapter ensure that the variables are identified and optimized to maximize hybridization. The detection of the hybridization process at a surface using electrochemical-based methods has attracted a great deal of interest to date (31). Regardless of which technique is used for detection of hybridization, the design of the probe modified surface and the choice of conditions are important to consider. The surface design influences the detection sensitivity (hybridization efficiency) and hybridization conditions will dictate the function and sequence selectivity. The current emphasis in oligonucleotide hybridization research is to develop robust detection methodologies that support analysis of individual micron-sized pixels in an array of different sequences of oligonucleotides and to avoid labeling target strands with an indicator molecule.

References 1. Southern, E. M. (1975). J. Mo!. Bio!., 98, 503. 2. Eckstein, E. (1991). Oligonucleotides and analogues, practical approach series. Oxford University Press. 3. Stryer, L. (1995). Biochemistry, 4th edn, p. 789. W.H. Freeman & Co., New York. 4. Shchepinov, M. S., Case-Green, S. C., and Southern, E. M. (1997). Nucleic Acid Res., 25, 1155.

5. Herne, T. M. and Tarlov, M. J. (1997). J. Am. Chem. Soc., 119, 8916. 6. Lemeshko, S. V., Powdrill, Y., Belosludtsev, Y. Y., and Hogan, M. (2001). Nucleic Acids Res., 29, 3051.

7. Kelly, S. O. et al. (1998). Langmuir, 14, 6781. 38

OLIGONUCLEOTIDE SENSITIVE ELECTRODES 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.

Chan, V. et al. (1998). J. Coll. Int. Sci., 203, 197. Levicky, R., Herne, T. M., Tarlov, M. J., and Satija, S. K. (1998). J. Am. Chem. Soc., 120, 9787. Sam, M. et al. (2001). Langmuir, 17, 5727. Huang, E. et al. (2001). Langmmr, 17, 1215. Hartwich, G., Caruana, D. J., de Lumley-Woodyear, T., Wu, Y., Champbell, C. N., and Heller, A. (1999). J. Am. Chem. Soc., 121, 10803. Watterson, J. H. et al. (2000). Langmuir, 16, 4984. Gillespie, D. and Speigelman, S. (1965). J. AM. Biol, 12, 829. Pease, A. C. et al. (1994). Proc. Natl. Acad. Sci. USA, 91, 5022. Maskos, U. and Southern, E. M. (1993). Nucleic Acid Res., 21, 2267. Armistead, P. M. and Thorp, H. H. (2000). Anal. Chem., 72, 3764. Halliwell, C. M. and Cass, A. E. G. (2001). Anal. Chem., 73, 2476. Korri-Youssoufi, H. et al. (1997). J. Am. Chem. Soc., 119, 7388. de Lumley-Woodyear, T., Caruana, D. J., Champbell, C. N., and Heller, A. (1999). Anal. Chem., 71, 394. Caruana, D. J. and Heller, A. (1999). J. Am. Chem. Soc., 121, 769. Anderson, M. L. M. (1999). Nucleic acid hybridization, p. 9. BIOS Scientific Publishers Ltd, Oxford, UK. Bej, A. K. (1996). In Nucleic acid analysis, principles and bioapplications, (ed. C.A. Dangler) p. 1, Wiley-Liss, NY. Bolton, E. T. and McCarthy, B. J. (1962). Proc. Natl. Acad. Sd. USA, 48, 1390. Breslauer et al. (1986). Proc. Natl. Acad. Sci., 83, 3746. Sugimoto et al. (1996). Nucleic Acids Res., 24, 4501. Anderson, M. L. M. and Young, B. D. (1985). In Nucleic acid hybridisation, (ed. B. D. Hames and S. J. Higgins) p. 73, A practical approach series. IRL Press. Oxford. Anderson, M. L. M. (1999). Nucleic acid hybridisation, p. 78. BIOS Scientific Publishers Ltd, Oxford UK. Gani, S. A., Mukherjee, D. C., and Chattoraj, D. K. (1999). Langmuir, 15, 7130. de Lumley-Woodyear, T., Campbell, C. N., and Heller, A. (1996). J. Am. Chem. Soc., 118, 5504. Kuhr, W. (2000). Nature Biotech., 18, 1024.

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Chapter 3 Screen-printing methods for biosensor production Xian-En Zhang Wuhan Institute of Virology, Wuhan, China.

1 Introduction Conventional enzyme electrodes use bulky electrochemical devices as transducers, such as the oxygen electrode and hydrogen electrode. Applications of the sensors are attractive but their market development is slow and limited due to the fact that these sensors, either electrodes or enzyme membranes, are made expensively and need frequent calibration and regular maintenance. In addition, the interferences from variation in oxygen tension or unspecific electro-active substances are also problems. Therefore, cheaper, more reliable and "friendly" biosensors are required to promote exploitation of the market. This had not been realized until the invention of the mediated enzyme electrode and introduction of screen-printing technology. The mediated enzyme electrode was first demonstrated in 1984 (1). The authors employed the ferrocene/ferricinium ion couple to replace molecular oxygen to shuttle the electron between an enzyme redox center and electrode. Its minimal sensitivity to oxygen concentrations, to change in pH and to nonspecific electroactive substances meant that it was possible to incorporate it in a device with a wide range of applications, especially in vitro or in vivo monitoring in whole blood and fermentation process, where oxygen tension fluctuate. This was the start of the second generation of biosensors, concluded by Professor Scheller in the First Congress on Biosensors (1990). The screen-printing technology is conventionally used in both the electronic and printing industries. It once became the core part of a patent for preparation of the thick film electrochemical sensor in 1981 (2). The technology has advantages of design flexibility, process automation, good reproducibility, a wide choice of materials, and reduced expense. It had been thus sought as an alternative method for mass production of biosensors at low cost. In 1987, a few groups reported their disposable enzyme sensors separately (3-5). All these sensors were based on the mediated and screen-printed

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XIAN-EN ZHANG

enzyme electrode. An early example was a pen-sized glucose meter that used ferrocence-mediated enzymatic electrochemistry and screen-printed technology to give a digital glucose readout only 30 s after the application of whole blood. The sensor was for "one-shot" measurement and was disposable. The authors claimed that "this pen marks the beginning of a new generation of devices involving direct electrochemistry which allows it to be fast, accurate, precise, and silent." At the time, two types of the ExacTech (later known as Medisense ExacTech) personal glucose monitors, "pen-type" and "credit card-type" soon became commercially available. "Within two-years of launching into this S billion world market, the product had achieved a 20% market share in the United States and were making substantial inroads into the market worldwide," claimed Higgins in the First World Congress on Biosensors (6), and the annual sales reached half a billion US dollars in the middle of 1990s as estimated by Turner of Cranfield University in the 4th World Congress on Biosensors (1996). As a result the personal blood glucose monitor for people suffering from diabetes is known as the best biosensor examples. This early stage progress was followed by a wide variety of investigations referring to the thick film biosensors, giving a strong push on the second generation biosensors. In 1994, 10 years after the publication of the first mediated enzyme sensor, the inventors shared the Mullard Prize awarded by the Royal Society for their most original contribution to the field. The progress of the field has been documented from various aspects (7-13). The aim of this chapter is to further outline the techniques and applications of screen-printed biosensors.

2 Screen-printing technology The screen-printing process may be a hand operation, a semi-automatic or automatic machine process. Due to the widespread establishment of screen-printing processes in industry, fabrication devices and various materials are commercially available.

2.1 Materials and methods 2.1.1 Substrate matrix The substrate matrix is the supporting material that provides surface for printing of the functional and constructional parts of the sensor. Since the electronic signal generated by the biosensors is small, the substrate must be inert. The cost of the substrate is also a consideration because the sensor is a "one-shot" or disposable measuring device. Some materials have been exploited for this purpose. PVC, which features dielectric properties, chemical inertness, low cost, and workability, is the most commonly used substrate (14-18). Ceramic (aluminum oxide, A12O3) is also frequently used for its excellent properties such as strength, hardness, heat resistance, and corrosion resistance (19-21). Other possible materials are polycarbonate (22, 23), nitrocellulose (24), and glass fiber (25). 42

SCREEN-PRINTING METHODS FOR BIOSENSOR PRODUCTION

2.1.2 Inks The inks or pastes are deposited sequentially through masks or screen on the substrate to form the functional and structural parts of the sensors. A wide range of inks with different physical and chemical properties (e.g. viscosity, conductivity, thermal resistance, and water resistance) can be found to meet diversified requirements in biosensor fabrication (13). They can be classified as two main categories, conductive inks and dielectric inks. The conductive inks form conductive tracks and the electrodes of the sensors. They are made of conductive materials, binding agent, solvent, and additives. The conductive material maybe gold, platinum, silver, or carbon (graphite) powder, which is dispersed in a binding agent. The carbon paste (CP) consists of a mixture of graphite powder and an organic binder. The electrodes made with CP show relatively low background current, a wide operating potential window, convenient modification, renewability, and low cost (26). Because of these fascinating properties, carbon paste electrodes (CPEs) are in extensive use in electroanalysis. Electrochemical performance of a series of CPEs was investigated for screen-printed sensors (27). The binding agent may be epoxy resin, alkyd resin, acrylic resin, polyurethane resin, or phenolic resin, which are dissolved in relevant organic solvents (e.g. mineral oil, ester, alcohol, ketone, etc.). Choice of a solvent is based on its solubility for the binding agent and volatility. Additives may be needed to improve the performance of the ink (such as viscosity, adhesion, curing time, etc.). For instance, a surfactant helps to disperse the carrier powders in the ink so as to prevent them from agglutination and sedimentation. Another example is the drier, which can shorten the curing process by stimulating oxidation of unsaturated bonds of oil molecules or inactivating the natural antioxidants (e.g. proteins, phospholipids, vitamins, which may coexist in the diluting agents). The drier is mainly composed of metal ions and organic acid. Cobalt, manganese, and zinc are the most important ones in this respect. The dielectric ink, usually made with polymer or ceramic, forms the insulation or encapsulating layer of the sensor. It has to be compatible with the substrate, that is, PVC ink is for PVC substrate and is not favorable to ABS substrate. The commercial inks are normally provided as concentrated packages, which need to be diluted before application. Diluents which usually come with the inks increase fluidity and decrease viscosity of the inks. Immobilization of inks after printing is called the curing process, which may be carried out at ambient temperature, by thermal curing or by ultraviolet radiation (UV). Curing at ambient temperature is regarded as an all-purpose method because it is a mild process, although it takes hours or days, longer than other methods. Thermal curing is employed when the substrate is ceramic, it makes stronger binding between inks and substrate and it reduces the curing time. However, the thermal curing is not suitable for the substrates that are not thermally resistant, such as PVC and other plastic materials. Ultraviolet inducible immobilization is a quick curing process and usually takes only a few seconds. It is usually composed of one or more UV curable

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binding agents (e.g. polyurethane resin and epoxy resin or alkyd resin), padding material (e.g. graphite powder), photosensitive agent, and adjuvant. The UV curable binding agent molecules adsorb the energy from radiation to cause so-called "thermal polymerization," the cross-linking reaction between its unsaturated carbon bonds, or "oxidation polymerization," the polymerization reaction between peroxides or hydroxides and the binding agent (28, 29). Photosensitive agents induce the immobilization of the binding agent, which takes a key role during the UV curing process. Benzophenone and benzoin dimethyl ether are the common photosensitive agents. Addition of a sensitizing agent (e.g. nitrile bases) may assist further in the curing. Valuable studies on the method have been carried out by some investigators during last few years (30-33). They showed that UV curing is a highly efficient method and that it needs no expensive facility and consumes less energy. Most importantly, its high printing speed allows the real online production of biosensors. However, some problems for UV curing remain to be solved. For example, quick immobilization of the ink brings about a high inside stress of the coating, which may lower the adhesion force to the substrate. Other drawbacks include costliness and short storage time of the ink prior to printing. Conducting and dielectric inks are well prepared by the commercial suppliers. They meet most of the requirements of the screen-printed sensors fabrication. Direct use of the commercial ink is recommended. However, in some cases, formulations have to be custom-made for specific purposes, for example, to modify the working electrodes, following indications in the literatures. Table 1 lists the basic information of the inks for biosensor fabrication, either commercial products or custom-made formulas.

2.1.3 Sensing element The sensing element is the analyte-specific part of biosensors. From a catalytic point of view, any enzyme could act as a sensing element. However, some enzymes are very expensive to obtain and are not affordable for disposable biosensors. And yet, most enzymes show poor thermal stability. Their shelf lives are too short to be used as sensing elements because the "one-shot" biosensor has no need to be calibrated by the customer, loss of enzyme activity during storage causes significant determination error. Additional stabilization treatment may need even for glucose oxidase that is widely used as a model enzyme for biosensors. Up to date, oxidases and dehydrogenase are the most frequently used sensing elements. Electrochemical properties of many of these enzymes have been well characterized on the electrodes. As mentioned above, a variety of mediators, such as ferrocenes, benzoquinones, and active dyes, may be added to reduce the working potential. As an option, some biological elements, other than enzymes, may be used as sensor elements, for example, antibodies (38-42) and microbial cells (43). The sensing element is immobilized onto the surface of the electrodes through covalent attachment, cross-linking or adsorption. Automated immobilization can be achieved either by ink jet techniques or screen-printing 44

Table 1 Inks that are typically used for screen-printed biosensors Composition or commercial product

Application

Immobilization

References or supplier

70% graphite powder and 30% paraffin, or 60% graphite powder and 40% light mineral oil (w/w). 1-4% (w/w) mediator, such as ferrocene (for oxidases) or Meldola's Blue (for dehydrogenases) may be added to reduce working potentials

Working electrode

Air dry or thermal curable if enzyme and mediator is not incorporated

(34)

70% graphite powder and 30% paraffin, or 60% graphite powder and 40% light mineral oil (w/w).

Padding for conducting track

Air dry or thermal curable

Carbon/graphite ink, SS, or Electrodag

Working electrode or conducting pad

71 °C, 2-5 mm

Acheson

Ag paste

Silver pastes, Electrodag series

Working electrode and conducting track

71-95 °C, 2-5 mm

Acheson

Ag-Pd paste

No. 7474 or QM 22

Reference electrode and conducting track

850 °C, 30 mm on AI203 substrate DuPont, Bad Homburg, Germany

Reference electrode

71-107 °C, 2-5 mm

Acheson

Reference electrode

Air dry

(35)

Ink

Conductive Carbon paste

Ag/AgCI paste Silver/Silver chloride pastes, SS, and PE series 0.2 g AgCI with 1 g Ag ink

Table 1 (Continued) Composition or commercial product

Application

Immobilization

References or supplier

PVC ink, SS seires

Insulating layer

Air dry

Zhongyi inks

Insulating layer, compatible with Au

80 °C, 20-25 min and UV exposure 1-3 s. Compatible with 96% alumina ceramic substrate and Au conductorz

DuPont

3-4% (w/v) hydroxyethyl cellulose added with Triton 100 (0.01%) and polyethylene glyco (3%)

Out dialysis membrane

Air dry

(35)

CA

2% (w/v) cellulose acetate

Out dialysis membrane

Air dry

Gafquat

4% cationic form of Gafquat. Gafquat 755N (Internations Specialty Products Ltd.)

Stabilize enzyme membrane

Air dry

Ink

Dielectric PVC paste

Ceramic paste Fodel 6050 dielectric paste

Encapsulating HEC

(36)

SCREEN-PRINTING METHODS FOR BIOSENSOR PRODUCTION

carbon-containing inks. The latter is thought to be a particularly favorable application for the production of biosensors (13) and is used for the glucose sensors produced by Medisense Inc. (Cambrdge, MA). To print the sensing layer, the sensing element is integrated into the ink to form the sensing element ink (30, 44-46). The ink has to be carefully prepared to ensure that the sensing element is dispersed in the ink properly. Otherwise, a determination error would occur. One drawback of the printing method is that the large amount of enzyme ink must be applied at a time, which may not be economical on the laboratory scale. The ink jet may be a more flexible method, it can be performed easily either in the mass production or laboratory scale. Both methods are applied in the later stage of fabrication so that the sensing element is not exposed to the high temperatures that may be needed during the initial thermal curing processes. In some cases, coatings of semipermeable membranes (e.g. Nafion or cellulose acetate) (47, 48) can be printed to the surface of the sensor so as to prevent the enzyme from leaking and to reduce the influence of possible interference from the sample solution.

2.2 Apparatus The screen-printing machine may be horizontal, vertical, or desk mounted. The printing surface may be flat, rotary, or cylindrical and the printing process may be direct, indirect, or electrostatic deposition. But the flat and direct printing machines are most convenient for biosensor fabrication. With the growing interests in screen-printing, the microprocessor-controlled automation process has largely replaced the hand-made process. An up-to-date automated screen-printer is equipped with advanced microcomputer controls with multi major functional program presettings. As shown in Figure 1, the main parts of the machine normally include a printing table with various printing sizes, pneumatic frame clippers making prepress preparation or printing frameshift much easier and faster, a squeegee that is driven by pneumatic element, a frequency regulator that controls the printing speed, and a micro adjuster that leads to high precision. Figure 1 is a diagram of the core part of the screen-printing machine.

Figure 1 Screen-printing process. Negative pressure is generated by an air compressor.

47

XIAN-EN Z H A N G

Figure 2 Diagram of screen-printing process for preparation of biosensors.

In InkJet printing, the liquid dispenser that delivers the sensor element to each sensor strip operates at controlled volumes. It is usually integrated into a platform to combine motion control with line and dot dispensing. This allows one program to control dispense speed, line concentration, drop size, and position. Biojet QuantiSOOO™ (BioDot Inc.) is an example used in the author's laboratory (Figure 2). The device is based on the combination of a solenoid valve to form drops and a syringe pump to meter reagent from a positive volume displacement. The system is capable of drop-on-demand dispensing precisely defined drop sizes (as low as 4.16 nl/drop) that are determined by the stepping resolution of the syringe pump (driven by a high resolution stepper motor) and synchronizing the stepper increments with the opening and closing of the solenoid valve. This state-of-the-art device provides non-contact dispensing of individual drops, which are quantitatively defined in either line or dot formats. Figure 3 shows the ink jet nozzle with a group of screen-printed electrodes on the substrate board.

2.3 Printing patterns The sensor may be a three-electrode (working electrode, counter electrode, and reference electrode) or a two-electrode configuration (without counter electrode). Conventional cyclic voltammetric experiments incorporate a three-electrode system avoiding current flow through the reference electrode. Instead, for small electrodes, the current flowing in the cell will not be large and therefore will not perturb the reference potential significantly, it may be convenient to use only a two-electrode configuration. If a multiple measurement is considered, the sensor can be designed as a multi-working electrode pattern. A group of printing masks is prepared using photolithographic technique, each mask provides a pattern for printing the relevant layer. The mask is made with nylon, polyester, or stainless steel meshes (100-300 meshes per inch with the fiber thickness 30-60 |j,m) that are stretched over a metal (e.g. duralumin) or wooden frame through an assembling machine. The mesh is coated with a photosensitive gel, 48

SCREEN-PRINTING METHODS FOR BIOSENSOR PRODUCTION

Figure 3 Ink jet nozzle with the screen-printed electrodes. The nozzle is jetting the enzymemediator solution onto each working electrode. The nozzle is fixed in position while the platform that carries the screen-printed electrode board moves from right to the left, driven by a high resolution stepper motor, ensuring the finely controlled volume size of enzymemediator solution to be deposited on the correct position of each electrode surface.

on which a master plate is then conglutinated tightly. The plate is made from a template by a photographic method. During exposure, the coating at the transparent area of the plate is solidified, leaving the covered area glutinous. The latter is to be removed by a solvent. After washing and drying, the mask is checked carefully. Additional modification steps may be required to ensure the quality of the mask, for example, in order to increase the solidity of the mask, repeated exposure or a hardening agent is applied occasionally.

2.4 Printing process Inks are deposited sequentially on a clean substrate by forcing them through a group of masks with the squeegee. Each layer is deposited following the corresponding mask. Drying or curing is performed between two printing stages. The ink that contains the sensing element generally forms the final layer, however it may be covered with a layer of semipermeable membrane when necessary. Figure 3 shows the diagram of a printing process, taking a two-electrode configuration glucose sensor as an example. Details are demonstrated in Protocol 1 that is routinely operated in our laboratory. The optimum screen-printing process may be different as it depends on the materials to be deposited, the intended application and the type of machine employed. Evaluation of the screen-printed 49

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electrochemical enzyme sensors can be performed with cyclic voltammetry and chronoamperometry methods.

Protocol

Preparation of a screen-printed glucose electrode A model S-600M screen printer (Ever Bright Printing Machine Ltd., Hong Kong) is used to print the electrodes throughout. All drying processes are undertaken at room temperature. 1 Clean the PVC substrate (150 mm x 150 mm x 0.5 mm) with tap water, anhydrous ethanol, and distilled water sequentially. Each sheet of the substrate allows a group of 45 sensors to be printed. 2 Fix the cleaned PVC substrate on the printing table of the printing machine by negative air pressure. 3 Place the first mask, which is designed for printing conductive strips, over the substrate on the table against registration stops, ensuring accurate pattern fitting. 4 Pour the silver ink onto the screen. Spread the ink over the surface of the screen with the squeegee so that the ink is pushed through the mask. 5 Let the ink solvent evaporate at ambient temperature. Print a carbon pad on to the silver strips by the same printing procedure with the second mask. Allow the ink to dry. The carbon pad protects the silver strip from oxidation through being exposed to air. 6 Print the reference electrode, with the third mask, by applying a silver ink mixed with finely ground silver chloride in the ratio of 2 g AgCl per gram of ink, onto one end of a graphite pad. The ink is again left to dry at room temperature. 7 Print the insulating layer, with the fourth mask, by applying a PVC ink, leaving terminals and active surfaces exposed. The active surface is a circular area that forms a planar, two-electrode electrochemical cell. One semicircle is the carbon electrode and the other is the silver/silver chloride reference electrode. 8 Prepare a saturated 1,1'ferrocenedimethanol solution in distilled water. Dissolve Aspergillus niger glucose oxidase into the ferrocene solution, making the final concentration 10 units GOD/|j,l. 9 Drop 1-2 |j,l of the enzyme-ferrocene solution onto each surface of the carbon electrodes by using the BioDot liquid dispenser. 10 The electrodes are then allowed to dry in a desiccator at room temperature. 11 Print an outer membrane (HEC) on the surface of the electrode cell to cover the enzyme and ferrocene layer. The HEC solution is prepared by mixing 3.5% (w/v) hydroxyethyl cellulose, 0.02% Triton XI00, and 3% polyethylene glycol in distilled water. 12 After the water had evaporated under ambient conditions, the sensors can be stored over silica gel at 4 °C.

50

SCREEN-PRINTING METHODS FOR BIOSENSOR PRODUCTION

3 Applications As they are inexpensive, reliable and rapid, screen-printed biosensors have been recognized so far as the most important technique pushing biosensors into practical use. During last 10 years, hundreds of screen-printed biosensors have been reported, with applications in a wide variety of analytical problems in medicine, pharmaceuticals, the environment, food, bioprocess, security, and defense. Some of them are made as pocket devices for home care of the patients and for field tests. Many of them are believed to soon become commercially available.

3.1 Clinical diagnosis Clinical importance of the screen-printed biosensor has been well demonstrated by the pocket blood glucose meter that can be found in the market in many styles. Most of the meters are designed as a one-step process. Only one drop of finger blood is needed for the detection. The result can be obtained in less than 1 min. With these devices millions of diabetics can test their blood glucose level at home, therefore, careful control of the disease is thus feasible so as to greatly improve their life quality. Subsequent studies were carried out for improving the sensor performance further. A detection limit to 0.02 mM was achieved by the enzyme electrodes prepared from platinized Vulcan XC-72 carbon particles (49), and an increased linear range up to 45 mM was achieved in the author's laboratory (to be published). A highly selective glucose sensor using copper (Il)-hexacyanoferrate and GOD with a carbon ink has been described (50). The dispersed metal-hexacyanoferrate catalyst offers a marked decrease in the overvoltage for the oxidation of the enzymatically liberated hydrogen peroxide, and gave a stable response at physiological pH. Such efficient catalytic activity allows tuning of the detection potential to a region (around — 0.1 V) where interfering reactions are negligible. Such an operation eliminates the need for an anti-interference membrane barrier, and along with the one-step dispersion of the enzyme and electrocatalyst, greatly simplifies the sensor fabrication. One problem encountered is the effect of temperature on the enzyme electrode. Since the enzyme electrodes provided to the customers were pre-calibrated by the manufactures at a certain temperature, errors will occur if the detection was performed at a different temperature, because the response signals varies with variation in temperature. Automatic temperature compensation was built based on careful investigation of the temperature coefficient of the enzyme electrode (51), making the device more reliable. Other important metabolites that have been measured with the screen-printed sensors are lactate (16, 34, 52), uric acid (53, 54), urea and creatinine (55), cholesterol (56), progesterone (57). In particular, sensors for urea and creatinine may fulfill a clinical demand in kidney dialysis, the treatment for renal disorder patients. During the dialysis process, concentration of blood urea and creatinine are used as control parameters. A bedside measurement sensor will provide the 51

XIAN-EN ZHANG

useful information to the doctor in a timely fashion and reduce the treatment costs significantly.

3.2 Food analysis bioprocess control In addition to the importance of glucose and lactate as major analytes in clinical diagnosis, they are also parameters in many foods, beverages, and bioprocess control. A screen-printed lactate sensor was constructed and applied with a flow injection system based on dialysis, to off-line and online monitoring of Geotridmm candidum cultivation producing lactate oxidase (58). By online injection analysis of undiluted fermentation broth over a period of 12 h prior to the inoculation, no significant loss of sensor response was observed which demonstrates the good operational stability of the system even with complex real samples. The stability of the sensors was due to use of UV polymerizable carbon ink and optimized working condition. Recently, a novel flow injection analytical system with a screen-printed enzyme sensor incorporating Os-complex mediator has been developed for continuous determination of glucose, giving a through put of about 40 samples per hour. The glucose biosensors retained their constant response after more than 100 injections and storage over a month. The design is suitable for automatic and rapid determination of glucose (59). Multipurpose biosensor is particularly useful in the analysis of foods and beverages. Its feasibility increased since the introduction of screen-printing technology, with which combination of multifarious sensing probes can be easily designed. The first example was a design for simultaneous determination of glucose and ethanol (60), followed by the sensors for determinations of biogenic amines for the fish freshness estimation (61, 62), maltose and glucose for dry beer quality control (35), starch and glucose for starch hydrolysis process monitoring (63), sucrose and glucose for analysis of soft drinks (48), and glucose and glucoamylase activity for control of glucoamylase production (64). The results showed great interests in the food and fermentation industries. Table 2 gives details of some applications.

3.3 Environmental monitoring With the growing concern of human beings in relation to their living environment, detection of toxic pollutants becomes more important than ever for environmental evaluation and protection. For instance, the effects of chemical pesticides on human health has drawn global attention due to their inappropriate use worldwide. Some pesticides are almost non-degradable, they can accumulate in the human body through the food chain, causing chronic poisoning. Some are very toxic, causing acute poisoning to animals and humans. Organophosphate pesticides, known as the strong nerve inhibitors, have dominated the pesticides market for many years and have caused numerous cases of acute poisoning due to careless intake of polluted vegetables, fruits, tea, or water. They are now prohibited in many countries. Field or local detection of 52

SCREEN-PRINTING METHODS FOR BIOSENSOR PRODUCTION Table 2 Some screen-printed biosensors and their performances Analyte

Sensor element

Electrode configuration

Performance

Lactose in milk

p-galactosidase, glucose oxidase

Pt working electrode Ag/Pd reference electrode

2-25 mmol/l SD: (65) 8.81% (n = 16) Stability, longer than 3 months

Lactic acid

Lactate dehydrogenase, NAD(+)

Meldola blue modified carbon working electrode and an Ag/ AgCI combined reference/counter electrode

1-20 mmol/l CV = 8.7% (n = 6)

(34)

Alcohol in beverages

Alcohol dehydrogenase, NAD(+)

Meldola's blue modified carbon working electrode and an Ag/ AgCI combined reference/counter electrode

35 mmol/l 90% residual activity after 49 days, stabilized by trehalose

(15)

NADH and lactate

NAD(+ ) and lactate dehydrogenase

NADH, 3-60 nmol/l Lactate, 0-20 mmol/l

(16)

Uric acid and hypoxanthine fish freshness

Xanthine oxidase

1-50 nmol/l CV: 2%

(62)

Maltose and glucose

Amyloglucosidase/ Carbon working glucose oxidase (A/G) electrodes Ag/AgCI reference electrode

Maltose, -20 mmol/l (35) Glucose, -40 mmol/l CV: 3.5-5.29%

Starch and glucose

GOD Glucoamylase

Carbon working electrode Ag/AgCI electrode

Starch, 0.4% (w/v) Glucose, -20 mmol/l

Lysine in fermentation

Lysine oxidase entrapment into polyurethane hydrogel

Pt working electrode Ag/AgCI pseudo reference electrode, Carbon counter electrode

Gentamicin in milk

Anti-gentamicin antibody labeled with GOD

Flow electrochemical cell

Carbon working electrode

Reference

(48)

(66)

0-10 ng/kg CV: = 13.2%

(67)

these pesticides is of particular importance. Sensors based on the screen-printed technology have been constructed to detect some of the important pesticides, such as 2,4-dichlorophenoxyacetic acid (68-70), organophosphate pesticides (71-79), carbamates (78, 80), polychlorinated biphenyls (81, 82). The sensors for organophosphate pesticides are based on the inhibition, through binding reactions, of cholinesterase or acetylcholinesterase that play an important role in nerve conduction. The principle was introduced to construct biosensors for more than 20 years but none of these sensors were reliable due to reasons of cost and poor reproducibility. The screen-printed biosensors may be the answer 53

XIAN-EN ZHANG

to the problem. Other applications using screen-printed biosensors include determination of nitrogen compounds which are one of the major factors causing eutrophication. Local determination of these compounds in water provides useful information for the control of eutrophication. A research team in England reported a group of screen-printed sensors used for the determination of ammonium ion (83, 84) and sulfite (85). The proposed biosensors were evaluated on samples of unspiked and spiked river water; the recovery and precision data indicated that the devices could be expected to give reliable data in these waters. Another example is quick estimation of biological oxygen demand (BOD). BOD indicates the level of organic pollution in the water. Conventional methods need 5 days to operate. With the BOD sensor, a combination of living microbial cells and oxygen electrode, the result can be obtained in just 20 min (86, 87). A disposable BOD sensor, based on mediated oxidation of organic material by the cells, was proposed (88). This protocol, however, needed further study to answer the question on how a chemical mediator interacts with the microbial cells.

3.4 Other approaches DNA sensor technology is one of the notable progresses in the biosensor field. A disposable electrochemical sensor for the detection of short DNA sequences is described. Synthetic single stranded oligonucleotides have been immobilized onto graphite screen-printed electrodes through binding of avidin-biotinylated oligonucleotide and adsorption at a controlled potential. The probes were hybridized with different concentrations of complementary sequences. The formed hybrids on the electrode surface were evaluated by differential pulse voltammetry and chronopotentiometric striping analysis using daunomycin hydrochloride as an indicator of the hybridization reaction. The DNA sensor was able to detect 1 ng/ml of target sequence responding to point mutation human diseases (89). Another DNA sensor was made of an ion-exchange film-coated screen-printed electrode adapted to the bottom of a polystyrene microwell. An alkaline phosphatase label was used to hydrolyze the monoester phosphate salt of [(4-hydroxyphenyl)aminocarbonyl]-cobaltocenium. This anionic substrate is transformed into a cationic electroactive product, which is then accumulated by ion exchange at the electrode surface to give an amplified electrochemical response. Detection limits of 10 amol/ml was thus achieved for amplified cytomegalovirus DNA. The sensor was also used to assay human chorionic gonadotropin hormone (hCG). The method was 10-40-fold more sensitive than the conventional absorption spectrophotometry using p-nitrophenyl phosphate as the substrate (90). Based on this work, it is likely that we can exploit the screen-printed oligonucleoitide array for mass DNA sample analysis. Biomimetic or molecular imprinting is being increasingly recognized as a versatile technique for the preparation of synthetic polymers containing tailor-made recognition sites. This is achieved by co-polymerizing functional and cross-linking monomers in the presence of an analyte, which acts as a molecular template. After elution of the template, complementary binding 54

SCREEN-PRINTING METHODS FOR BIOSENSOR PRODUCTION

sites are revealed that allow specific rebinding of the analyte (91, 92). The method was proposed for biosensor purposes in 1990 (93) and has shown its utility in recent years (94, 95). However, such sensors need to be mass-produced cost effectively. Attempts were made by a few investigators. A biomimetic sensor based on the combination of a screen-printed peroxidase electrode and an iron porphyrin complex iron(III)-meso-tetrakis-(pentafluorophenyl)-beta-tetrasulfonatoporphyrin chloride was proposed for aliphatic hydrocarbons, using octane as the model analyte. The biomimetic compound was immobilized in a polyelectrolyte matrix. Catalytic reaction was described in the homogeneous system and on the sensor surface (96). The binding principle was used to build an imprinted polymer-based sensor for herbicides (2,4-dichlorophenoxy acetic acid). The method involves a competitive binding step of the herbicides with a unrelated electrochemically active probe, 2,4-dichlorophenol that showed very high non-specific binding to the imprinted sites. The imprinted polymer particles were directly coated onto the screen-printed carbon working electrode. Following incubation of the modified electrode in a solution containing the analyte and the probe, the bound fraction of the probe was quantified by differential-pulse voltammetry. This system provides a cheap, disposable sensor for rapid determination of environmentally relevant and other analytes (91).

4 Conclusion The screen-printing technology has shown its great utility to biosensor researchers. During the last 10 years, it has become a mature, widespread sensor technology, drawing biosensors out of "the tower of ivory," for use by the people. With the technology, biosensor design can be more flexible: disposable or reusable, single or multiple purpose, planar or spherical sensing surface, mediated or direct electrode process. Therefore, further application of the technology in the biosensors field is very promising. It is, however, pointed out that, at present, most of the screen-printed biosensors are disposable; successful developments of the sensors largely rely on the cost and stability of the sensing elements. This is an intrinsic problem of biological nature. Many genetic expression vectors have been developed for high-level expression of the enzymes or other functional proteins, and protein engineering provides a powerful tool for improving the enzyme toward the desired properties, which may be a potential resolution to the problems. (See Chapter 9, this volume.)

Acknowledgment The author thanks the Comet Bioelectronic Tech Company (Wuhan, China) for its consultancy in the preparation of screen-printed biosensors, and Zhi-Ping Zhang, Wei Li, and Li-Qun Chen for valuable discussions.

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58

Chapter 4 Kinetic modeling for biosensors Philip N. Bartlett and Ghee-Seng Toh Department of Chemistry, University of Southampton, Southampton, S017 1BJ, UK.

1 Introduction 1.1 The purpose and practice of modeling The purpose of a kinetic model of a biosensor is to identify the key experimental factors (such as the rates of reactions, rates of mass transport, loading of the bio-recognition component, etc.), which determine the response, or output, of the sensor and to then provide a link between these key experimental factors and the concentration of the analyte and the sensor response. Thus, modeling provides a mathematical description of the physical processes occurring within the system. By applying kinetic modeling to enzyme electrodes and biosensors, we can derive a number of advantages. It is clear that several different kinetic processes are involved in the overall functioning of any enzyme electrode or biosensor. In an enzyme electrode these include the reactions between the enzyme and its substrate, between the enzyme and the mediator, and between mediator and electrode, as well as the various mass transport processes which bring reactants to the electrode and take products away. On its own, any single measurement cannot give any insight into the relative significance of the different kinetic steps. If, however, a set of data from the sensor, obtained under a sufficiently wide range of different conditions, is analyzed and compared to a kinetic model for the sensor, this will yield more intimate knowledge of the system. In turn, this will provide the experimenter with the tools with which to plan future experiments or with which to rationally improve sensor performance for a specific application. In contrast, in the absence of a suitable kinetic model for a particular sensor, it will be necessary to carry out a large amount of trial and error experimental work in order to achieve a comparable understanding of the system. This is very time consuming and it will have to be repeated, effectively re-inventing the wheel, each time a new but related system is studied. Developing a kinetic model is not only an efficient experimental approach, it also gives insight into the mechanisms and processes involved in the operation of 59

PHILIP N. BARTLETT AND CHEE-SENG TOH

the sensor. This is especially true when we approach the analysis using approximate analytical methods (see below). This is a highly satisfying experience for the individual, and it can produce a rich reward in terms of the new knowledge that can be obtained from such a pursuit. In this chapter, we will take the reader through a basic understanding of enzyme kinetics, electrochemistry, and the basic mathematical tools necessary for an understanding of kinetic analysis found in the literature on enzyme electrodes. Toward the end of the chapter, the reader will find a table (Table 2) summarizing much of the published work in this field from 1992 to the present. This will be useful for those who wish to take the subject further and to apply these approaches to their own data for enzyme electrodes. In what follows we concentrate heavily on amperometric enzyme electrodes. This is because there is a much greater amount of literature on the modeling of this type of biosensor than others. However, the same general principles will apply in developing models for other biosensor types including potentiometric and conductimetric biosensors as well as optical and gravimetric sensors. In all cases it is necessary to consider the interplay of mass transport to, or from, the sensor surface and its interaction with the kinetics of the biorecognition process and the transduction mechanism. At the end of the chapter we briefly review the modeling of other biosensor types.

1.2 Enzyme kinetics 1.2.1 Equilibrium and the steady state At equilibrium, the concentrations of reactants, intermediates, and products are constant; however, this does not mean that there is no reaction going on. Equilibrium is a dynamic state. At equilibrium, the rates of the forward and reverse reactions (i.e. the products of the appropriate rate constant and reactant concentrations for each step) must be in balance. In general, enzyme electrodes do not operate at equilibrium, rather they operate in the steady state where there is a constant supply of fresh reactant to the electrode. In the steady state, the rates of change in the concentrations of the reaction's intermediates are negligible so that the rates of reaction are unchanging with time. Under these conditions we can equate the fluxes (i.e. the number of moles of reaction occurring per unit area in unit time) for the different reaction steps in order to derive useful kinetic information about the system. For biosensors these reactions generally occur at or near the sensor surface rather than uniformly throughout the solution; consequently, the concentrations of reactants and products at the sensor surface will be different from those in the bulk. As a result, there will be concentration gradients set up and net fluxes of material to and from the electrode surface.

1.2.2 Michaelis-Menten kinetics The simplest description of steady-state enzyme kinetics is based on the work of Michaelis and Menten (1). It assumes that the substrate first forms a complex 60

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with the enzyme in a reversible step and that equilibrium is maintained between the enzyme, E, and substrate, S, and the enzyme-substrate complex, ES. The irreversible breakdown of this enzyme-substrate complex then yields the product, P.

The second assumption in this model is that the concentration of the enzymesubstrate complex is constant, so that it is a steady-state process. Clearly this is not true immediately after the reactant and enzyme are first mixed together when the concentration of intermediate is building up (called the pre-steady state phase) and will only be true as long as the concentration of substrate is not significantly depleted by the course of the reaction. The following, more generalized, mechanism in which the forward and backward rate constants for the formation of the ES complex are explicitly included, was proposed by Briggs and Haldane (2):

If eE is the total concentration of enzyme and eES is the concentration of the enzyme-substrate complex, then the concentration of uncomplexed enzyme is (eE — eEs). Assuming that the concentration of substrate is much greater than the concentration of enzyme (which is generally the case), then the concentration of uncomplexed substrate can be taken as equal to the initial concentration of substrate, s. Then we can write

At steady state, deES/dt = 0 (strictly the approximation we make is that deES/dt is small compared to the reaction flux k^s). Then,

The velocity of reaction, v, is given by

so that which can be rewritten in the form of the Michaelis-Menten equation

where kcate^ is the maximum reaction velocity and KM — (k_i+ kcat)/ki is the Michaelis constant. 61

PHILIP N. BARTLETT AND CHEE-SENG TOH

Figure 1 Normalized plot of steady-state velocity against substrate concentration for an enzyme reaction obeying Michaelis-Menten kinetics. The dotted lines show the point at which the concentration of substrate equals KM.

This model reduces to the simple form of Michaelis-Menten kinetics, with

1.2.3 Analysis of enzyme kinetic data Analyses of enzyme kinetics are often based on the Michaelis-Menten equation (7). Figure 1 shows a typical plot of reaction velocity, v, against substrate concentration, s. From Figure 1 we can see that at low concentrations the reaction velocity increases linearly with substrate concentration. This is because when s KM) or that the substrate concentration is much smaller than KM (i.e. s KM all the way through the membrane. 76

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We now turn to the other approximation. When the substrate concentration is much smaller than KM, the enzyme kinetic term reduces to (kcatCz/KivOs and Equation (38) becomes

This is a linear second-order differential equation for which we can write a general solution of the form: where A2 and B2 are integration constants and

is the kinetic length, that is, it is the distance that the reactant S can diffuse within the enzyme layer before its concentration is reduced to 1/e (36.79%) of its original value by reaction with the enzyme in the unsaturated enzyme kinetic regime. Differentiation of Equation (48) gives

Applying the same boundary condition as before, so that at x = 0, ds/dx = 0, we find that A2 — 0. Then applying the boundary condition for the concentration of substrate at the outside of the enzyme layer, that is, at x — I, s — sbuik, we find that

Thus, we obtain the following expressions for substrate concentration profile:

and substrate concentration gradient:

Again, the steady-state current density is given by

so that for this case

Equation (55) itself has two limiting forms. When J/XkK M and sK]v[> Equation (58) becomes

where the reaction rate is zero order with respect to the substrate concentration. At the boundary between the two cases, where s — KM, we find that although these two approximate solutions are equivalent (i.e. if we substitute s = KM in Equation (59) we obtain Equation (60)), the two approximate expressions do not agree with the full equation. On the other hand, away from the boundary the approximate expressions are very good approximations to the full equation. As a more complex example consider the enzyme membrane | electrode problem considered briefly in Section 2.4.3. Starting from the approximate analytical solutions we can derive a case diagram which describes the different solutions, this is shown in Figure 10. For this system there are four limiting cases. Three of these correspond to the simple limiting cases derived in Section 2.4.3 and given by Equations (44), (56), and (57). For thin films, when 1 < Xk, the reaction occurs uniformly throughout the enzyme layer. If s < KM (case I), the reaction is first order in S, if s > KM (case III), the enzyme is saturated and the reaction is zero order in S. For thicker films, when 1 > Xk, the enzyme-substrate reaction occurs in a thin layer at the outside surface of the enzyme layer and the concentration of substrate falls substantially as we go into the film. For low substrate concentrations, when s < KM (case II), the

Figure 10 Case diagram for the enzyme membrane|electrode problem showing the four cases with the corresponding concentration profiles. 80

KINETIC MODELING FOR BIOSENSORS

reaction is first order in S and so the concentration of S falls exponentially as we go into the enzyme layer. When s > KM, we find a new, fourth, case not discussed in Section 2.4.3. In this case (case IV), the reaction kinetics will be zero order in S at the outside of the film but as the concentration of S falls, it will change to first order at some point within the film. The corresponding concentration profiles are shown in Figure W. This type of model has been used by Bartlett et al. (27-29) to describe the oxidation of nicotinamide adenine dinucleotide (NADH) at polyfaniline) electrodes. Other examples of case diagrams and their applications can be found in the literature (14, 30, 31).

2.6 Experimental verification of approximate analytical kinetic models Deriving kinetic models without subsequent verification against real experimental data is a theoretical exercise of little value for real applications. Quite often, it is the comparison of the model to experimental data that helps to refine the model and to produce an improved understanding of the factors which determine the performance of the real biosensor. Before rushing ahead with experimental measurements, the researcher needs to think about the design of the experiments. Although one can derive a considerable amount of information from a single experiment if one already has a valid theoretical model, a single experiment can never be sufficient to "prove" or "disprove" the validity of a particular model. In order to do this, a systematic series of experiments must be carried out. This series of experiments should be designed to systematically test the dependence of the biosensor response on each experimental variable over as wide a range as feasible. That is, if factors such as enzyme loading, rotation speed, and film thickness are variables in the kinetic model, it will be necessary to carry out series of experiments to investigate the dependence of the biosensor output on each of these variables in turn. This immediately implies that this type of study can only be carried out on biosensors which can be reproducibly fabricated so that the results from one sensor can be compared quantitatively with those from another sensor. The approach toward experimental measurements which will allow us to establish a meaningful kinetic model is as follows. (a) Set up an initial model for the biosensor and define those experimental variables which are likely to affect the performance of the electrode (e.g. enzyme loading, substrate concentration, membrane thickness). (b) Choose a starting point—it is best, if possible, to start with conditions close to those for similar biosensors already described in the literature. If there is no appropriate equivalent, one has to be guided by the initial model and the results of preliminary experiments. (c) Compare the values from the first exploratory experiments with those derived from the initial model. Try to find out which step is rate limiting. 81

PHILIP N. BARTLETT AND CHEE-SENG TOH

If the initial model does not fit, it will need to be modified to include new factors or assumptions which were not considered in the initial model. Further exploratory experiments may then be required to explore the revised model. (d) Once one is happy that the model appears to be the correct one, carry out a detailed series of experiments systematically varying each experimental parameter. Try to vary each parameter over as wide a range as practicable so that all the different cases are explored. (e) Analyze the data using the model to produce a set of quantitative results for the model parameters (e.g. rate constants, mass transport coefficients, etc.). (f) Critically compare the quantitative results obtained from the model both from one experiment to another and with existing values available in the literature. Only when all the experimental data fits the model, when the quantitative values obtained from each series of experiments are consistent, and when the quantitative values for rate constants, diffusion coefficients etc. are reasonable in view of the published literature should one be satisfied that the model is reasonable. (g) Even at this stage one should only accept the validity of the model with caution. If there are other possible experimental ways to test the model these should be investigated if at all possible.

2.7 Numerical simulation methods So far we have concentrated on the use of approximate analytical solutions. A second, complementary, approach is to use numerical simulation techniques to model the processes involved in the system. Numerical techniques can provide accurate treatments for the behavior close to the boundaries between limiting cases. The disadvantage is that these numerical approaches do not provide as much insight into the behavior of the biosensor. Thus, the combined use of the two approaches is often more powerful than either on its own particularly for complex situations, for example, see ref. (14). Below, we will briefly describe the general principles behind the use of numerical simulation. A full discussion of the different techniques is beyond the scope of the present chapter. For further information, the reader should consult some of the more specialized texts (32).

2.7.1 Explicit numerical methods The first use of digital simulation in electrochemistry was based on the explicit finite difference method, pioneered by Feldberg (33). In many ways this is the simplest method to understand and to implement but it also suffers a number of limitations, particularly when one wishes to simulate diffusion and coupled kinetics. The first step in setting up a numerical simulation is to transform the problem into a discrete form. The explicit finite difference method treats the problem as an array of discrete "boxes" where the boxes are sufficiently small such that the concentration of species within each box is considered to be constant, see Figure 11. 82

KINETIC MODELING FOR BIOSENSORS

Figure 11 Schematic diagram showing the relationship between the change in concentration (s t+it -s t ), over a discrete time, St, and the net flux, (J\ + i — J\-i), across a discrete distance, x.

As an illustrative example of the method, we will consider a potential step at an electrode in a solution of the reduced form of an electroactive species, S, where the potential is stepped from a potential at which there is no reaction to one at which the oxidation of S is mass transport limited, this is the same situation considered in Sections 1.3.2 and 2. The potential step brings about a sharp change in the concentration near the electrode surface. If we divide the space in front of the electrode into a number of boxes then the concentration differences between the boxes create the driving force for diffusion of species into and out of the boxes. We can estimate the change in concentration over time within each box by calculating the net flux of species moving into or out of the box.

where c5sf is the change in concentration in the ith box over a time interval §t, A is the area of the electrode, V—ASx is the volume of the box, andjjinet the net flux into the box. Hence, for the box at a distance i$x from the electrode, where all the boxes are assumed to be of the same width, 8x, the change in concentration over time §t will be

83

PHILIP N. BARTLETT AND CHEE-SENG TOH

where Jj+1 andjj_! are the fluxes across the boundaries with the two adjacent boxes. Then using the discrete form of Pick's first law, we have

and

Thus, from Equation (62)

Rearranging gives

Equation (66) gives an explicit expression of the concentration in the ith box from the electrode surface at time step (t + §t) in terms of the concentrations in the ith box and the two boxes on either side of it at time t. In practice it is useful to recast Equation (66) in a dimensionless form by defining a dimensionless diffusion coefficient, DM such that

and introducing a dimensionless concentration, , defined as the ratio of the concentration to the bulk value (<j> — s/sbuik). Equation (66) then becomes

Thus by starting from the known concentration profile at time zero we can use Equation (68) to calculate the evolution of the concentration profile with time. This is the basis of the explicit finite difference method. Using the dimensionless formulation in Equation (68) has the advantage that we do not need to carry out a new simulation every time we change the bulk concentration or the substrate diffusion coefficient; rather, we can carry out the dimensionless simulation once and then calculate the specific results for particular concentrations and diffusion coefficients by appropriate scaling of the result. So far we have only considered the simulation of the diffusion part of the problem. In the explicit finite difference method, reaction kinetics can be included by adding a second calculation at each time step in which we calculate the change in concentration within each box as the result of the homogeneous kinetics. Full details can be found in the literature (32). A limitation of the explicit method is that the simulation is only stable for restricted values of the dimensionless diffusion coefficient, DM. For a 84

KINETIC MODELING FOR BIOSENSORS

one-dimensional simulation, the condition is that DM be less that 0.5. From Equation (67) we can see that this imposes a restriction on the relative sizes of the time step, 8t, and box size, 8x, that we can use. In turn this can be a severe limitation when we include reaction kinetics in our simulations. For this reason a number of more sophisticated simulation methods are often applied and we briefly introduce these below.

2.7.3 The Crank-Nicholson method Fully explicit methods, although simple to understand and to program, have the disadvantage that they are not unconditionally stable. The Crank-Nicholson method (34) uses a semi-implicit approach which calculates concentrations at time (t + §t/2) by linear interpolation between the known concentrations at time t and the unknown concentrations at time (t + §t). A comparison of this method to the fully explicit method described above is shown in Figure 12. As an illustration, consider three points describing the concentrations of a species at a particular point in space but at different times. Given an approximate value for the rate of change of the concentration of S in the ith box at time t, dSj(t)/dt, we can estimate the value of the concentration in the same box at time (t + St)

As we saw in the previous section (Equation (66)), dsf(t)/dt can be approximated from the discretized form of the second derivative (over space) in the diffusion equation. It is obvious from Figure 12 that this method suffers from poor accuracy when the concentration of S is changing rapidly, that is, when dsf(t)/dt is large. In

Figure 12 Schematic diagram showing the estimation of s,,t+itfrom s/,t using the explicit method, and the estimation of s/j+st from s/j+st/2 using the Crank-Nicholson (CN) method. 85

PHILIP N. BARTLETT AND CHEE-SENG TOH

the Crank-Nicholson method, the gradient at (t + 8t/2), that is, dsf(t + 8t/2)/dt, is used to estimate st(t + §t) from st(t) as follows:

As shown in Figure 12, this gives a more accurate estimate of st(t + §t) as compared to the fully explicit method. In the Crank-Nicholson method, the value of Sj(t + §t/2) is approximated by linear interpolation as follows:

so that Sj(t + c5t/2) is expressed in terms of st(t) and st(t + 8t) and is not calculated, but simply used as a point of reference to obtain a better estimation of st(t + 8t). As in the explicit method, c5t(dsf(t+c5t/2)/dt) is used to approximate for the second derivative (over space) in the diffusion equation, expressed in terms of both the known concentration, st(t) and unknown concentration, st(t + 8t), using the above expression for s4(t + 1 Gfi) (17, 37).

Protocol 2

Dry cleaning and activation of silicon chips Equipment and reagents • Oxygen Plasma Asher/Cleaner Tegal Plasmaline 415 (Petaluma, CA, USA)

• Fluoroware® boxes, 2 (Entegris, Chaska, MN, USA).

Method 1

158

Separate the chips from the sawed wafer by cutting the supporting foil with a scalpel blade.

FUNCTIONAL ANALYSIS OF ION CHANNELS

Protocol 2 continued 2 Immerse the chips (still attached to the supporting foil) in a bath of acetone. Chips will lift off from the foil after 10-20 s. 3 Transfer the chips in a new bath of acetone. 4 Rinse the chips with a stream of acetone and transfer them into two additional, fresh baths of acetone. 5 Rinse the chips with a stream of acetone, and dry them with a stream of nitrogen. Store the chips in hermetic Fluoroware® boxes. 6 Clean and activate the chips in an oxygen plasma (pressure of 800 mTorr, power of 100 W) for 2-3 min. In case the chips are not immediately modified according to Protocol 4, they can be stored in Fluoroware® boxes and re-activated using step 6 just prior to surface modification.

Protocol 3

Wet cleaning and activation of silicon chips Reagents • NH4OH solution (28% NH3, Fluka, Switzerland)

• SC-2 solution (6:1:1 H 2 O 2 : 32% HC1)

• SC-1 solution (5:1:1 H 2 O 2 : NH4OH solution)

• Dilute HF (100:1 vol. H 2 O 2 :48% HF).

vol.

H2O : 30%

vol.

H 2 0:30%

Warning: HF is extremely dangerous, take all necessary safety precautions!

Method 1 2 3 4 5 6 7 8 9

Separate the chips from the sawed wafer and clean them in acetone following steps 1 to 5 of Protocol 2. Immerse the chips for 20 min in SC-1 solution heated to 75-80 °C. Rinse the chips with deionized water. Dip the chips for 10 s in dilute HF. Rinse the chips with deionized water. Immerse the chips for 20 min in SC-2 solution at room temperature. Rinse the chips with deionized water. Dip the chips for 10 s in dilute HF. Rinse the chips with deionized water and dry them with a nitrogen stream. In case the chips are not immediately modified according to Protocol 4, they can be stored in Fluoroware® boxes and re-activated using step 8 and 9 prior to surface modifications.

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The cleaning method described in Protocol 2 is recommended, however, if an oxygen plasma cleaner is not available, a wet cleaning protocol (Protocol 3) can be used. Stable planar membranes with giga-seals are obtained by appropriate electrostatic attraction between the chip surface and the negatively charged giant vesicles. Therefore, the chips are pretreated with positively charged poly-L-lysine (see Protocol 4).

Protocol 4

Surface modification of the silicon chips Reagents •

Poly-L-lysine (Mr = 70,000-150,000) from Sigma, USA.

Method 1

Immerse freshly plasma- or wet-cleaned chips (Protocol 2 or 3) in a solution of 0.01-0.1% poly-L-lysine for at least 5 min until use. 2 Rinse the chips thoroughly in a stream of deionized water for at least 1 min. 3 Dry the chips in a stream of nitrogen. 4 The chips can now be stored in air (in a Fluoroware® box) or used immediately for the measurements.

Figure 3 Laser scanning microscopic fluorescence images of giant unilamellar vesicles, prepared according to Protocol 5. (a) Vesicle preparation before removal of vesicles smaller than 5-10 \m\ by dialysis, (b) Vesicle preparation after 18 h dialysis over a nylon net filter. Note that small vesicles inside giant vesicles (multivesicular vesicles) cannot be removed by dialysis. However, these enclosed vesicles do not interfere with the process of vesicle-derived seal formation. The vesicles were stained with TRITC DHPE. Excitation at 543 nm, fluorescence detected using a LP560 nm filter. Scale bars: 50 \im. From ref. (8), with permission.

160

FUNCTIONAL ANALYSIS OF ION CHANNELS

2.2.2

Formation of giant unilamellar vesicles

Vesicle-derived lipid bilayers spanning the chip aperture can only be obtained with giant vesicles (Figure 3), which have diameters larger than the aperture. Therefore, the preparation of vesicles with diameters >10 (am is crucial for efficient seal formation. Generally, the formation of unilamellar giant vesicles is promoted by a high percentage (> 10%) of negatively charged lipids (38, 39), by gentle lipid film hydration (40) at elevated temperature (30-70 °C) (41,42), and by hydration of the lipid film in a solution of low ionic strength (41). To adjust the osmotic properties of the vesicles, sorbitol can be added to the hydration solution in concentrations up to 0.4 M. Protocol 5 was optimized to yield vesicles with diameters >20 (am.

Protocol 5

Preparation of giant unilamellar vesicles Equipment and reagents • Rotary evaporator (Rotavapor R114, Biichi, Switzerland). • Sorbitol, asolectin, and cholesterol were obtained from Fluka, Switzerland. l-Palmitoyl-2-oleoyl-sn-glycero-3-[phosphorac-(l-glycerol)] (POPG) was purchased from Avanti Polar Lipids Inc., Alabaster, USA and N-(6-tetramethylrhodaminethio-

carbamoyl)-!, 2-dihexadecanoyl-sn-glycero3-phosphoethanolamine (TRITC DHPE) from Molecular Probes, Eugene, USA. Stock solutions in chloroform methanol (7: 3): 10 mg/ml asolectin; 7 mg/ml POPG; 10 mg/ml cholesterol; 0.1 mg/ml TRITC DHPE. Store the stock solutions at - 20 °C.

Method 1

2

3

4 5

6

Mix the lipid stock solutions (total amount of lipids: 1.25 mg) in a thoroughly cleaned 10 ml round flask to a molar ratio of 63.7% asolectin, 25% POPG, 11% cholesterol, and 0.3% TRITC DHPE. Dry the lipids at 45 °C in a rotary evaporator at 500 mbar until no more solvent is visible (this takes usually less than 30 min), then dry at room temperature at 20 mbar for at least 2 h to obtain a sufficiently solvent-free, homogeneous lipid film. Pre-hydrate the lipid film for approx. 2 min with a stream of nitrogen, which is enriched with water vapor. To obtain N2 enriched with water, bubble N2 through a heated flask (80 °C) filled with deionized water. Hydrate the lipids with 10 ml of 0.2M sorbitol in deionized water overnight at 37 °C. Harvest the turbid and slightly rose-colored vesicle cloud with a pipette (avoid excessive shear stress). The total volume of the harvested cloud varies between 1.5 and 4 ml. Store the vesicles at 4 °C.

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2.2.3

Size separation of vesicles

Even an optimized protocol for the formation of giant vesicles yields a heterogeneous size distribution of vesicles (Figure 3(a)). Vesicles < 5 (am diameter inhibited seal formation presumably due to contamination of the highly attractive chip surface (these vesicles diffuse faster and are present in large numbers). Therefore, a size separation method is required to remove vesicles with diameters < 5 (am. Efficient and gentle size separation of giant vesicles (see Figure 3(b)) is obtained by dialysis. The raw vesicle suspension (1 ml) is filled into a tube made of a nylon net filter (20 (am mesh size, Millipore, Bedford, USA) and dialyzed at 4°C for at least 16 h in 0.6 1 of the same sorbitol solution as used for lipid film hydration (0.2 M). The sorbitol solution is agitated gently (to avoid excessive convective flow through the nylon mesh) during dialysis.

2.2.4

Automated seal formation

By using negatively charged vesicles, properly directed electrical fields can provide precise electrophoretic positioning. Here, concentric electrical fields (see Figure 4) are used for positioning, because they result in a focused movement of charged objects toward the point of the highest electric field. A concentric electrical field is created around the small aperture in the chips upon application of a potential between the cis and the trans compartment (Figure 2(d)). By accessing the buffer compartments with Ag/AgCl electrodes, the main voltage drop after application of a potential occurs within and near the aperture. The resulting radially symmetrical field directs the electrophoretic movement of charged objects to the spatially fixed aperture, as illustrated in Figure 5(a). Upon delivery of a giant vesicle to the aperture, the strong electrostatic interaction with the chip surface leads to vesicle spreading (Figure 5(b)) (8). The result is a solvent-free unilamellar lipid bilayer spanning the aperture. Reconstitution of channel-forming peptides or ion channels into this planar bilayer allows single-channel recording with a current resolution superior to standard PLB recordings and comparable to patch clamp.

Protocol 6

Formation of PLBs on a silicon chip Equipment and reagent • Bilayer Lipid Membrane Amplifier data acquisition card, National InstruBLM-120, equipped with a 10 mV/pA headments Corp., Austin, USA). stage, Bio-Logic Science Instruments SA, • Buffer: 85 mM KC1, 2 mM Hepes pH 7.4 Claix, France. (adjusted with KOH). • Oscilloscope (e.g. TDS 210, Tektronix, Inc., USA) or PC (equipped with AT-MIO-16E-1

162

FUNCTIONAL ANALYSIS OF ION CHANNELS

Protocol 6 continued

Method 1

2 3 4 5 6 7 8 9 10

Put the chip with the cavity side upward in a clean Fluoroware® box. Place a Sylgard® pad (see Protocol 7) on the chip such that the hole in the pad is positioned over the cavity. Fill the cavity with a droplet of buffer. Fill the liquid channel below the chip (trans compartment, Figure 2(d)) with buffer (electrolyte). Place the chip on the channel with the cavity side facing the channel. Add 10 [d of buffer to the as side of the chip. Immerse the Ag/AgCl electrodes in the as and trans compartment and connect them to the amplifier. Adjust the voltage offset and evaluate the series resistance of the chip. Apply —60 mV to the as side. Add 1-4 |al dialyzed vesicle suspension to the as side. After seal formation (see Figure 5(b)) wait for 1 min to evaluate the seal resistance and the stability of the planar bilayer.

AITSYS 5 . 5 . 3

Figure 4 Finite element simulation of the electric field E around a 2 jxm aperture in a diaphragm with a thickness of 100 nm and an applied potential of 100 mV. The gray part in the center of the aperture indicates that the electric field exceeds IT.OOOVm" 1 . Due to the walls of the cavity on the bottom side of the chip (Figure 2 ( a ) ) , the electric field on each side of the chip is slightly different. The simulation was done with ANSYS software from ANSYS Inc. (Canonsburg, PA, USA).

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11 If desired, the buffer in the as and trans compartment can be replaced by a buffer with higher ionic strength (e.g. 0.5 M KC1, 10 mM Hepes pH 7.4). 12 After a seal resistance >1 Gfi and a stable baseline is reached, reconstitute channelforming peptides or proteins of interest. 13 Depending on the current amplitude and the kinetics of the recorded current traces, the low-pass filter of the amplifier should be adjusted to 0.1-10 kHz. A good starting range for the filter cutoff frequency is 1-3 kHz (the sampling rate should be at least four times higher than the cutoff frequency) (43). More detailed information about data acquisition can be found in literature (44) or online (http:// www.axon.com/MR_Axon_Guide.html) in the "Axon Guide" (45).

Figure 5 Electrophoretic vesicle positioning in two different configurations, (a) Coulter counter-like current modulations /(t) due to small vesicles (undialyzed sample) passing through the aperture of a chip with a "non-attractive" surface (unmodified Si3N4 diaphragm), (b) When negatively charged and dialyzed giant vesicles are added to a positively charged chip surface, an abrupt step to zero current is observed due to closure of the aperture by a single bilayer. Resulting seal resistances are typically 1-200 GS1. From ref. (8), with permission.

164

Next Page FUNCTIONAL ANALYSIS OF ION CHANNELS

Protocol 7

Fabrication of Sylgard

pads

Equipment and reagents • Oven at 60 °C

• 30 and 90 mm plastic Petri dishes.

• Sylgard® 184, base and curing agent (Dow Corning, Midland, USA)

Method 1 Prepare ca. 6 g of Sylgard® elastomer in a 30 mm Petri dish, with a base to curing agent ratio of 10:1. 2 Devide the elastomer into two 90 mm Petri dishes (~3 g each). Store this sample for 30 min at room temperature to allow complete coverage of the bottom and removal of air bubbles. 3 Cure the elastomer at 60 °C overnight. 4 Cut the desired patterns (ca. 7 x 7 mm) with a scalpel blade. Punch a hole (diameter ca. 1.3 mm) in the center of the pad using a clean metal tube with a sharpened tip opening. 5 Peel off the pads with tweezers when needed.

Chip-based planar bilayers: single-channel measurements of alamethicin pores Ion channels are only functionally active if they are embedded in an appropriate lipid membrane. An important parameter influencing ion channel function is the chemical composition of the lipid bilayer (e.g. the proportion of cholesterol or charged lipids) (46). PLB experiments not only make it possible to study the function and the characteristics of reconstituted single ion channels but also to assess the effect of the lipid composition on ion channel activity. Additionally PLBs are very useful to study those ion channels that are not accessible with patch pipettes, like channels located in intracellular membranes or ion channels from bacteria. Although there is extensive BLM literature dealing with the functional reconstitution of ion channels into planar bilayers (46-51) this task requires experience in handling fragile membrane proteins. The principle of single-channel recording can, however, be demonstrated with channel-forming peptides or self-integrating ion channels. Both, channel-forming peptides and self-integrating ion channels have recently gained increasing attention for biosensor development (8, 37, 52-58). Examples of channel-forming peptides are melittin (a component of the bee venom) and the antibiotic alamethicin (Figure 6(a, b)). These peptides self-integrate into bilayers and form voltage-gated ion channels by oligomerization (Figure 6(c, d)) (59-61). In the case of alamethicin, well-defined 165

Previous Page MICHAEL MAYER ETAL

Figure 6 Alamethicin structure and channel formation, (a) Alamethicin structure according to Fox and Richards (59), showing an a-helical structure of the peptide. (b) Space-filling model of the same structure, (c) Model of voltage-gated alamethicin activation (59). Upon application of a voltage V, alamethicin peptides integrate in the lipid bilayer. (d) Aggregation of alamethicin peptides leads to the formation of pores. The pore diameter (and consequently the single-channel conductance) increases with increasing number of assembled monomers (60, 61).

Figure 7 Chip-based single-channel recording of an alamethicin pore, (a) The l-t diagram recorded at a potential of -130 mV in 0.2 M KCI and 8 mM Hepes pH 7.4 shows the typical activation pattern comprising up to seven conductance states. The dotted line represents the current baseline while the channel is closed, (b) Current histogram derived from alamethicin traces recorded under conditions given in (a). The conductance states Oi to 07 are shown. C reflects the current baseline. From ref. (8), with permission.

multiple open states of single pores are found resulting in up to nine conductance states (21, 62, 63). Figure 7 shows typical alamethicin events recorded with a vesicle-derived lipid bilayer on a chip. Upon seal formation, alamethicin was added to the cis side of the bilayer (final concentration in the cis compartment: 3 ng/ml) and after 10-50 s voltage-dependent activation with up to seven conductance states was observed (64). 166

FUNCTIONAL ANALYSIS OF ION CHANNELS

Single-channel recording provides unique information about the function and the kinetic properties of ion channel proteins. Important parameters, which are accessible by PLB or patch clamp experiments, are: (a) the single-channel conductance (related to the amplitude of the ionic current flowing through a single open channel) (b) the existence of sub-conductance levels (c) the open state lifetime (d) the closed state lifetime (e) and the opening and closing probability. The single-channel conductance is an important parameter since mutated channels might have an altered conductance, which can cause diseases, like a subtype of cystic fibrosis (14). The open and closed state lifetimes and consequently the opening probability might depend on the applied voltage as in the case of voltage-gated ion channels. On the other hand, ligand-gated ion channels have a higher opening probability in the presence of specific ligands (e.g. the neurotransmitter acetylcholine increases the opening probability of the acetylcholine receptor) (17). The third category of ion channels comprises mechanosensitive channels, whose opening probability is altered by mechanical stress applied to the cell membrane (65). In the case of alamethicin, the opening probability of ion channels increases strongly with increasing applied potential, and the single-channel conductance of the various open states are listed in Table 1. As the example of alamethicin demonstrates, single-channel recordings can yield detailed information about the function of channel proteins. Table 1 Conductance states of alamethicin pores, measured at - 170 mV in 0.5 M KCI in an asolectin/POPG/cholesterol (70%: 25%: 5%) bilayer, compared with previously reported conductance values in asolectin and l,2-diphytanoyl-sn-glycero-3-phosphocholine (DphytPC) bi layers Open state

Conductance (planar patch clamp) (pS)

Conductance (PLB) in bilayers of Asolectin (pS)a

DphytPC (pS)b

1

129

40

88

2

423

370

499

3

0

824

880

7

1430 0

1868 8

1990 0

2611 1

4

1247

5

1694 4

1160

(Votes a

According to Schlue and Hanke, recorded at +100 mV (21).

"Accoring to Sansom, recorded at -125 mV (63).

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Ion channels take part in vital functions of the cell and we only begin to understand their involvement in genetic and autoimmune diseases (1). We believe that the planar patch clamp technology presented here will open new possibilities to investigate the function of ion channel proteins. Due to the miniaturized format, the automated formation of giga-seals and the potential for parallel recordings, this chip technology should be particularly useful for high-throughput assay formats.

3 Impedance spectroscopy of tethered lipid membranes There is a fast growing interest in the realization of long-lasting, stable lipid layers on solid supports for application in molecular (bio)electronics and (bio)sensors, as well as for fundamental studies using surface-sensitive techniques (10, 66, 67). In cases where only the physical and chemical properties of a lipid membrane/water interface are of interest, supported lipid monolayers are the system of choice. Such lipid monolayers are most easily produced by self-assembly on hydrophobic supports (e.g. gold surfaces covered by a monolayer of thioalkanes or silanized oxide surfaces). After incorporating certain receptor molecules in the outer lipid monolayer, specific ligand-receptor interactions may be detected by measuring changes of the optical or the electrical properties of the molecular layer (9, 68). A classical example where ligand receptor interactions using biological components were measured on the lipid membrane surface, is the binding of cholera toxin to its ganglioside receptor (9). This concept was some time later adapted and commercialized by a surface plasmon resonance biosensor company (69). In this context, selective detection of Ca2+ by ionophores in a supported hybrid lipid layer is reported. Tethered lipid Mayers are required to preserve the function of integral membrane proteins. In addition to their biological importance, ion channel proteins are particularly interesting in the field of biosensors because they provide an intrinsic amplification of ligand binding (52, 53). The modulation of the electrical resistance of a tethered lipid bilayer by ligand binding to a particular membrane receptor protein is illustrated with the pore-forming protein OmpF. Antibody detection with tethered lipid layers containing a novel synthetic ligand-gated channel (SLIC) is described at the end of this chapter (70). The formation of molecular layers, which are attached chemically to a gold electrode either by the phospholipids or by the proteins, is of particular importance and techniques to create hybrid lipid layers and tethered lipid bilayers are described in detail in the different protocols.

3.1 Basics of impedance spectroscopy The use of a supporting inert electrode excludes a simple amperometric measurement as presented above, unless ions can be reduced or oxidized at the electrode. Experiments with redox proteins on conductive supports have been reviewed recently (71). In the absence of faradic reactions, molecular interactions can be investigated with alternating current methods. The basics of electrical 168

FUNCTIONAL ANALYSIS OF ION CHANNELS Table 2 Definitions of the impedance Z and of the admittance Y.

Resistor Capacitor RC parallel RC series

Notes "Real part of the impedance. "Absolute value of the imaginary part of the impedance. °Real part of the admittance. d

Imaginary part of the admittance.

The impedance Z is defined as the ratio of the applied voltage to the resulting current, where Vm is the amplitude of the voltage, /m the amplitude of the current, 6>m the phase delay, uj, the radial frequency and / the imaginary number ( — I)0'5.

impedance spectroscopy have been already discussed in this series (72). We will therefore concentrate on aspects related to supported lipid membranes. In a typical impedance spectral analysis, a small-amplitude sinusoidal voltage is applied between two electrodes at successive frequencies and the current response is measured. If the response is linear, the resulting current has the same frequency as the stimulus and the ratio of the amplitude Vm/Im is constant at a given frequency. The impedance Z, and the admittance Y, are usually described by complex numbers, whose real and imaginary parts represent their components for a phase delay of 0° and 90°, respectively (Table 2). The convention of plotting the absolute value of the imaginary part of the impedance is used throughout this text (the imaginary part of the impedance of a capacitor is actually negative). The impedance depends on the frequency and this spectrum can often be fitted by an electrical equivalent circuit composed of resistors and capacitors. The real and imaginary parts of the impedance and admittance of basic circuit elements are given in Table 2. A supported lipid mono- or bilayer is classically modeled by a capacitor and a resistor in parallel. The capacitance describes the dielectric properties of the electrically insulating lipid layer sandwiched between two conductive phases, namely the electrode and the electrolyte. The resistance critically depends on electrically conducting defects in the lipid layers. More complicated models consisting of a larger number of circuit elements or using non-ideal circuit elements such as the constant phase angle element have been proposed but will not be considered here (9, 53, 73). Data analysis and interpretation are central issues in impedance spectroscopy. In particular, there is an intrinsic ambiguity in data fitting in the sense that many equivalent circuits have exactly the same mathematical forms and cannot be distinguished from a single spectrum. Spectra taken under different conditions are required to establish a unique model (74).

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3.2 Measuring technique and electrochemical cell There are several ways to record an impedance spectrum as discussed in detail elsewhere (75, 76). Recent technical developments improved and facilitated such measurements considerably. Impedance modules are now offered on standard electrochemical analyzers such as the BAS 100 from Bioanalytical Instrument and software for sophisticated data analysis is available commercially. One of the major improvements of modern instrumentation, as far as the investigation of insulating molecular layers is concerned, is the extension of the measurable impedance limit to the Teraohm range as in the Solartron 1296 dielectric interface. This is of particular importance if it is desired to decrease the electrode size to the micrometer range. Impedance measurements can, however, be performed by simply using a 2-phase-channel lock-in amplifier such as SR 850 from Stanford Research Systems. Sinusoidal voltages are applied to the electrochemical cell by the internal function generator in a programmable automated frequency sweep. The currents in phase and 90° out of phase with the applied ac voltage are directly recorded on the two channels of the lock-in amplifier and a highly accurate phase-sensitive detection is performed digitally. Our set-up includes a homebuilt current-to-voltage converter with a gain of 104-107 and the lock-in amplifier measures the input voltage. An additional external function generator or a dc offset can be used. It is important to realize that the current, which is proportional to the admittance (Table 2), is actually measured and that the impedance is then calculated. An impedance spectrum will include contributions from the whole electrochemical cell, that is, the electrodes, the interfaces, and the electrolyte. It is good practice to minimize the effect of all elements except the working electrode. Therefore, a large (ca. 1 cm2 effective area) and highly conductive counter electrode should be used. If the electrolyte contains chloride ions, an AgCl-coated Ag wire is recommended. A high concentration of the electrolyte (usually KC1 > 0.1M decreases the effect of the electrical resistance of the aqueous phase and of the interfacial capacitances. Under these conditions, the impedance of a small working electrode (in our case: 3.34 mm2) coated with an electrically insulating lipid layer will dominate the spectrum. An electrochemical system with two electrodes is sufficient because only a negligible current will actually flow through the Ag/AgCl electrode.

3.3 Hybrid lipid layer 3.3.1 Description A hybrid lipid layer consists of a phospholipid monolayer deposited on a hydrophobic monolayer grafted to an underlying support as shown in Figure 8. Monolayers of remarkable electrical insulation have been produced by the chemisorption of thioalkanes of sufficient hydrocarbon chain length on gold electrodes (77). Phospholipids are then self-assembled on this hydrophobic su face. Formation and characterization of thioalkane monolayers and hybrid lipid 170

FUNCTIONAL ANALYSIS OF ION CHANNELS

Figure 8 Schematic view of a two-electrode electrochemical cell with an Ag/AgCI counter electrode and a gold working electrode coated with a chemically attached monolayer of tetradecanethiol and a second phospholipid (POPC) monolayer. An ion-selective electrode is obtained if an ionophore, such as the Ca2+ -ionophore ETH1001, is incorporated in the phospholipid monolayer.

layers have been described in detail in recent reviews (10, 78, 79). Hybrid lipid layers are typically used for investigating the binding of proteins and polypeptides to the lipid membrane surface (80). In the absence of electrical amplification (e.g. the introduction of charges in a low-dielectric-constant layer), electrical (capacitance) measurements are, however, less sensitive than optical methods for the detection of hydrated protein layers at these surfaces. This was shown for the binding of cholera toxin to its receptor ganglioside GM1, incorporated into a phospholipid monolayer by simultaneous surface plasmon resonance and electrical impedance measurements (9). The coating of the thioalkane surface with phospholipids can be performed according to different methods (10, 81). Self-assembly techniques from vesicles or from a detergent solution are distinguished by their simplicity. In the following protocol, a very simple dilution technique from ethanol produces vesicles in situ that will spontaneously cover the hydrophobic thioalkanes in the presence of water. The formation of a mixed layer of phospholipids and ionophores is described hereafter.

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Protocol 8

Preparation of a Ca2+-sensitive hybrid lipid layer electrode Equipment and reagents • Gold electrode • Solution of ca. 1 mM tetradecanethiol (Fluka, Switzerland) in ethanol • Solution of 1 mM palmitoyl-oleoyl phosphatidyl choline, POPC (Fluka) in ethanol

• Solution of 1 mM Ca2+ ionophore, ETH 1001 (Fluka) in ethanol.

Method Incubate a clean gold electrodea in the thioalkane solution in ethanol for about 1 h to form a self-assembled thioalkane monolayer. Overnight immersion results in better insulating layers and is recommended. 2 Remove the excess of thioalkane by washing with ethanol. 1

3 Mount the electrode in the electrochemical cell and cover the electrode with a 1 mM solution of POPC and ETH1001. In our electrochemical cell as little as 10 (il of solution is required. 4 Agitate the solution by using a syringe and wait for 5 min. 5 Dilute with several milliliters of water. a

We used freshly evaporated gold electrodes on glass supports which we transferred immediately from the vacuum chamber into the thiol solution. Cleaning procedures with a plasma discharge or with an oxidizing cleaner such as piranha solution (3:2 H2SO4/H2O2) or chromic acid (saturated potassium dichromate in concentrated sulfuric acid) are recommended if an aged gold electrode is used. Warning: These are dangerous cleaning solutions and care must be taken in solution handling. Methods for preparing gold surfaces are described elsewhere in detail (78).

3.3.2 Impedance spectroscopy data The impedance spectra shown in Figure 9 are typical of an electrically blocking electrode and can be modeled by the equivalent circuit shown in the inset. The capacitor C accounts for both the hybrid lipid membrane and the electrode/ electrolyte interface. Under usual conditions of high electrolyte concentration, the capacitance of the interface is negligible as compared to the membrane capacitance CM. The serial resistor Rs accounts mainly for the resistance of the electrolyte. The resistance R in parallel to the capacitor is dominated by the charge transfer resistance RCT. Its value is very large and cannot be obtained with precision under our measuring conditions because practically no Faradic currents are flowing in this electrochemical cell in the absence of redox ions. The simple model of Figure 9 describes the electrochemical cell equally well when the 172

Figure9 Real (o) and imaginary (G) part of the impedance spectrum of a 3.34 mm2 disk gold electrode covered with a hybrid lipid membrane (composed of a first tetradecanethiol and a second POPC monolayer). The electrical equivalent circuit is presented in the inset. The hybrid lipid membrane and the electrode/electrolyte interface are modeled by the same RC parallel circuit. The capacitance of the membrane, dominates the imaginary part of the impedance spectrum at all measured frequencies while the resistance of the membrane is masked by the extremely high value of the charge-transfer resistance. The resistance of the electrolyte Rs, can be obtained from the real part of the impedance at high frequencies.

Figure 10 Traces of the imaginary part Y, of the admittance of a hybrid lipid bilayer membrane containing the ionophore ETH 1001 measured at 120 Hz as a function of time. The admittance is modulated by the Ca2+ concentration: (a) 10~5 M, (b) 10~4 M, (c) 10~ 3 M,(d) 10~ 2 M, (e) lO^M MgCI2 in the surrounding buffer. The working electrode is a disk n-doped silicon electrode (ca. 1 mm2) covered by a 3.5 nm Si02 layer derivatized by (3,3'dimethylbutyl)dimethyl (dimethylamino)silane. The sensing membrane is formed by self-assembly on the hydrophobic silane monolayer by diluting an ethanol solution of POPC:ETH1001 (1:1) with buffer. From ref. (82), with permission.

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gold electrode is coated with a self-assembled thioalkane monolayer or with thioalkane and an additional phospholipid layer. Therefore, the changes of the measured capacitance can be used to quantify the layer formation. Substantial changes of the capacitance are obtained by incorporating ionophores into the supported membrane. By ion complexation of the ionophore, electrical charges are incorporated in the low-dielectric-constant environment of the sensitive layer. The number of charged complexes and thus the membrane capacitance depend on the ion composition of the electrolyte. Figure 10 shows the real-time response of a membrane prepared by self-assembly of equimolar amounts of POPC and ETH1001 (82). The measured admittance is directly pro portional to the capacitance of the molecular layer at a sufficiently low frequency. The membrane is initially in contact with 0.1 M MgQ2, which is then replaced by a solution of 0.1 M MgCl2 containing the indicated amount of Ca2+ . The capacitance changes abruptly to a new value depending on the calcium content of the electrolyte solution. After washing with the calcium-free 0.1 M MgQ2, the signal returns to its original value. Crown-ethers derivatized with alkyl chains have been used to give K + sensitivity to supported phospholipid monolayers (83, 84). Similarly, the change of capacitance of a chelator-modified gold electrode has been used to detect traces of divalent metal ions (0.5 nM Cu2 + ) (85).

3.4 Tethered lipid bilayers 3.4.1 Description Phospholipid bilayers chemically bound to the gold support are obtained by selfassembly of sulfur containing synthetic lipids. These thiolipids are phospholipids comprising at their polar head groups a hydrophilic spacer, which is terminated by a -SH group (Figure 11) (52, 86-88). The hydrated spacer decouples the lipid bilayer from the gold surface. The resulting aqueous phase between the electrode surface and the lipid bilayer has been designed to accommodate extracellular parts of transmembrane proteins. G protein coupled receptors and ion channels have been reconstituted in a functional active form into these gold supported lipid bilayers (11, 53, 87). Protocol 9 has been optimized for the incorporation of the trans-membrane protein OmpF in tethered lipid bilayers. OmpF is a pore-forming protein from the outer membrane of Escherichia coli whose conductance can be modulated by binding the bacterial toxin Colicin N (53). The proteolipid layer is formed in two steps. First, a mixed monolayer of thiolipid and a short i^hydroxy (or) carboxythioalkane is formed by coadsorption from a detergent solution. The small hydrophilic thiols serve to dilute the thiolipid molecules laterally and to prevent direct contact and thus denaturation of membrane proteins at the gold surface. Second, the proteolipid bilayer is then completed by incubating the monolayer-covered gold electrode with a mixed detergent solution of dioleoyl phosphatidyl choline (DOPC) and OmpF, and finally diluting this solution below the critical micelle concentration of the detergent.

174

FUNCTIONAL ANALYSIS OF ION CHANNELS

Figure 11 Schematic view of a two-electrode electrochemical cell with an Ag/AgCI counter electrode and a gold working electrode coated with an OmpF-containing lipid bilayer, which is tethered to the gold electrode via synthetic thiolipids. The membrane protein OmpF forms ion channels in the bilayer, which are closed by the selective binding of the toxin Colicin N. From ref. (53), with permission.

Protocol 9

Preparation of an OmpF-containing lipid bilayer tethered on a gold electrode Reagents • Gold electrode (45 nm thick gold film evaporated on a glass slide) • Buffer 1 (0.3 M NaCl, 20 mM sodium phosphate, pH 7.4) • Buffer 2 (48 mM octylglucoside (Bachem) in buffer 1)

• Solution of 1 mM sulfanylpropionic acid (Fluka) in buffer 2 • Stock solution of approximately 10 ~ 5 M OmpF in buffer 2 (purification protocol described elsewhere) (89) • Solution of DOPC (0.5 mg/ml) in buffer 2.

• Solution of thiolipids (0.5 mg/ml buffer 2) (synthesis of thiolipids described elsewhere) (87)

Method 1

Leave the gold electrode overnight in a mixture of thiolipids and sulfanylpropionic acid at a molar ratio of 2 :1 in buffer 2. 175

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Protocol 9 continued

2 Wash with buffer 2. 3 Mount the gold electrode in the electrochemical cell and wash again with buffer 2. 4 Add a solution of 0.5 mg/ml DOPC and 5 (xg/ml OmpF in buffer 2 and incubate for about 30 minutes. 5 Dilute stepwise with buffer 1, thereby forming a tethered lipid bilayer at subcritical micellar concentrations of DOPC and OmpF.

Figure 12 Real (O) and imaginary (D) part of the impedance spectrum of a 3.34 mm2 disk gold electrode covered with a tethered lipid bilayer. An extra RC parallel circuit element has to be added in series to the equivalent circuit presented in Figure 9 in order to account for a second time constant. The resistances of the gold/water interface (charge transfer) RCT, of the membrane RM, and of the electrolytic solution Rs, are spectrally well resolved. CM and CDL denote the capacitance of the membrane and the interface, respectively. The electrical resistance RM of the bilayer can be obtained from the difference between the values of Zr in the plateau region around 100 Hz and at high frequencies. From ref. (53), with permission.

3.4.2 Impedance spectroscopy data The impedance spectra of a tethered lipid bilayer are shown in Figure 12 (53). As a main difference to the spectra of Figure 9, two charging processes are recognized. The impedance of the lipid bilayer is well separated from the interfacial impedance. This is believed to be due to the aqueous phase between the electrode and the lipid bilayer. As a main consequence, the resistance of the lipid bilayer is separated from the extremely high charge transfer resistance at the gold electrode and can be measured in the frequency range between 10 Hz and 1 KHz. (A more complex circuit was used in the original literature (53) but left the resistance of the bilayer, R M > virtually unchanged.) The obtained 176

FUNCTIONAL ANALYSIS OF ION CHANNELS

Figure 13 Interaction between OmpF in lipid bilayers and Colicin N added to the surrounding buffer solution. Conductance changes of OmpF in BLMs (•) and in tethered membranes by impedance spectroscopy (G); changes of the SPR signal due to the binding of Colicin N to OmpF in tethered membranes (o). In the impedance experiments, the initial and the final conductances were 1.54 x 10 ~ 3 SI"1 cm2 and 1.35 x 10~3 SI"1 cm2, respectively. From ref. (53), with permission.

resistance depends on the protein content of the supported membrane and a low protein-to-lipid ratio is required. It should be noted that the measured layer resistances are still a few orders of magnitude lower than the electrical resistances reported for PLB experiments (see first part of this chapter). Nevertheless, the modulation of the resistance of supported lipid layers containing a channel or a pore by a biological function, such as ligand binding, has been demonstrated by different groups (52, 53, 90). The conductance decrease of a supported lipid bilayer containing OmpF by the selective binding of the toxin Colicin N is shown in Figure 13. The membrane protein OmpF forms ion channels in the bilayer, which are closed by the selective binding of the toxin Colicin N. The specificity of the effect is confirmed by parallel SPR and PLB measurements.

3.5 Lipid bilayer tethered via surface-attached proteins 3.5.1 Description In order to create a robust device, both the lipid bilayer and the functional molecule might be chemically attached to the supporting gold surface. To immobilize proteins in an oriented manner, functional groups have to be introduced into the protein, which will then be recognized by the complementary binding partner on the sensor surface. Histidine-tags are commonly used to bind proteins to surfaces derivatized with the nitrilotriacetic group (10). 177

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Figure 14 Schematic representation and amino acid sequence (one-letter-code) of a synthetic ligand-gated ion channel (SLIC) chemically bound to a gold electrode and then mixed with a supported lipid bilayer. "X" is used for the amino acid ornithine that allows the synthesis of branched polypeptides. The conductive pathway can be closed reversibly and specifically by the antibody Sp3E9.

Another possibility is the site-specific modification of proteins by biotin, which are then bound to surface-immobilized streptavidin (10, 11). Here we report on the direct immobilization of an artificial protein to a gold surface via cysteine groups. An example of a new class of synthetic ligand (antibody)-gated channel protein (SLIC) made of branched polypeptides is shown in Figure 14. The amino acid sequence is shown in one-letter-code. Conceptually, a SLIC comprises a ligand-binding and a channel-forming region. As a ligand-binding region we have chosen the peptide (NANP)3, which is recognized by the specific monoclonal antibody Sp3E9. As will be demonstrated below, antibody binding modulates the channel activity of SLIC. This opens the door for new highly sensitive immunosensing on the basis of simple electrical detection methods. The NANP sequence is the major B cell epitope of the circumsporozoite protein of Plasmodium falciparum, the parasite causing human malaria. The channel-forming part is built by four melittin peptide segments, which are chemically attached to the branched spacer. The melittin sequence was chosen because of its well-known channel-forming properties (58, 70, 91).

Protocol 10

Formation of a proteolipid bilayer tethered by SLIC to a gold electrode Reagents • Gold electrode (45 nm thick gold film evaporated on a glass slide) . Buffer 1 (0.1 M KC1, 10 mM sodium phosphate, pH 7.4) • Buffer 2 (48 mM octylglucoside (Bachem) in buffer 1)

178

. Stock solution of SLIC (10 ~ 5 M) in 0.1 M KC1, 100 mM sodium phosphate, pH 9.0. • Solution of thiolipids (0.5 mg/ml buffer 2) • Solution of phospholipids (0.5 mg POPC/ml buffer 2).

FUNCTIONAL ANALYSIS OF ION CHANNELS

Protocol 10 continued

Methods 1 Mount the gold electrode in the electrochemical cell. 2 Add a dilute aqueous solution of 10 ~ 8 M SLIC at pH 9 and let the molecules selfassemble for about 1 h.a 3 Add the thiolipid from buffer 2 and wait until a steady-state impedance value is obtained (ca. 1 hour). 4 Wash with buffer 2. 5 Add POPC from buffer 2 and wait at least 15 min. 6 Dilute stepwise with buffer 1 to remove the excess of detergent. a

Note that most membrane proteins have to be introduced from a detergent solution at a neutral pH. A prior coating of the gold electrode with a short hydrophilic spacer is often needed to avoid direct contact of the protein with the gold surface, which might induce protein denaturation.

3.5.2 Impedance spectroscopy data The impedance spectra are qualitatively similar to those presented in Figure 12 and can be fitted with the same equivalent circuit (70). Only the real part of the impedance is plotted in Figure 15. Increasing the thiolipid-to-protein ratio to approximately 2200 increases the membrane resistance, R M > sufficiently so that the charging current of the membrane dominates the intermediate frequency range. The resistances of the solution, of the membrane and of the interface (charge transfer) are then well separated in the impedance spectra. Here, the important finding is the increase of RM in the presence of nanomolar concentrations of the antibody Sp3E9 in the bulk solution. The major part of the response can be reversed by washing with buffer because the fraction of rebound antibodies Sp3E9 is very small at this low density of surface-bound antigens. Unspecific adsorption of IgG to these layers is found to be negligible.

3.6 Highly insulating tethered lipid bilayers for single-channel experiments 3.6.1 Description We recently obtained gold electrode-tethered lipid bilayers with exceptionally high electrical resistances (92). We reached values above 2 x 108 fi, which translate into a membrane resistance of 7 Mficm2. For comparison, the highest electrical resistances of unsupported lipid bilayers have been reported for PLB with values around 10 Mficm2. Due to the low density of defects of the tethered membrane, the effect of a few channels can be resolved thus opening the way to single-channel experiments on this highly stable and versatile platform. In turn a minute quantity of analyte, here antibodies, can be measured which is of great interest for bioanalytics. 179

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Figure 15 Real part of the impedance spectra of a SLIC-containing lipid bilayer at various concentrations of antibody Sp3E9 (CAb, from bottom to top): O M , 1 0 ~ 9 M , 3 x l O ~ 9 M, 10~8 M, and 3 x 10~8 M. The increase of the layer resistance as a function of the antibody concentration in the bulk solution is seen at frequencies between 10 Hz and 1 KHz. From ref. (70), with permission.

The increase in membrane resistance is mainly due to an optimized lipid composition. A new thiolipid consisting of a single phytanoic acid coupled to a hydrophilic spacer terminated by a thiol group is used to tether the first lipid monolayer to a gold electrode (92). The lipid bilayer is then completed by a additional monolayer of 1,2-diphytanoyl-srt-glycero-3-phosphocholine (DphytPC) and cholesterol formed by dilution of a detergent solution of DphytPC/cholesterol (9/1) below the critical micelle concentration of octylglucoside. In order t maintain a high membrane resistance, a very small number of channel molecules should be incorporated in the lipid bilayer. In the experiments reported here, the SLIC of Figure 14 inserts into a preformed highly insulating tethered lipid bilayer at low concentration from the aqueous phase, in analogy to protocols used in classical PLB experiments. SLIC has such a high affinity to the tethered lipid bilayer that no dc voltage is required for its bilayer insertion.

3.6.2 Impedance spectroscopy data The impedance spectra are qualitatively similar to the ones presented in Figure 12 and can be fitted with the same equivalent circuit using similar parameter values. The only difference lies in the substantial increase (more than four orders of magnitude) of the membrane resistance (92). In Figure 16(a) the incorporation of SLIC in a tethered lipid bilayer is reflected in a drop in membrane resistance measured on the real part of the impedance at 0.01 Hz. Upon addition of 0.7 |oM SLIC in the aqueous phase, the layer resistance decreases immediately and saturates in about 30 min. The slow kinetics allows interruption of the bilayer 180

FUNCTIONAL ANALYSIS OF ION CHANNELS

Figure 16 (a) Real part of the electrical impedance (measured at 0.01 Hz) of a tethered lipid bilayer after addition of 7 x 10~7 M (i) of SLIC to the aqueous phase. Single-channel recordings on PLBswere done at -30 mV in 1 M NaCI, 10 mM Tris-HCI, pH 7.4 as described in refs. (24, 58). The single-channel events shown correspond to a conductance level of 90 pS at buffer conditions used for IS. (b) Real part of the electrical impedance (measured at 0.01 Hz) of the tethered lipid bilayer containing SLIC after the addition (i) of 10~6 M of the antibody Sp3E9. From ref. (92), with permission.

insertion process by washing with buffer and thus keeping the number of SLIC molecules in the membrane as low as possible in a controlled way. Single conductance states of up to 90 pS are measured for SLIC in freestanding PLBs. The decrease of the membrane resistance after incorporation of SLIC in tethered lipid membranes shown in Figure 16(a) can be explained by the presence of only about 200 open SLIC channels with a conductance of 90 pS. Selective antibody binding to SLIC in the lipid bilayer increases the membrane resistance as a function of the concentration of the antibody Sp3E9 in the aqueous phase (Figure 16(b)). The observed response corresponds to a total closure of 95 SLIC channels with a conductance of 90 pS. With the presently achieved 181

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signal-to-noise ratio, the closure of a few individual channels could be detected. The kinetics of opening and closing of single-channels, however, cannot be measured with an ac voltage at low frequency. Further optimization such as a decrease of the time constant of the membrane using, for example, microelectrodes will be required to record channel dynamics in tethered lipid bilayers.

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Chapter 9 Protein engineering for biosensors Gianfranco Gilardi Imperial College, London, UK.

1 Introduction Protein engineering is defined here as the ensemble of the methods that allow the modification of the gene that codifies for a protein with the purpose of generating mutants or variants (multiple mutants generated by random mutagenesis) with the desired properties. Traditionally the area of biosensors has been the domain of chemists who already have explored chemical modification of proteins over the past few decades. The advantage of protein engineering over traditional chemical modification lies in its ability to introduce controlled modifications in specific positions. Molecular biology has revolutionized the way we can design proteins for biosensing: it allows a high degree of control over the amino acid sequence by precise variations at the DNA level. This enables one to design properties such as binding, catalytic turnover rates, substrate specificity, and stability. Recombinant DNA technology also allows for the creation of fusions of proteins and peptides with combined functions such as tagging for immobilization and binding or catalysis. The early 1990s saw the first applications of protein engineering specifically for application to biosensors. The increased knowledge of protein structure-function, combined with the larger number of structures available and genes cloned have allowed increasingly wider applications to biosensing. An early example is the engineered S337C mutant of the maltose binding protein where the surfaceexposed serine 337 was mutated into a unique cysteine able to covalently link thiol-specific flurophores such as LANBD (1, 2). These acted as reporter groups for maltose binding that resulted in an increase in the fluorescence emission signal (Figure 1). This approach was soon applied to other binding proteins for detection of other ligands such as phosphate ions (3) and glucose (4), and it has recently been extended to engineered antigen-antibody systems (5). Mutagenesis has now become a widely used strategy, applied to both fundamental and applied fields of research oriented toward a variety of purposes. 185

Figure 1 Rational protein engineering combined with fluorescence labeling of the maltose binding protein (MBP) mutant S337C. (a) A unique cysteine was engineered by site-directed mutagenesis in position 337, where the maltose binding protein undergoes a large conformational change upon binding its ligand. (b) The thiol-specific fluorophores IANBD and acrylodan are covalently linked to the unique cysteine 337 where they act as reporter group for ligand binding, (c) Upon maltose binding, the fluorescence emission at 519 nm of IANBD (on excitation at 480 nm) increases and a binding curve is obtained (see Protocol 17).

PROTEIN ENGINEERING FOR BIOSENSORS

On the basis of these, different branches of protein engineering can be identified. The most traditional branch is that of analytical protein engineering that uses mutagenesis for understanding fundamental issues of protein structure and function of existing proteins. Most relevant to biosensors is rational protein engineering that uses the established knowledge of well-studied proteins to create site-directed mutants displaying the desired functions, and directed evolution that uses random mutagenesis and screening or selection methods to identify variants with new properties. Another emerging branch is the de novo protein engineering that uses the established knowledge on protein folding and structure prediction to create new proteins from first principles. This chapter will cover selected aspects of protein engineering relevant to the area of biosensors. It will illustrate the fundamental concepts and provide the basic protocols to enable the non-expert to design and set up mutagenesis experiments for various purposes.

2 Rational protein engineering Rational protein engineering relies on the existing information about the threedimensional structure of the target protein and the implication of specific residues in its function. When this information is not available, a possible alternative is the directed evolution of the target protein. Directed evolution is based on random mutagenesis and it is most applicable when a suitable selection or screening method can be identified (Figure 2). This approach will (a)

(b)

Structure-based design Analysis of three-dimensional structures/models Site-directed mutagenesis

Random mutagenesis Error prone PCR/homologous gene family

DMA sequencing

Recombination DMA shuffling

Expression

Functional screening of library Selection (in vivo)/ Screen ing (in vitro)

Functional characterization

DMA sequencing

Structure determination

Functional characterization

Structure determination

Figure 2 Flow chart showing the experimental stages involved for (a) rational design and (b) directed evolution of proteins. The need for structural and functional information for the rational design and an efficient functional screening are respectively evidenced for the two approaches to guide the iterative cycles shown by the arrows. Ideally the two approaches are interconnected.

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be described in Section 3, but as shown in Figure 2, it can be used in combination with rational design in series of iterative cycles. When a three-dimensional structure of the protein is not available, rational protein engineering can still be based on a three-dimensional model of the target. The model can be validated by traditional biochemical studies on the native protein or by the lengthy process of establishing the role of different residues by alanine scanning mutagenesis. The final output is the generation of a structural and functional model that allows the identification of functionally important residues to be mutated by site-directed mutagenesis to achieve the desired property.

2.1 Modeling and calculations on protein structures Protein modelling is a discipline in its own right and only aspects relevant to the design of biosensors will be covered in this section. The reader is referred to critical reviews in this area (6-8 and references therein); this section will provide the fundamental principles and protocols necessary for the construction of a three-dimensional model of a target protein and the procedures for calculations on the three-dimensional structures. The models derived by using the procedures described in this paragraph are generally good enough for at least initial engineering studies. Protein modelling generally relies on the vast amount of structural and functional information stored in databases available on the web. Based on these, the non-expert can easily have access to programs that allows them to build threedimensional models of target proteins. One example of such program is the comparative model building service of SWISS-MODEL available at http://www. expasy.ch/. The service allows the user to build a three-dimensional model of a protein based on the amino acid sequence that is submitted by the user by email. The server uses sequence alignment and homology or comparative modelling methods (9) to produce the three-dimensional coordinates of a model of the target protein that are sent back to the user by email in the form of a protein data bank (pdb) file. The standard format of the pdb file allows the user to visualize and study the three-dimensional model with a large number of molecular graphics programs, for example, the Swiss-PdbViewer (9) or Insight II by Biosym/MSI (http://www.accelrys.com/). This latter program was used to produce the three-dimensional protein structures shown in this chapter. The quality of the three-dimensional model built by SWISS-MODEL on the basis of the published structures of similar proteins can then be checked for the deviations from ideal bond geometry, angle, and length. The quality of the three-dimensional model can be assessed using the Biotech Validation Suite for Protein Structures (10; http://biotech.ebi.ac.uk:8400). The output of this server is a series of Ramachandran plots that show the occurrence of the residues in the most geometrically favored regions. The G factor is given as an overall indicator of quality of the stereochemical properties of the model, including torsion angles and covalent geometry. When necessary, the quality of the 188

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model can be improved by energy minimization by using programs such as the module Discover 3 of Insight II. Protocols for the preparation of three-dimensional models for refinement by molecular dynamics and energy minimization are available on the Accclrys web site (http://www.accelrys.com/). Once a reliable three-dimensional structure or model of the protein of interest has been generated, a wide number of possibilities exists to study and interrogate the structure to allow informed and quantitative considerations on the position and functional roles of its residues. The three-dimensional structure can be represented in a number of convenient ways to suit the purpose of the investigation. Figures 3 and 4 show the sequence alignment and the three-dimensional models obtained in this laboratory for the three main human cytochrome P450 (CYP) enzymes. These enzymes are part of an ongoing research program in this laboratory that aims at the construction of optical and electrochemical sensors for environmental and pharmaceutical purposes (11-13). CYP form a large class of haem-thiolate monoxygenases involved in xenobiotic metabolism in human and nearly all living organisms. The three-dimensional models of Figure 4 have formed the basis for protein engineering studies, both by rational design and directed evolution.

Protocol 1

Protein sequence alignment Equipment A standard computer connected to the web with web-browser software.

Method 1

Download the amino acid sequences for the bacterial CYP102 (P450 BM3 from Bacillus megaterium), rat CYP2C5, and human CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 from the SwissProt database (http://ca.expasy.org/sprot). These sequences are chosen on the basis that only the X-ray crystal structure of CYP102 and CYP2C5 are known and published to date. They will be used to validate the results of the three-dimensional models of the unknown human CYP.

2 Submit the sequences in fasta format to Multialign (http://prodes.toulouse.inra.fr/ multalin/multalin.html). This creates a multiple sequence alignment from a group of related sequences using progressive pair-wise alignments as described in (14). The alignment obtained is shown in Figure 3, where the key stretches of sequence involved in substrate binding and catalysis are identified by comparison with the known CYP102 and CYP2C5 structures. 3 Download the pdb files of the three-dimensional X-ray structures of CYP102 and CYP2C5 from the Brookhaven National Laboratory (http://www.rcsb.org) and open them using the SwissPdbviewer program (9) available at (http://ca.expasy.org/spdbv/). Derive the secondary structure elements for CYP102 and CYP2C5 for these structures. 189

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Protocol 1 continued 4 Predict the secondary structure elements for the human CYPs using the 3D-PSSM service available at (http://www.sbg.bio.ic.ac.uk/~3dpssm/). This predicts the secondary structure for the aligned amino acid sequences and it also provides a confidence level for the predictions. Alternatively, the PSIPRED service available at (http://bioinf.cs.ucl.ac.uk/psipred/) can be used, but no confidence score is provided as feedback. 5 Compare the information on the secondary structure available for CYP102 and CYP2C5 with that predicted for the human CYPs to validate the sequence alignments. Intervene editing where necessary.

Figure 3 Sequence alignment between class II cytochrome P450s. The sequences of cytochromes of known three-dimensional structure, the bacterial CYP102 (P450 BM3) and the rabbit CYP2C5, are aligned with the human CYP2C9, 2C19, 2E1, 2D6, and 3A4 of unknown structure to date. The validity of the alignment is supported by the position of the highly conserved structural elements such as the cysteine ligand and the ExxR motif.

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Figure 4 Carbon-a three-dimensional models for the human CYP2D6 (black) and CYP2C9 (grey), superimposed to the three-dimensional structure of rabbit CYP2C5 (light grey). The models were built as described in the text, following the structure alignment shown in Figure 3.

Protocol 2

Creation of three-dimensional models Equipment Computer connected to the web and equipped with the Insight II Biosym/MSI software with the Builder and Discover modules. Other similar molecular modeling software will be equally good, but the procedure will need to be modified according to the specific program used.

Method Step 1. Creation of the three-dimensional model 1 Submit by email the amino acid sequences of human CYP2C9, CYP2C19, and CYP2D6 to the comparative model building service SWISS-MODEL available at the expasy site at http://www.expasy.ch/spdbv/. The server will build models for the submitted sequences by sequence alignment and homology or comparative modeling using a program called PROMOD II (9). 2 The SWISS-MODEL service will email back to you a pdb file along with any relevant information or problems for the sequence submitted. The models are built by the server using the published CYP X-ray crystal structures, mostly the closely related CYP2C5 (17) and the haem domain of CYP102 (15). 3 Submit the pdb file to the WHATCHECK service available at http://biotech.ebi.ac. uk:8400/. This will check the models for any geometry and bond length problems. 4 Address the potential problems, then place the haem from the CYP2C5 structure (17) in the human CYP models by using the SwissPdb viewer program (9) available through the same website as the SWISS-MODEL service.

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Protocol 2 continued 5 Proceed to refinement of the model by energy minimization. Step 2. Energy minimization of the three-dimensional model

6 Load the pdb files into Insight II and open the Builder module. 7 Break the bonds between the haem pyrrole nitrogens and the haem iron, as well as that between the iron and the cysteine sulfur. Bonds are broken by selecting them and pushing delete. 8

Select the consistent valence forcefield (CVFF) with the forcefield/select command and clear the potentials and charges. Note that in Insight II, the residue library has to be set to "cvffa.rlb" by using the session/environmental variables command, before the forcefield CVFF is selected.

9 Replace the iron in the haem with a sodium atom using the atom/replace command of the Builder module. 10 Add the hydrogens to the protein model using the modify/hydrogens command and select a pH of 7, switch off the lone pairs option and set the capping mode to charged. 11 Delete the two hydrogen atoms attached to the carboxyl groups of the haem's propionates. 12 Change the bonds in the outer ring of carbons on the haem from Kekule structures to partial double bonds using the modify/bond command of Builder. Change the bonds of the haem's pyrrole nitrogens to partial double bonds. 13 Fix the potentials, partial charges and formal charges using the CVFF residue library by selecting the forcefield/potentials command. 14 Select the forcefield Extensible systematic forcefield (ESFF) from the forcefield/ select command and clear the potentials but keep the charges. 15 Restore the haem's iron together with the bonds to the pyrrole nitrogens and the cysteine ligand by using the modify/bond command. 16 Assign the ESFF parameters by using the forcefield/potentials command and setting the potential action to fix, the partial charge action to accept and the formal charge action to accept. 17 Change the porphyrin's pyrrole carbons to have an atom potential type of c5p using the atom/potential command. 18 Check the partial charge assignments by using the forcefield/potentials command and fixing the potential action to accept, the partial charge action to fix and the formal charge action to accept. 19 Select the Discover 3 module and start the refinement. 20 Initially fix the haem, alpha carbons and side chains to minimize the hydrogens previously added. Minimize the models for 300 iterations (called steps by the program) followed by 10 fs of molecular dynamics. Further minimize for 300 steps. Then fix the haem and the alpha carbons and minimize the side chains of models for 300 steps. 192

PROTEIN ENGINEERING FOR BIOSENSORS Protocol2 continued

21 Submit the model to the Procheck service (http://biotech.ebi.ac.uk:8400/), that will check the models for bond length and geometry problems. Residues without geometry problems are then tethered and the haem is fixed before further minimization and molecular dynamics are carried out. This is repeated for many cycles until energy minimized models are obtained. Once the three-dimensional X-ray or NMR structure or a reliable three-dimensional model for the target protein has been obtained, molecular graphics is an invaluable instrument to investigate the structure and decide on the mutants to be constructed. The following protocols provide guidelines on how to display and perform calculations on three-dimensional protein structures, taking as an example CYP102. For example, the three-dimensional structure of the haem domain of CYP102 can conveniently be represented as a ribbon displaying the secondary structure of the carbon-a backbone to allow visualization of the buried haem prosthetic group and the conformational change that occurs upon substrate binding (Figure 5(a)). The structure can be used not only to locate key residues of interest as targets for mutagenesis, but also to perform calculations to interpret or extrapolate principles for design of new mutants. One example is the Connolly algorithm (18) that allows calculatation of the solvent-accessible surfaces of proteins. This can be performed within the Insight II program by choosing the radius of a probe that is rolled over the whole surface of the protein. A radius of 1.4 A is typically chosen to define a sphere that approximates a water molecule. The algorithm provides values of areas (in A2) for the whole or (b) 25-,

Figure 5 (a) Super-imposition of carbon-abackbone of the substrate-free and palmitoleatebound structures of the haem domain of CYP102. The haem and the palmitoleate are shown in CPK, respectively. The cysteines400 (axial ligandtothe haem iron), 62, and 156 are shown in CPK respectively, (b) Example of results from the accessible surface area (ASA) calculated for the three cysteines by using the Connolly algorithm (18) using a probe of radius 1.4 A. Dark and light bars correspond to calculations performed on the substrate-free and palmitoleatebound structures, respect ively. The level of details of the information is shown by the areas for the whole residues (C62, C156, and C400) as well as for the sulfur atom only (C62-S, C156-S, and C400-S).

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re-entrant area for all or for the selected residues. For example, the surface accessibility of the three cysteine residues of the haem domain of CYP102 are shown in Figure 5(b). The following protocols describe the procedure to be followed to display a protein structure using Insight II, to overlay two structures and to calculate the surface accessibility of specific residues.

Protocol 3

Molecular graphics: display and basic calculations on protein structures Equipment This protocol illustrates how to display and perform basic calculations using the program Insight II by Biosym/MSI (http://www.accelrys.com/) installed on a suitable Silicon Graphics workstation, such as an SGI Indigo2 IRIX 6.2. Most of the other molecular graphics program will perform the same calculations, operating in a very similar manner, often using a PC or Mac environment. In all cases, the pdb files containing the three-dimensional coordinates of the atoms of the protein under investigation must be available. The pdb files are available for download from the PDB web site (http://www.rcsb.org/pdb/). The substrate-free (2hpd.pdb) (15) and palmitoleate-bound (Ifag.pdb) (16) structures of haem domain of CYP102 are chosen as examples. The results are shown in Figure 5.

Method Step 1. How to load and display a molecule 1 Open a Unix shell of an SG workstation. 2 Type: >InsightII—You are now in the Insight II environment, Viewer module. 3 Select the Molecule pulldown, >get. 4 Click on file type PDB under the PDB Directory User. Select one of the two following pdb files. 5 2hpd.pdb: substrate-free structure; call the object FREE, 6 Ifag.pdb: palmitoleate-bound structure; call the object BOUND. 7 Set the heteroatom on to display "non-protein" moieties, such as the haem and the substrate, called "haem" (residue 460) and "pam" (residue 465) in the pdb files, respectively. 8 Later, if you want to load the two structures together, use the connect object to select the object you want to move. Click connect object first, and then click on the molecule to be selected. 194

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Protocol 3 continued Step 2. How to display and color selected residues 9 Under molecule pulldown:

10 render to display atoms/residues as CPK, sticks and so on (NB: selecting CPK will slow down the program), 11 display to show or hide part of the structure. 12 colour to selectively color part of your molecule. 13 ribbon turns the backbone in a ribbon. 14 To select part of your structure set Molecule pick level to molecule and type the selected residue behind the molecule name in the command window as "molecule name: residue name or number." For example, type "BOUND:pam" to select the palmitoleate. 15 Using display you can show only the ribbon (set atoms off)', specific residues can then be selectively shown. To color different parts of the molecule choose color pulldown, type: 16 FREE: 1-455 for the substrate-free structure shown in yellow ribbon; 17 FREE: 460,400 for the haem and cysteine axial ligand shown in red and green CPK respectively (note that Insight II does not require a space between commas and numbers); 18 BOUND: 1-455 for the palmitoleate-bound structure shown in blue ribbon; 19 FREE: 460,465,400,62,156 for the haem, palmitoleate, cysteine axial ligand, and surface cysteines 62 and 156 shown in red, gray, green, magenta, and cyan CPK, respectively. Step 3. How to calculate surface accessibility 20 Open a Unix Shell under the Desktop menu. 21 Type >jot and minimize the window: you will need this window to check the value of Connolly surface calculations. 22

Select surface under the molecule menu.

23

Set create on.

24

Select surface type Connolly or van der Waals.

25 Display as Dots at quality Medium. 26 The probe radius can be changed to check the level of exposure of the residues. The surface can be quantified (in A2); values can be seen in jot (open the jot window you should have set up at point A) as molecule name_surf.out. Be careful: the data will be overwritten in the same file every time you calculate a surface, even if different residues or part are selected. Write down the values displayed as these will be reset at each single step. 27 The values found for the three cysteines 62,156, and 400 for the FREE and BOUND structures are shown in Figure 5(b).

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Protocol 3 continued Step 4. How to superimpose molecules 28 To compare conformational changes occurring upon substrate binding, it is possible to use the command superimpose under the transform pulldown, taking as a reference the haem (called "hem" in the pdb files):

29

Set superimposition mode to atom and label mode off.

30

Source spec.: type "BOUND:hem;

31

Target spec.: type "FREE:hem.

32 Execute and then click on end definition and execute to visualize the superimposition the program just calculated. The information line at the bottom should give you the output root mean square deviation (RMSD) in Angstrom. Step 5. How to measure distances 33 Set the measure pulldown to distances. 34 Set the monitor on. 35

Set monitor mode on add.

36 The atom can be specified as described (molecule name:residue number:atom type i.e. BOUND: 400: CB for the C|3 of residue 400) or they can be selected by clicking on the atom with the mouse left button. 37 The distance will be reported on the screen. Step 6. How to replace a residue 38 Select the Biopolymer module from the star pulldown at the top left corner. 39 Select residue pulldown and replace. 40 Type the number of the residue to replace (molecule name: residue number) in the box. 41 Click on residue and select the amino acid desired from the list. Another useful operation that can be performed to guide the design of mutants is the calculation of a map of electrostatic potentials on a protein surface. It is widely recognized that electrostatics play a key role in molecular interactions such as protein/protein, protein/ligand, enzyme/substrate as well as protein/surface interactions in electrochemical or other immobilized systems. Therefore, the characterization of such interactions is often an important element in the rational protein design process. Electrostatic potentials can be calculated using programs such as the DelPhi module within Insight II. This can calculate the electrostatic potentials on the points of a cubical three-dimensional grid that is defined around a given object containing a number of pre-defined point charges. This grid is divided into a solvent and a solute space. The main feature of this system is that the solvent space (water) has a much higher dielectric constant (g) than the solute (protein). This is because the peptide dipoles in the protein have much less orientational freedom than those in the solvent. DelPhi models this system using the 196

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Poisson-Boltzmann equation together with a finite difference approximation (19). Protocol 4 illustrates how to calculate the surface electrostatic potentials for the haem domain of CYP102 using DelPhi. The results are shown in Figure 6.

Protocol 4

Calculation of a map of electrostatic potentials on a protein surface Equipment Same as Protocol 3, with the addition of the module DelPhi to the Insight II software. To perform a DelPhi calculation, a number of variables need to be defined. The dielectric constants for the solute (protein) and the solvent (water) can be set to 2.00 and 78.6, respectively; the ionic strength of the solvent can be set at 0.1 OM or any other suitable value, and the solvent radius of 1.40 A with an ionic radius of 2.00 A. All radii are set to their van der Waals values as follows: H (1.10), C (1.55), N (1.40), O (1.35), S (1.81), P (1.88), Fe (1.95). Only formal charges are taken into account: Glu, Asp ( — 0.50 on each carboxyl-oxygen), Lys (+1.00 on NQ, Arg (+ 0.50 on each of NH1 and NH2), +/ - 1.00 on N- and C-terminus, Fe ( + 2.00), porphyrin nitrogens ( — 0.50), and phosphate oxygens ( — 0.67). The haem propionates taken to be fully protonated. These formal charges are to be implemented manually in a *.crg file (* refers to object name).

Method 1

Load the molecule into Insight II by going to the molecule menu and selecting get. Select pdb as file type and select the molecule from the left-hand side of the menu, select heteroatom and press execute. For the substrate-free of the haem domain of CYP102, use the pdb file Ifag.pdb.

2 Open the Builder module. 3 Open the environment menu and select environmental variables. Select the residue library, select get and press execute. Type cvffa.rlb in the interpretation box and press set and execute. The residue_library = cvffa.rlb should appear at the bottom of the Insight II window. 4 Open the modify menu and select hydrogens. Enter the molecule name, set a pH of 7 and set the capping mode to charged. Press execute: the hydrogens should appear on the molecule in white). 5 Open the DelPhi module. 6 From the DelPhi setup menu pick the boundary option and select full-columbic, press execute. 7

From the DelPhi setup menu select the grid option. Switch on the molecule region option and add the name of the molecule. The grid size boundary space option should be on with a size of 15 A.

8

Select the grid resolution point spacing option and use a grid point size of 1 A. Press execute (a white box should appear around the molecule). 197

Next Page GIANFRANCO GILARDI Protocol 4 continued

9 This will result in a grid resolution of 1.0 A, with a minimal distance between the molecular surface and the grid boundary of 15 A. Note that for such resolution, the number of grid points increases rapidly with object size, which in turn has its effect on the computing time needed to complete the calculation. The origin is centered on the grid, and not the object. The potential values on the boundary points (edge of the grid) are approximated according to Debye-Hiickel theory. This approximation is denoted as "full_coulombic," as selected in point 6. The need for this approximation stems from an essential feature of an Poisson-Boltzmann computation: it calculates the potential value of each grid point in respect to the surrounding ones. This is not possible for the points that lie on the edge of the grid. Hence the need to approximate their values. However, the quality of the approximation has an effect on the entire computation. The best method for doing this is known as "focussing": first a computation is carried out on a crude grid that encompasses the grid of interest. The results of this computation are then incorporated in a second, more accurate, one. The approximation according to Debye-Hiickel is slightly more crude than the one mentioned above, but was deemed to be sufficient for this application. 10 Return to the DelFhi setup menu and select the solute option. Switch on the charge (current charges) and radius options (vdw radii) and set the solute dielectric to 2. Press execute. 11 From the setup menu, select solvent and set the solvent dielectric to 78.6, the solvent radius to 1.4, the ionic strength to 0.1 or another suitable value, and the ionic radius to 2. Press execute. 12 Select iterations from the setup menu and enter 2000 in the non-linear iterations option, switch on the converge criteria and enter an RMS difference of le-05 and a max difference of 0.0001. Press execute. 13 This will apply the Poisson-Boltzmann algorithm in its nonlinear form with a 2000 iterations limit and convergence limit of 1.0 E — 5; these settings should be more than sufficient to reach convergence. The computed potential energy (kcal mol ~ a e ~ a ) is the total electrostatic energy, which represents the energy that is involved in moving the molecule in a given charged state from vacuum to the solvent environment. This form of potential energy is denoted as "total_plus_grid". 14 Go to the templates option and select charge edit. Switch on the get option and select protein formal. Press execute. 15 Go to the charge edit menu and select the list option. Press execute. A list of the rules used to assign formal charges to the protein should appear in the Unix shell window. 16 Return to Insight II and select the add option from the charge edit menu and for the haem of the CYP102 add the following charges: +2 for Fe and -0.5 for the prophyrin nitrogens (named N A, N B, N C, and N D). Press execute. 17 Go to the charge edit menu and select the list option. Press execute. The rule list will reappear in the Unix shell window, ensure the charges you have added are included. 18 In the DelFhi menu select run, enter an appropriate job name. Press execute. 198

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Protocol 4 continued 19 The output of the computation consists of three files: *.prm that contains the parameter set of the computation; *.atm that contains the atomic coordinates of the object; *.grd that contains the potential values per grid point. 20 Once the DelPhi has completed the calculations, the results are displayed using the Connolly surface of the molecules colored in the DelPhi spectrum.

21 To do this, select the grid menu and go to get. Enter the name of the grid file you just created (*.grd). Press execute. 22

In the Insight II menu, select molecule and go to the surface option. Select the Connolly surface type, enter the name of your molecule, a solid style in display surface, and a low quality surface. Press execute.

23 Once the Connolly surface has been calculated and rendered, go to the molecule menu and select colour. Switch on the attribute surface. Enter the name of your molecule and select grid as the colour method, select the DelPhi spectrum and enter the name of the grid file you created with DelPhi in the scalar grid name option. Press execute. 24 The surface of your molecule should be colored in blue for the positive charges, in red for the negative charges, and in white for the neutral regions.

An example in which electrostatic calculations have played a key role in understanding the interactions between two redox proteins is shown in Figure 6. The haem domain of CYP102 from Bacillus megaterium is able to form an electron transfer complex with flavodoxin from Desulfovibrio vulgaris through electrostatic interactions of a patch of positively charged residues located near the haem ligand cysteine 400 and the negatively charged residues located around the FMN. These are evidenced in the open complex shown in Figure 6( b). The electron transfer complex is stabilized by five H-bonds between the residues Glu 16, Phe 107, Tyr 17, Asp 129, Glu 110 of flavodoxin and HislOO, Arg 131, Ser 108, Gin 109, ArglSl of CYP102, respectively. The model of the complex has guided the engineering of a DNA gene fusion, where the flavodoxin is linked to the CYP102 P450 domain through a suitable loop. The fusion is exploited for the electronic coupling between the P450 domain and the surface of electrodes via the flavodoxin module (12). 2.2 Site-directed mutagenesis On the basis of the knowledge of the structure/function relationship of the protein under investigation, the rational design cycle may begin according to the scheme of Figure 2. Rational design relies on controlled amino acid changes in precise positions. These changes are achieved by site-directed mutagenesis. The pre-requisite for site-directed mutagenesis is the availability of the cloned gene of the target protein and its expression at high levels, typically tens to hundreds of milligrams per liter of culture, of the mutants in a host cell that does not produce the wild-type protein. Site-directed mutagenesis can be used to replace, delete, or insert one or more amino acids. In all cases, a DNA polymerase is used to insert an 199

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Figure 6 (a) Ribbon diagram of the complex between the haem domain of CYP102 from Bacillus megaterium and flavodoxin from Desulfovibrio vulgaris. (b) View of the open complex obtained by a ±90° rotation of the two molecules to show the interface of contact between the proteins. The Connolly surface shows the electrostatic potentials calculated using DelPhi. Positive potentials are shown in black, negative potentials in grey, and neutral in white with a contour scale of ±5 kcal/mol.

oligonucleotide primer containing one or more mismatches in reference to the wild-type DNA template. Several methods have been developed over the last 15 years to achieve these objectives; all of these have advantages and disadvantages related to their yields, costs, or length and complexity of the procedure. In general, all site-directed mutagenesis methods share the need for a mutagenic oligonucleotide that encodes for the desired mutation(s). Figure 7 shows a schematic diagram for site-directed mutagenesis. The gene encoding for the protein of interest is cloned in a suitable plasmid vector by using unique restriction sites, that are specific DNA sequences recognized by commercially available endonucleases. The dsDNA is then used to produce a ssDNA template either by propagation in a Ml 3 phage (Kunkel or phosphorothioate method) or simply by heath denaturation (polymerase chain reaction, PCR method). The ssDNA template is annealed with the mutagenic oligonucleotide that functions as a primer for the synthesis of the complementary mutant strand by a DNA polymerase. An ideal DNA polymerase should not have a 5'- to 3'-exonuclease or strand displacement activity, so that the mutagenic primer is not displaced once the mutant strand synthesis is completed; moreover, the use of a high fidelity enzyme with only one mis-incorporation per 107 bases will decrease the risk of undesired secondary mutations. The T4 DNA polymerase meets these requirements and for this reason it has been used extensively for site-directed mutagenesis. The T4 DNA ligase is then used to link the nicks at the 5'-ends of the primers and the resulting heteroduplex DNA is propagated by transformation in Escherichia coli. At this point the theoretical 50% efficiency of mutagenesis is lowered by host repair mechanisms, therefore, a selection strategy must be used to improve the efficiency of mutant strand recovery at rates as high as possible (80-90%). Various methods of selection have been developed over the years; here I will illustrate some alternatives including the use of 200

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Figure 7 Schematic diagram showing the overall steps necessary to perform a sitedirected mutagenesis.

uracil-containing DNA (Kunkel method), incorporation of dNTP analogs (phosphorothioate method), as well as two examples of the more recent and widely used PCR method (megaprimer and QuikChange™ methods). The great advantage of the PCR method is that selection is not necessary due to the rapid and exponential synthesis of the mutant strand by a heat stable DNA polymerase.

2.2.1 Kunkel method This is one of the earliest mutagenesis methods developed in 1985 by T. A. Kunkel (20, 21). The procedure consists of a combination of in vitro DNA synthesis and in vivo selection steps. Based on the incorporation of deoxyuridine, the Kunkel method relies on the in vivo degradation of a uracil-containing template strand. It can be used with any vector that can produce ssDNA, requiring the isolation and manipulation of ssDNA and a dut~ung~ Escherichia coli strain. For this reason, the wild-type ssDNA is replicated in vivo by a dut~ung~ Esdierichia coli host strain such as BW313 that lacks the dUTPase (dut~) and uracil N-deglycosidase (ung~) activity, allowing the incorporation of a fraction of dUTP instead of dTTP. This ssDNA is used as template for the in vitro mutagenic oligonucleotide-directed 201

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synthesis of the mutant DNA strand, resulting in a dsDNA. This is then transformed in a dut + ung + Escherichia coli strain, for example, JW101, that will degrade the wild-type template strand that contains uracil (U) instead of thymidine (T).

2.2.2 Phosphorothioate method The phosphorothioate method is purely in vitro, allowing a more direct control of the experimental conditions. It is based on the resistance to restriction enzyme digestion by a mutant DNA strand containing a thiol-modified dNTP analog (22, 23). The mutant strand is synthesized from the mutagenic oligonucleotide in the presence of dCTPaS using a ssDNA wild-type template in the presence of T7 DNA polymerase and ligase. The heteroduplex DNA is then nicked by the Nci I restriction endonuclease that will produce nicks only on the CCCGG repeats of the wild-type strand. The T5 DNA exonuclease is used to completely digest the nicked wild-type strand. The remaining DNA fragments are used as primers for the repolymerization by DNA polymerase I in the presence of T4 DNA ligase, creating a heteroduplex mutant DNA with a mutagenesis efficiency greater than 80%.

2.2.3 PCR methods The method based on PCR is the preferred approach available today. It provides a quick and efficient strategy to generate mutants by amplification of a dsDNA template using synthetic oligonucleotide primers that contain the desired mutation(s). The PCR methods use a heat stable DNA polymerase such as the Taq polymerase from Thermus aquaticus or the Vent from Thermococcus litoralis or the Pfu from Pyrococcus furious. The choice of the best heat stable DNA polymerase depends on the needs of the specific experiment, as different enzymes have different optimal temperature, level of fidelity, and proof-reading activity (Table 1). Most protocols for PCR site-directed mutagenesis are based on the method of Higuchi et al. (24). In all cases the enzyme carries out the synthesis of a complementary DNA strand of DNA from the 5'- to the 3'-end using a ssDNA template, starting from a dsDNA region, where a synthetic oligonucleotide primer has annealed. At least one of the primers must contain the sequence for the desired deletion(s), insertion(s), or replacement(s). Two primers are necessary, each complementary to opposite strands of DNA, initiating the synthesis of the region flanked by them. The elements necessary in the mix for a PCR reaction are an excess of the deoxynucleotides (dNTPs) that provide both the energy and the nucleosides required for the synthesis of the DNA, an excess of the oligonucleotide primers, and a very small amount of the DNA template. The reaction is carried out by first heating the sample to separate the DNA strands (denaturation), followed by cooling to allow binding of the primers on the DNA template (annealing) and setting to the temperature for optimal polymerase activity (extension). The heating/cooling process is repeated in cycles by using an automated thermocycler able to achieve the required temperatures very rapidly. The major advantage of the PCR method is that the new DNA strand containing the 202

Table 1 Properties of the DNA polymerases most often used in protein engineering Polymerases

Organism origin

Optimal temp. (°C)

Error rate ( x l O 6)

Exonuclease activity

Comments

DNA polymerase 1

Escherichia coli CM 5199, a lysogen carrying \polA transducing phage

37

9

5'->3'

Nick translation of DNA

3' — > 5'( proof-reading activity)

Second strand synthesis of cDNA Can withstand up to 95 °C

Hot Tub DNA Polymerase

Thermus ubiquitous

75

Klenow fragment of DNA polymerase

Escherichia coli carrying a plasmid in which two-thirds of the 3'-end of the Escherichia coli DNA polymerase 1 gene (Klenow fragment) is cloned

37

40

3'->5'

DNA sequencing by the Sanger dideoxy method and oligolabeling. In vitro mutagenesis. Generates blunt ends.

T4 DNA polymerase

Escherichia coli carrying a T4 DNA polymerase overex pressing plasmid

37

3' 3' — > 5'( proof-reading activity)

VentR DNA polymerase

Escherichia coli that carries the Vent DNA polymerase gene from the archaea Thermococcus litoralis

75

57

3' —>5'( proof-reading activity)

Thermal cycle sequencing.

High temperature dideoxy-sequencing.

PROTEIN ENGINEERING FOR BIOSENSORS Table 2 Exponential growth of the new dsDNA as a function of the number of PCR cycles Cycles

No. of mutated dsDNA

1

0

5

8

10

256

15

8192

20

262,144

25

8,388,608

30

268,435,456

mutagenic primers will accumulate exponentially at each cycle, whilst the wildtype DNA template will remain in trace amounts as initially introduced by the experimentalist. For this reason it is crucial to use only minimal amounts of template DNA. A quantitative example of number of mutant strands generated at each cycle is given in Table 2. Some key aspects of the PCR reaction must be carefully planned. For example, although increasing the number of cycles leads to an exponential increase in the amount of the product, typically 20-25 cycles are sufficient to generate amenable quantities of the products. Increasing the number of cycles also increases the probability that errors are introduced in a random fashion and they are subsequently amplified by the polymerase. Modern PCR thermal cycles are provided with a hot-top assembly that prevents condensation on the thin walls and lid of the PCR tubes. This will cause changes in the reaction conditions that can result in undesired error prone amplification. In the event that no hot-top assembly is available, a typical 50 jol PCR reaction can be covered with 30 [d of mineral oil to achieve the same results. Care must be taken during the following steps in not pipetting any of the oil present on the surface. The design of mutagenic oligonucleotide primers is crucial for the success of the PCR reaction. The parameters to be evaluated in the primer design process are the length of the sequence, the degree of similarity to the template and the presence of secondary structure. The length of the primers varies depending on the number of variations introduced. A primer of 20 base pair (bp) is generally sufficient for a single base mismatch. The primer must be long enough to hybridize the target DNA at a unique position. There are a number of software packages freely available on the web that allow one to automatically check the sites for hybridization of a primer to a given template (see Table 3). A 100% identity over 10-15 bases must be present on both stretches at the 5'- and 3'-ends of the DNA surrounding the mismatch. A full match between template and primer is particularly important at the 3'-end of the primer because the DNA polymerases typically will not extend from a mismatched or poorly hybridized 3'-end. On the other hand, non-complementary bases may be added to 205

Table 3 Some useful web sites for protein engineering and PCR applications Web address

Information

www.expasy.ch

Range of proteonomic tools, from translation to homology modeling

http://biotech.embl-heidelberg.de:8400/

Checking protein models for problems

www.bmm.icnet.uk/~3dpssm/

Recognition of protein folds

www.firstmarket.com

Restriction map generator

http://www.chemie.uni-marburg.de/ becker/welcome.html

Provides useful links to useful software for PCR and primer design

http://www.ebi.ac.uk/software'software.html

Archives of software for molecular biologists

http://endeavor.med.nyu.edu/download/molbio/ http://bioinformatics.weizmann.ac.il/mb/ software.html http://alces.med.umn.edu/VGC.html

Useful for oligonucleotide Tm and protein coding regions

http://bimas.dcrt.nih.gov/sw.html

Useful links to software sites and molecular biology protocols

http://www.yk.rim.or.jp/~aisoai/soft.html and http://iubio.bio.indiana.edu/soft/molbio/ Listings.html

Useful collection of www links for information and services to molecular biologists

http://www.ncbi.nlm.nih.gov/

Links to GenBank® and sequence databases

Table 4 Codon usage by Escherichia co// calculated out of a total of 1,363,279 codons. Each group shows the count, % frequency, and codified amino acid. Note that AUG and UUG are the most frequently used start codons. UAA, UAG, and UGA are stop codons. 3rd position

1st position

2nd position U

C

A

G

U

30,407 (2.23) Phe

11,523 (0.85) Ser

22,048 (1.62) Tyr

7,062 (0.52) Cys

c

U

22,581 (1.66) Phe

11,766 (0.86) Ser

16,669 (1.22) Tyr

8,846 (0.65) Cys

C

18,943 (1.39) Leu

9,793 (0.72) Ser

2,706 (0.20) Stop

1,260 (0.09) Stop

A

18,629 (1.37) Leu

12,195 (0.89) Ser

326 (0.02) Stop

20,756 (1.52) Trp

G

15,018 (1.10) Leu

9,569 (0.70) Pro

17,631 (1.29) His

28,458 (2.09) Arg

U

15,104 (1.11) Leu

7,491 (0.55) Pro

13,272 (0.97) His

29,968 (2.20) Arg

C

5,316 (0.39) Leu

11,496 (0.84) Pro

20,912 (1.53) Gin

4,860 (0.36) Arg

A

71,710 (5.26) Leu

31,614 (2.32) Pro

39,285 (2.88) Gin

7,404 (0.54) Arg

G

A

41,375 (3.03) lie 34,261 (2.51) lie 5,967 (0.44) lie 37,994 (2.79) Met

12,223 (0.90) Thr 31,889 (2.34) Thr 9,683 (0.71) Thr 19,682 (1.44) Thr

24,189 29,529 45,812 14,076

(1.77) (2.17) (3.36) (1.03)

Asn Asn Lys Lys

11,982 (0.88) Ser 21,907 (1.61) Ser 2,899 (0.21) Arg 1,694 (0.12) Arg

U C A G

G

24,910 20,800 14,850 35,979

20,808 34,770 27,468 45,862

43,817 25,996 53,780 24,312

(3.21) (1.91) (3.94) (1.78)

Asp Asp Glu Glu

33,731 (2.47) Gly 40,396 (2.96) Gly 10,902 (0.80) Gly 15,118 (1.11) Gly

U C A G

206

(1.83) (1.53) (1.09) (2.64)

Val Val Val Val

(1.53) (2.55) (2.01) (3.36)

Ala Ala Ala Ala

PROTEIN ENGINEERING FOR BIOSENSORS

the 5'-end of either or both primers. The possibility of these additional mutations offers the opportunity to add unique restriction sites to the PCR products to facilitate subsequent sub-cloning steps. In this case, extra bases must be added at the 5'-end to allow the direct digestion of the PCR product by restriction endonucleases. The length of the primer is also important because it determines the melting temperature, Tm, of the oligonucleotide. This parameter is usually determined experimentally by the supplier of the oligonucleotide, but at the stage of the design of the primers a rough estimate can be made by using the formula: Tm = 4 x (nG + nC) + 2 x (nA + nT),

where nA, nT, nG, and nC indicate the number of these basis in the primer sequence. Most primers exhibit Tm between 55 °C and 65 °C, but ideally the length of both primers should be adjusted to have a Tm as similar as possible. Another important parameter is the presence of a secondary structure in the primers. The presence of a secondary structure interferes with the hydridization of the primer with the template, and therefore should be avoided. It arises from the self-complementarity within the primer that anneals to itself (self-annealing). As this is often caused by a high content of G and C bases, the GC content should not exceed 50% of the total number of bases. A number of software packages are freely available to guide the design of primers and the calculation of Tm (Table 3). Moreover, the codon chosen for the design should be checked against the list of codons used by Escherichia coli, to ensure it is not in the list of the rarely used codons that would impair translation of the gene into the mutant protein (Table 4). Protocol 5 describes a typical PCR experiment.

Protocol 5

Hot start PCR This protocol illustrates a general PCR experiment for amplification of a 1 kb DNA fragment flanked by two oligonucleotide primers n.l and n.2.

Equipment and reagents 1

Plasmid DNA template, 50 ng/nl. This is the typical concentration of plasmid DNA template obtained from a commercial plasmid purification kit such as the QIAprep Miniprep (Qiagen Ltd, UK). As a rule of thumb, the concentration of dsDNA can be estimated from the absorbance at 260 nm using the formula: A2eo x 50 = Hg/ml.

2 The purity of the dsDNA preparation can be estimated from the ratio: A260/A280>1.8.

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GIANFRANCO GILARDI Protocol 5 continued

3 Oligonucleotide primers at 50 pmol/|ol The concentration of the stock solution of synthetic oligonucletides is provided by the manufacturer. As a rule of thumb, the concentration of ssDNA can be estimated from the absorbance at 260 nm using the formula: A26o x 33 = (Xg/ml. 4 Mix of the four deoxynucleotides, ATP, TTP, CTP, GTP, generally called dNTP mix, at 2.5 mM each. These are commercially available as a mix already made at 20 or 25 mM (Amersham Biosciences Ltd, UK). 5 DNA polymerase at 2.5 U/|ol (U, units). Depending on the specific experiments, different DNA polymerases can be used. Taq DNA polymerase (5 U/nl, Amersham Biosciences Ltd, UK) can be used for standard mutagenesis or error prone PCR experiments. Pfu (2.5 U/nl, Stratagene) or Vent (2 U/nl, New England Biolabs Inc) can be used to achieve DNA amplification with high fidelity. 6 DNA polymerase buffer. A 10 x solution of this buffer is supplied by the manufacturer together with the specific DNA polymerase. 7 PCR block. Various types of PCR thermocyclers are commercially available. They can be programmed to perform the desired number of denaturation, annealing, extension cycles. More sophisticated blocks allow to program gradients of temperatures so that different annealing temperatures can be tested at once. In all cases the use of a heated cover is recommended to avoid condensation of the reaction mixture. When this option is not available, 30 |ol of mineral oil should be added on top of the reaction mixture.

Method For a 50 |ol reaction: 1 Add in a 0.5 ml PCR tube: 1 |ol plasmid template (50 ng) 1 |ol oligonucleotide primer n.l (50 pmol/|ol) 1 |ol oligonucleotide primer n.2 (50 pmol/|ol) 4 nl dNTP mix (2.5 mM each) 5 [d 10 x DNA polymerase buffer 37 |ol water. 2 Mix the reagents and briefly spin in a microcentrifuge to collect the mix at the bottom of the tube. 3 Incubate in the PCR block at 94 °C for 5 min. 4 Chill on ice for 2 min. 5 Add 1 nl of DNA polymerase (2.5 U).

208

PROTEIN ENGINEERING FOR BIOSENSORS

Protocol 5 continued

6

Perform 25 cycles as follows:

7 denaturation: 94 °C for 30 s, 8

annealing: 60 °C for 1 min,a

9 extension: 72 °Cb for 1 min,c 10 At the end of the cycles incubate at 72 °C for 7 min, then place on ice. 11

Load 10 (il of the PCR product on an agarose gel (See Protocol 6) and visualize the bands of the product with a UV transilluminator.

12 When the conditions are optimized,11 load all the PCR products on an agarose gel. After separation, excise the bands from the gel and purify the PCR products from the agarose (see Protocol 7). a

This should be 2 °C lower than the lowest Tm of the oligonucleotide primers.

b

Set the original extension temperature according to the DNA polymerase used (see Table 1). c

This depends on the length of the DNA strand to be synthesized. A typical extension rate is 1 min/kb of target length.

d

lf no PCR product is formed, lower the annealing temperature 2-3 °C from the previous set value and run the reaction again. If nonspecific PCR products are present in the form of many bands or smear on the agarose gel, raise the annealing temperature by 2-3 °C and run the reaction again, until a clean band of the desired product is observed.

Protocol 6

Agarose gel electrophoresis of DNA Submarine agarose gel electrophoresis is a widely used method for the separation and purification of DNA fragments of 100-10,000 bp in length. The range of the separation is determined by the percentage of agarose used in the preparation of the gel (Table 5). The DNA is visualized with ethidium bromide, a highly toxic compound; all operations must be carried out using gloves and with the precautions listed by the supplier.

Equipment and reagents 1

50 x TAB buffer stock solution: 242 g Tris, 57.1 ml concentrated acetic acid, 100 ml of 0.5M EDTA at pH 8.0, make to 11 with water. To obtain a 1 x , dilute 20 ml of the 50 x TAB stock with 980 ml of water. This corresponds to 40 mM Tris-acetate, 1 mM EDTA, pH.

2

Stock ethidium bromide solution, 10 mg/ml. 209

GIANFRANCO GILARDI Protocol 6 continued

3 1% agarose gel stock solution: 1 g agarose in 100 ml of 1 x TAB buffer. Other agarose percentage can be prepared varying the amount of agarose. Generally the solution is prepared in heat-resistant bottles, so that when the gel is solidified it can be re-dissolved warming the bottle in a microwave oven. The agarose stock solution will be usable for 1 week. 4

6 x loading buffer: 0.25% (w/v) bromophenol blue, 40% (w/v) sucrose. Make to volume with water. 5 Submarine gel tank, for example, the GNA100 by Amersham Biosciences Ltd, UK.

Method 1 Place masking tape to block the two open edges of the gel tray. 2 Mix a sufficient volume (12 ml for a 5 x 7.5 cm standard gel tray with 25 jol wells) of 1% melted agarose stock solution with 2 jol of stock ethidium bromide in a 15 ml tube. 3 Pour the gel in the tray and place the comb. 4 Allow the gel to cool and set. 5 Remove the masking tape and place the gel tray in the gel tank. 6 Fill the tank with 1 x TAB buffer until the gel is fully covered by a 3 mm layer of buffer. Gently remove the comb avoiding formation of air bubbles in the wells. 7 Prepare the DNA samples by mixing with the appropriate volume of 6 x loading buffer. For a typical 25 jol well, mix in a tube 20 jol of DNA sample with 4 jol of loading buffer in a tube. Use a suitable pipette to load the mix at the bottom of the well. Also prepare suitable molecular weight markers to be loaded for reference. 8 Run the samples at a constant voltage of 120 V for approximately 30-40 min. 9 To visualize the separation, place the gel tray on a UV transilluminator (use a protection shield). The DNA will be made visible by the intercalated ethidium bromide and appear as bright orange bands.

Table 5 Range of separation of agarose gel electrophoresis

210

% Agarose gel

Optimum resolution of linear DNA (kb)

0.5

30-1.0

0.7

12-0.8

1.0

10-0.5

1.2

7-0.4

1.5

3-0.2

2.0

1-0.1

PROTEIN ENGINEERING FOR BIOSENSORS

Protocol 7

Purification of PCR products from agarose gels Equipment and reagents Isolation of PCR products and of DNA in general from agarose gels is a straightforward procedure which is greatly facilitated by the availability of commercial kits that allow the whole procedure to be carried out in less than 20 min. Some examples of these kits are the GENECLEAN by BIO101, the QIAquick by Qiagen, and the Wizard by Promega. Low melting point agarose must be used for this procedure, as all the kits rely on melting the agarose and binding the DNA to silica in high concentrations of a chaotropic salt. This is followed by a washing step and elution of the DNA in low salt or pure water. It is thought that high concentrations of chaotropic salt disrupt the water molecules structured around the negatively charged silica, allowing the formation of a cation bridge between the silica and the DNA phosphate backbone. Removal of salt causes rehydration of the silica and release of the DNA. Commercial kits are capable of to isolating a wide range of sizes of DNA, from less than 100 bp to more than 10 kbp. However, different sizes of DNA will bind to silica under various salt concentrations, pH, and wash conditions, therefore it is recommended to look carefully at the different kits available and assess which is most suitable for the particular size of DNA you are using.

Method There are the many variations on the procedure, depending on the kit used. However, all kits proceed as follows: 1

Excise the DNA band from the agarose gel stained with ethidium bromide and transfer it to an eppendorf tube. This must be done very rapidly using a razor blade over a UV transilluminator. As the UV light is mutagenic, the speed of the process ensures that no unwanted mutations are introduced at this stage on the DNA to be used. An appropriate face shield must be used.

2 Weigh the excised band to determine the volume of chaotropic salt to be added. Generally DNA will bind to silica when the salt concentration is above 4M and the pH is kept between 6.0 and 7.4. Smaller DNA strands (< 200 bp) bind at lower pH, therefore in this case 10% acetic acid can be added to keep the pH around 6.0-6.5. For example, the GENECLEAN kit provides a 6M Nal solution and its protocol requires 3 volumes of this solution to 1 volume of agarose. 3 Melt the low-melting temperature agarose by incubating the tube at 55 °C in a water bath for 5 min. Mix the content of the tube to help melting. 4 Add the silica suspension. Generally this is a 1:1 water: silica suspension provided in the kit. For example, GENECLEAN provides the so-called GLASSMILK. Take care to resuspend the silica before pipetting 10 [d of the GLASSMILK. This should be more than sufficient, as typically 10 [d of GLASSMILK will bind 1-2 [ig of DNA.

211

GIANFRANCO GILARDI Protocol 7 continued

5 Incubate 5 min at room temperature to allow the DNA to bind to the silica. Mix every 1-2 min to ensure that the silica remains resuspended. 6 Separate the DNA bound to the silica from the bulk solution. This can be done either by spinning the tube 5 s at full speed in a microfuge, or by using small spin columns provided with the kit. In the former case the silica must be carefully recovered at the end of the tube, in the latter it is retained by the spin column. The QIAquick kit columns already contain the silica in the column itself. 7 Wash the silica with a high salt concentration solution. This varies between the kits. For example, GENECLEAN provides a concentrated solution of NaCl, Tris, and EDTA with 50% ethanol. The silica must be washed 2-3 times with 10-50 volumes of wash solution. When spin columns are provided, they can either be inserted on an eppendorf tube and spun, or they can be fixed on a vacuum manifold apparatus and the wash removed by suction. 8 Elute the DNA. Add 50 (il of pure sterile water or TEA buffer to the silica, wait 2 min, then spin the columns on clean and sterile eppendorf tubes to recover the solution containing the DNA. Usually more than 80% of the DNA is recovered in one step. This is generally sufficient, as a second elution will result only in an additional ^10% of DNA. The silica and the columns are then discarded.

PCR megaprimer method The megaprimer PCR is a widely used method for site-directed mutagenesis. Generally three oligonucleotide primers are used in two steps of amplification (Figure 8). The first step uses one mutagenic and one flanking primer. The mutagenic primer contains the mismatches necessary to introduce the desired mutation(s). The flanking primer should be designed to include a restriction site (R1) to allow future sub-cloning of the product of mutagenesis. The PCR product of the first step (PCR1) is purified from agarose gel electrophoresis. It is then used as "megaprimer," given its unusually high length, for the second step of PCR (PCR2) together with a second flanking primer designed to allow amplification of the entire gene or to the nearest restriction site (R2). This second restriction site will be used in combination with R1 to allow sub-cloning. If the mutagenic primer can be designed to contain a restriction site, only the first step of PCR will be necessary and the product of the first PCR reaction can be cloned directly in the expression vector. The insertion of a new restriction site (R3) associated with the presence of the specific sequence of the desired mutation will allow an easy screening procedure to select for the clones that contain the mutant plasmid. In all cases it is strongly recommended to confirm the presence of the desired mutation and check the absence of undesired ones by DNA sequencing of the full length of the gene. The size of the megaprimer varies depending on the requirements of the specific gene and mutation to be introduced. Generally it is much longer than a typical oligonucleotide primer, varying between ~100 and ~900 bp. The difficulty of this method lies in achieving good amplification in the second 212

PROTEIN ENGINEERING FOR BIOSENSORS

Figure 8 (a) Schematic diagram of a PCR experiment. Two oligonucleotide primers are designed to anneal on opposite strands flanking the sequence to be extended by a thermostable DNA polymerase. In order to facilitate the sub-cloning of the DNA products, two restriction sites R 1 and R2 are included, (b) Steps for a megaprimer PCR experiment. The oligonucleotide primer containing the desired mutation is used to give a PCR product, that is then used as a megaprimer to extend the full length of the target sequence.

PCR step using the megaprimer. For this reason, the experiment should be designed so that PCR1 is as short as possible, to avoid exceedingly high Tm and the risk of secondary structure causing self-annealing. On the other hand, the size of PCR1 should be such that the recovery from the agarose gel electrophoresis is not a limiting factor. A typical experiment is reported in Protocol 8 (25).

Protocol 8

Megaprimer PCR Equipment and reagents See Protocol 5.

Method Step 1. PCR1.

1

For a 100 ul reaction, add in a 0.5 ml PCR tube: 1 ul plasmid template (50 ng) 2 ul oligonucleotide primer n.l (50 pmol/ul) 2 ul oligonucleotide mutant primer n.2 (50 pmol/ul) 8 ul dNTP mix (2.5 mM each)

213

Next Page GIANFRANCO GILARDI Protocol 8 continued

0 |ol 10 x DNA polymerase buffer 75 |ol water.

2 Perform the PCR experiment as described in Protocol 5, but add 2 jol of DNA polymerase (5 units). 3 Load all the PCR product on a 1.5% agarose gel (use a wide comb to obtain large bands that are easier to excise from the gel).a 4 Perform the separation as described in Protocol 6. 5 Excise the band corresponding to the megaprimer (PCR1 product) by cutting the band with a sharp blade. Minimize the exposure of the megaprimer to the UV light to avoid undesired mutations. 6 Purify the megaprimer from the agarose as described in Protocol 7. 7 Determine the concentration of pure megaprimer as described in Protocol 5. Step 2. PCR2 8 For a 100 |ol reaction, add in a 0.5 ml PCR tube: 1 |ol plasmid template (50 ng) 2 |ol oligonucleotide primer n.3 (50 pmol/|ol) 40 |ol megaprimer (50 pmol/|ol)b 8 |ol dNTP mix (2.5 mM each) 10 |ol 10 x DNA polymerase buffer 75 |ol water. 9 Perform the PCR experiment as described in Protocol 5, but add 2 [d of DNA polymerase (5 units). 10 Cool the PCR products on ice. 11 Load 10 |ol of the PCR product on a 1% agarose gel to check that the correct product has been formed. 12 When the Taq DNA polymerase is used, add 1 |ol of the Klenow fragment (5 U/|ol) directly to the PCR reaction and incubate at 37 °C for 1 h. 13 Perform a restriction digest as described in Protocol 9, using the suitable restriction enzymes flanking the mutated amplified region.0 14 After separation by agarose gel electrophoresis (Protocol 6), purify the product of digestion from the gel (Protocol 7). 15 Perform a ligation in a vector suitable for expression (Protocol 10). 16 Transform (Protocol 11) the resulting plasmid in a strain of Escherichia coli compatible with the promoter carried by the chosen expression vector (e.g. Escherichia coli BL21 (DE3) for a pT7 plasmid carrying the T7 promoter). 17 Spread on LB plates containing the appropriate antibiotic and incubate at 37 °C overnight.

214

Previous Page PROTEIN ENGINEERING FOR BIOSENSORS

Protocol 8 continued

18

Pick 5-10 colonies and grow overnight at 37 °C on liquid LB medium with the appropriate antibiotic.

19 Isolate the plasmid from the colonies (Protocol 12), perform a restriction digest (Protocol 9), and separate on agarose gel electrophoresis (Protocol 6) to verify that the plasmid contains the correct DNA insert. 20

Submit the positive clones to DNA sequencing to verify the presence of the mutation. This can be done routinely by sending the plasmid with the relevant primers to DNA sequencing services.

a

In some cases it may be better to first load only 10 (il of the PCR reaction products, to check the efficiency of the reaction and the purity of the megaprimer prior to large scale separation. In an ideal case, all the primers of the reaction have been converted in the megaprimer only. In this case one may proceed directly to the second step of PCR, without purification an agarose gel. When this convenient short cut is chosen, one must be aware of the risk of recovery of the very small amount of the wild-type used as template in the first step.

b

Ideally the concentration of the megaprimer should be determined spectrophotometrically from the absorbance at 260 nm. Then 100 pmol should be added to achieve equal concentration with primer n.3.

C

A sufficient number of bases must be present at the terminals to allow restriction endonucleases to function. When this proves to be difficult, an intermediate cloning step maybe necessary. This consists in the blunt end ligation of the PCR product into a high copy number plasmid such as the pBluescript vector. The fragment can then be easily digested using the relevant restriction endonucleases.

Protocol 9

Restriction digest of DNA Equipment and reagents • 1.5 ml microcentrifuge tubes and standard bench microcentrifuge • Heating block at 80 °C • Restriction endonucleases from various suppliers, for example, Amersham

Biosciences, New England Biolabs, Promega, Stratgene • Water bath at 37 °C • Ice bucket • Reaction buffer provided with the restriction endonucleases.

Method 1

For a 20 [d reaction, add in a 1.5 ml microcentrifuge tube: 16 nl DNA (0.1-1.0 ^g)a 2 |ol 10 x buffer for the chosen restriction endonucleaseb 1 |ol each of the two restriction endonucleases (5-10 units of each).c

215

GIANFRANCO GILARDI Protocol 9 continued 2 Make sure that the enzymes are added last and the tube is always kept on ice except for the incubation period.

3

Mix gently using a pipette.

4

Briefly spin using a microcentrifuge to collect the mixture at the bottom of the tube.

5

Incubate for 1-3 h at 37 °C.d

6

Stop the reaction by incubating 3 min at 80 °C.

7 Cool on ice, briefly spin, and load 5-10 ul on an agarose gel to check the products (Protocol 6). a

A typical DNA miniprep should contain this concentration of DNA that is easily visualized on agarose gel.

b

Consult with the requirements of the suppliers of the enzymes. Usually the buffer is supplied with the enzymes. The correct concentration and buffer compatibility between different enzymes is crucial to avoid nonspecific cleavage of DNA.

C

10% of the total volume of the reaction mixture, because the glycerol used to store the enzymes will promote nonspecific cleavage of DNA.

d

Length and temperature of incubation varies depending on the efficiency of the enzyme. Consult the recommendations of the enzyme suppliers.

Protocol 10

Ligation of a DNA insert in a vector Equipment and reagents • 0.5 ml microcentrifuge tubes and standard bench microcentrifuge

• Sterile water. • Heating block at 65 °C

• Linearized vector

• PCR product or insert • 10 x T4 ligase buffer containing 10 mM ATP . T4 ligase (3-6 U/ul)

Method 1

For a typical 30 ul experiment reaction, add in a 0.5 ml microcentrifuge tube: 2 ul of linearized vector (approx 50 ng) 20 ul of PCR product or insert (approx 250 ng) 3 ul 10 x T4 ligase buffer containing 10 mM ATP 1 ul T4 ligase (3-6 U/ul) 4 ul sterile water.

2 In parallel, set up a control reaction as follows: 2 ul of linearized vector (approx 50 ng) 3 ul 10 x T4 ligase buffer containing 10 mM ATP

216

PROTEIN ENGINEERING FOR BIOSENSORS

Protocol 10 continued

1 |il T4 ligase (3-6 U/nl) 24 (il sterile water. 3 Proceed for the two reactions as follows: 4 Mix the water, vector, and PCR product (insert) in a 0.5 ml microcentrifuge tube. 5 Heat the sample at 65 °C for 10-15 min to uncoil any supercoiled DNA. 6 Cool the sample on ice and touch spin to collect all the liquid to the bottom of the tube. 7 Add the buffer followed by the ligase enzyme. Incubate the sample overnight at 16 °C for blunt-end or 4°C for sticky-end ligations. 8 Use half the reaction mixture to transform competent cells (Protocol 11) and store the remainder at — 20 °C in case a second transformation is needed. For blue-white screening of the insert, see the note in Protocol 11.

Protocol 11

Transformation of a plasmid in a bacterial host Equipment and reagents Standard LB media are made using per liter 10 g of tryptone, 10 g of sodium chloride, and 5 g of yeast extract; the pH was then adjusted to seven using 0.1M sodium hydroxide. For agar plates 15 g of agar is added per liter of LB. Ampicillin stocks contain 100 mg/ml of ampicillin and are used to give a final concentration of 100 (xg/ml and stored at — 20 °C when not in use. IPTG stocks contained 1M IPTG and were diluted to give a working concentration of 1 mM and stored at — 20 °C when not in use.

Method Step 1. Competent cell preparation An easy procedure to produce Escherichia coli competent cells is the so-called calcium chloride method. This method yields around 2 x 107 transformed colonies per microgram of plasmid DNA. 1 Pick one Escherichia coli colony from a freshly grown (overnight at 37 °C) plate, and transfer it into 10 ml of LB broth. Incubate over night at 37 °C with vigorous shaking. 2 Pick 7 |ol of the overnight culture and inoculate 7ml of fresh LB broth, which is then incubated at 37 °C with vigorous shaking for 2 h. 3 Spin the cells at 4000 rpm for 10 min at 4 °C. 4 Remove the supernatant and keep the cell pellet on ice at all times. 5 Resuspend the cells in 3.5 ml of ice cold 50 mM CaCl2.

217

GIANFRANCO GILARDI Protocol 11 continued

6 Leave on ice for 30 min. 7 Spin the cells at 4000 rpm for 10 min at 4°C and remove the supernatant. 8 Resuspend the pellet with 700 |ol of ice cold 50 mM CaCl2 and store on ice. 9 The cells will be competent within a couple of hours, with a peak after 24 h. Step 2. Transformation procedure 10 Add 1 |ol (50 ng) of the desired plasmid to 200 [d of the competent cells. 11 Incubate on ice for 30 min. 12 Quickly heat shock the cells for 90 s at 42 °C. 13 Immediately transfer the tube to an ice bucket and incubate for 2 min. 14 Add 800 |ol of fresh LB broth and incubate for 45 min at 37 °C to allow the bacteria to recover and express their antibiotic resistance marker encoded by the plasmid. 15 Spin the cells at 4000 rpm for 10 min at 4°C and remove the supernatant. 16 Resuspend the pellet with 200 |ol of fresh LB. 17 Plate the cells onto agar plates containing the antibiotic for which the plasmid confers resistance. 18 Incubate the plates overnight at 37 °C to allow the colonies to grow.a a

When the transformants are expected to contain a plasmid that contains a ligated insert, a so-called blue-white colony screen may be helpful in identifying the colonies containing the insert. This can be achieved by using the vector pBluescript that contains the cloning site on the fS-galactosidase gene. When the insert is present, the fS-galactosidase is not expressed and the colonies will appear white. When the insert is not present, the fS-galactosidase is expressed and the colonies will yield a blue precipitate. The screen is achieved by adding 80 (ig/ml of 5-bromo-4-chloro-3-indolyl-|3-D-galactopyranoside (X-gal, prepared in dimethylformamide), plus 20 mM isopropyl-1-thio-fS-D-galactopyranoside (IPTG prepared in sterile water) to the agar plate. Alternatively, 100 (il of 10 mM IPTG and 100 (il of X-gal can be spread on the agar plate 30 min prior to plating the transformations. Do not mix the IPTG and the X-gal before pipetting them onto the agar plates because they may precipitate.

Protocol 12

Isolation of a plasmid from a bacterial host Equipment and reagents Plasmids must be isolated from bacterial cultures derived from one single colony picked from fresh agar plate, not older than one week. The Luria-Bertani (LB) broth is usually preferred to Terrific Broth (TB) because the latter would yield 2-5 times the number of cells that may present problems in the efficiency of alkaline bacterial lysis, purity of plasmid DNA (contamination from genomic DNA) and overloading of the DNA binding membranes used in DNA purification.

218

PROTEIN ENGINEERING FOR BIOSENSORS

Protocol 12 continued

LB broth, 1 1 10 g NaCl 10 g tryptone 5 g yeast extracts 1 ml of 1M Tris-Hcl at pH 7.0. TB broth, 1 1 12 g tryptone 24 g yeast extracts 8 ml of 50% glycerol 2.31 g KH2PO4 2.54 g K2HPO4. Generally the protocols for plasmid isolation are adjusted for culture volumes of 1-5 ml. One single colony is grown overnight for 12-16 h, in the presence of a suitable antibiotic (see Table 6). The antibiotic selection must be kept at all stages of growth and it is important to realize that long incubations cause depletion of the antibiotic. For example, the resistance to ampicillin is caused by the enzyme p-lactamase encoded by the plasmid-linked bla gene. As p-lactamase hydrolyzes ampicillin, the levels of this antibiotic are continuously depleted from the culture medium. This is evidenced by the presence of satellite colonies in agar plates. Escherichia coli DH5a and XLl-Blue are highly recommended for plasmid preparations. Strain HB101 and its derivatives such as the TG and JM series are known to produce large amounts of carbohydrates that are released during the plasmid isolation procedure and can inhibit subsequent enzyme activities if not completely removed. Another important factor to be considered is the copy number of plasmid per cell that is dependent on the origin of replication born by the plasmid itself (see Table 7). Copy numbers higher than 200 per cell are classified as "high," whilst lower than 100 are "low." This must be kept in mind when expecting a high yield of plasmid preparation. Traditional methods for plasmid isolation are based on the alkaline lysis of the bacteria followed by phenol extraction and ethanol precipitation of the plasmid DNA. The tedious steps of lysis, repeated extraction, and precipitation are today replaced by kits that require three simple steps: lysis, adsorption of the plasmid DNA to a membrane, washing, and elution of the plasmid DNA. The protocol reported here is based on the widely used QIAprep® kit provided by Qiagen. Although the DNA yields depend on many factors such as plasmid copy numbers, bacterial growth, size of the culture, a typical preparation by QIAprep® yields 5-20 (ig of dsDNA in 50 jol of final elution volume. This is good enough for most applications. Interestingly, Qiagen can also provide kits for high throughput plasmid preparations in 96-well plates; 96 preparations can be achieved in less than 45 min.

Method Step 1. Alkaline lysis of bacteria 219

Protocol 12 continued

1

Spin down the 1-5 ml overnight culture at maximum speed for 5 min using a conventional microcentrifuge. 2 Resuspend the pellet in 250 |ol of lysis buffer PI by vortexing, taking care that no lumps of cells are observed. The buffer contains RNase to improve the purity of the plasmid DNA (usually RNA co-purifies with the plasmid). 3 Add 250 |ol of buffer P2, mix gently by inversion, allow to lyse for no more than 5 min. This buffer contains sodium hydroxide that causes bacterial lysis due to the alkaline conditions.

4 Add 350 |ol of buffer N3 to terminate the lysis, mix gently by inversion. This buffer contains guanidine hydrochloride to fully precipitate the proteins and acetic acid to neutralize the pH. The solution should become cloudy. 5 Centrifuge for 10 min at full speed. Step 2. DNA adsorption to membranes 6 Carefully remove the supernatant and load it to the centrifuge spin columns containing the DNA binding membrane. 7 Centrifuge for 30-60 s, discard the flowthrough. 8 Wash with 500 |ol of buffer PB, centrifuge for 30-60 s, discard the flowthrough. This buffer contains guanidine hydrochloride and isopropanol and it is necessary for removing the nuclease activity and the carbohydrates from strains such as HB101, TG, and JM series (see above). Step 3. Washing and elution of the plasmid DNA 9 Wash with 750 ul of buffer PE, centrifuge for 30-60 s, discard the flowthrough. This buffer contains ethanol that ensures that the DNA remains bound to the membrane while being washed. The same considerations made on DNA binding to resins made in Protocol 7 are valid here. 10 Centrifuge again at full speed for 1 min to remove any residual buffer. This is very important to elute a clean plasmid DNA in the following step. 11 Place the spin column on a clean and sterile eppendorf tube. 12 Add 50 ul of sterile water to the top of the spin column, let stand for 1 min to promote DNA release, centrifuge for 1 min. Maximum elution efficiency is obtained at pH 7.0-8.5. 13 Collect the flowthrough containing the pure DNA plasmid and store at — 20 °C. Table 6 Preparation of most commonly used antibiotics. All solutions must be stored at -20°C Antibiotic

Stock solution

Working concentration (|xg/ml)

Dilution

Ampicillin sodium salt

50 mg/ml in water

100

1:500

Chloramphenicol

34 mg/ml in ethanol

170

1:200

Kanamycin

10 mg/ml in water

50

1:200

Streptomycin

10 mg/ml in water

50

1:200

Tetracyclin HCI

5 mg/ml in ethanol

50

1:100

PROTEIN ENGINEERING FOR BIOSENSORS Table 7 Origin of replication and copy numbers of the most commonly used plasmids and cosmids Plasmids

Origin of replication

Copy number

pUC vectors

ColEl

500-700

pBluescript vectors

ColEl

300-500

pGEM vectors

pMBl

300-400

pTZ vectors

pMBl

>1000

pBR322 and its derivatives

pMBl

15-20

pACYC and its derivatives

p!5A

10-12

pSClOl and its derivatives

pSClOl

~5

SuperCos

ColEl

10-20

pWE15

ColEl

10-20

Cosmids

QuikChange ® PCR method The QuikChange® is the most rapid and efficient method available to date to produce site-directed mutants. It can be performed in one day and it consists of a PCR reaction carried out directly on the plasmid DNA template, without the need of sub-cloning steps or specialized vectors, or unique restriction sites or multiple transformations. The procedure consists of three steps: PCR reaction, digestion and transformation. QuikChange® is provided in a kit by Stratagene® (www.stratagene.com) and the principle of the procedure is shown in Figure 9. The first step consists in a PCR reaction that uses the high fidelity PfuTurbo DNA polymerase with two 5'-phosphorylated primers containing the desired mutations, each complementary to the same region of opposite strands of the vector. The incorporation of the oligonucleotide primers by PCR extension generates a mutated plasmid containing staggered nicks. The mutated strands are selected by digestion of the template wild-type, methylated plasmid with the restriction endonuclease Dpn I. This enzyme digests the sequence 5'-Gm6ATC-3' and is specific for methylated DNA as the starting plasmid isolated from the commonly used dam+ Escherichia.coli strains. The final step consists of the transformation of the mutated nicked dsDNA into the XL-1 Blue supercompetent cells that will repair the nicks in the mutated plasmid. The QuikChange® kit comprises a control reaction to be carried out with the oligonucleotide primers and the pWhitescript™ plasmid provided. This plasmid contains a stop codon (TAA) at the position of glutamine 9 (CAA) of the p-galactosidase gene. This results in an inactive p-galactosidase that gives white colonies on LB-ampicillin agar plates containing IPTG and X-gal. The mutagenesis control reaction is designed to introduce oligonucleotide primers that restore the CAA codon, resulting in an active enzyme. Following transformation, the colonies containing the successful mutagenesis can be screened for the p-galactosidase 221

G I A N F R A N C O GILARDI

TM

Figure 9 Schematic procedure for the QuikChange

PCR mutagenesis method.

blue phenotype. The mutagenesis efficiency (£) can be calculated with the formula: E — (No. of blue colonies)/(Total No. of colonies) x 100. A typical successful experiment should result in a 80% efficiency of mutagenesis.

3 Directed evolution Directed evolution is a new and powerful approach of protein engineering particularly suitable when little information is known on the structure/function relationship of the target protein/enzyme. It relies on iterative cycles of random mutagenesis, recombination, and functional screening of a library of variants from which mutants with improved or new properties are identified (Figure 2). The possibility of generating enzymes with high chemical and thermal stability, solubility in organic solvents, activity toward new substrates, specificity and enantio- or regie-selectivity in catalysis is a very attractive target for the biosensors of the future. Essentially this approach mimics in vitro the process of natural molecular evolution that is able to generate a potentially infinite plethora of new proteins with 222

PROTEIN ENGINEERING FOR BIOSENSORS

new functions. Therefore, it becomes obvious that there is a great potential that directed evolution offers to the bioanalytical area, where the experimentalist will no longer be limited by the availability and the properties of natural proteins.

Protocol 13

QuikChange

PCR mutagenesis

Equipment and reagents See Protocol 5. The Stratagene kit contains all the reagents except the plasmid template and the oligonucleotide primers. An important step in all PCR experiments is the choice of the oligonucleotide primers. Some variations from the general procedure given in Section 2.2.3 are necessary when using the QuikChange® method. Both primers should contain the desired mutation and must anneal to complementary sequences on opposite strands of the plasmid. They should be 25-45 bases long with the desired mutation in the middle, with a minimum GC content of 40% and terminating with one or more C or G bases. They must be purified by FPLC or PAGE and their Tm should be equal to or greater than 78 °C. Two different empirical formulae can be used to calculate Tm: Tm = 81.5 + 0.41 (%GC) - 675/N - %mismatch can be used for substitutions, where N is the length in bases, %GC and %mismatch are whole numbers; Tm = 81.5 + 0.41 (%GC) - 675/N can be used for insertions or deletions, where N does not include the bases that are being inserted or deleted.

Method Step 1. PCR reactions 1 For a 50 jol reaction, add in a 0.5 ml PCR tube: 1 |ol plasmid template (125 ng) 2 |ol oligonucleotide primer n.l (125 pmol/|ol) 2 |ol oligonucleotide mutant primer (50 pmol/|ol) 4 nl dNTP mix (2.5 mM each) 5 |ol 10 x DNA polymerase buffer 35 |ol water 1 |ol Pfu DNA polymerase (2.5 U/|ol). 2 Perform the following cycling reactions. 3 1 cycle at 95 °C for 30 s. 4 12-18 cyclesa as follows: denaturation: 95 °C for 30 s annealing: 55 °C for 1 minb extension: 68 °C for 2 min/kb of plasmid length. 223

GIANFRANCO GILARDI Protocol 13 continued

5 Place on ice for 2 min to cool the reaction to < 37 °C. Step 2. Dpn I digestion of the wild-type template 6 Add 1 [d of a 10 U/|ol stock of Dpn I directly to the reaction mix. 7 Mix gently using a pipette. 8 Briefly spin using a microcentrifuge to collect the mixture at the bottom of the tube. 9 Incubate for 1 h at 37 °C. Step 3. Transformation 10 Transform a 2 jol aliquot of the reaction mix according to the procedure described in Protocol 9. If possible use the commercial XLlO-Gold Ultracompetent Escherichia coli cells that are capable of highly efficient transformations as they are both endonuclease (endAl) and recombination (recA) deficient. This will increase the yield of transformants. If standard competent cells are used (prepared as described in Protocol 9), use the whole reaction mix or increase the concentrations of DNA by ethanol precipitation. a

Adjust the number of cycles according to the following: 12-16 cycles = point mutations, single amino acid changes; 18 cycles = multiple amino acids deletions or insertions. b This should be 2°C lower than the lowest Tm of the oligonucleotide primers. Directed evolution is based on the generation of genetic diversity by DNA recombination, also called DNA shuffling. This process mirrors the Darwinian evolution where random mutagenesis, recombination, and selection allow the addition of beneficial mutations and the removal of deleterious ones. This section will describe the three key steps of the iterative cycles of directed evolution: random mutagenesis, recombination, and functional screening. As indicated in Figure 2, once a trend in the structure/function properties of the target enzyme has been rationalized, the directed evolution approach can be set to work in tandem with the rational design one.

Random mutagenesis: error prone PCR Molecular diversity can be created in a number of ways, for example, by error prone PCR and combinatorial cassette mutagenesis. In combinatorial cassette mutagenesis, a relatively short DNA sequence is mutagenized by synthetic oligonucleotide primers (26). This method is relatively straightforward, but it is useful only when the targeted amino acids are in the same stretch of primary sequence. It relies on the synthesis of a large number of oligonucleotide primers, and therefore it is combinatorial only within the limits of the synthetic oligonucleotides, that may be labor-intensive and expensive. Error prone PCR is by far the most used approach to produce random mutants. It was developed by Leung in 1989 (27, 28) who exploited the ability of thermostable DNA polymerases lacking proof-reading activity, for example, Taq polymerase (see Table 1), to introduce random mistakes during the extension 224

PROTEIN ENGINEERING FOR BIOSENSORS

of the new DNA strand. Taq has the highest known error rate, in the range of 0.1 x 10~ 4 to 2 x 10~ 4 per nucleotide per pass of the polymerase. The rate of mis-incorporation depends on the specific reaction conditions, therefore, the standard PCR protocol needs to be modified to control the error rate. For example, the MgCl2 concentration is increased to 7 mM to stabilize non-complementary pairs, MgCl2 is added at 0.5 mM to diminish the template specificity of the polymerase, the concentrations of dCTP and dNTP is increased to 1 mM to promote mis-incorporation, and the amount of Taq DNA polymerase is increased to 5 U to promote the chain extension beyond the positions of base mismatch. Generally the procedure proposed in Protocol 14 works well for DNA templates up to 1 kb; longer target sequences can be divided into two or more fragments.

Protocol 14

Error prone PCR Equipment and reagents See Protocol 5. Stock reagents are: 10 x PCR buffer: 70 mM MgCl2 500 mM KC1 100 mM Tris-HCl pH 8.3 0.1% gelatin 10 x dNTPs: 2 mM dGTP, 2 mM dATP, 10 mM dCTP, 10 mM dTTP MnCl2 : 5 mM.

Method 1

For a 100 |ol reaction, add in a 0.5 ml PCR tube: 1 |ol DNA template (50 ng) 2 |ol oligonucleotide primer n.l (50 pmol/|ol) 2 |ol oligonucleotide primer n.2 (50 pmol/|ol) 10 nl 10 x dNTP mix 10 [d 10 x DNA polymerase buffer 63 [d water.

2 Mix well. 3 Add 10 [d of 5 mM MnCl2, mix well ensuring that there is no precipitate. 4 Add 5 U of Taq DNA polymerase to a final volume of 100 |ol. 5 The mixture is heated for 5 min at 94 °C and then subjected to thermal cycling as follows (30-80 cycles depending on the specific requirements): 94 °C for 1 min 55 °C for 1 min 225

Next Page GIANFRANCO GILARDI Protocol 14 continued

72 °C for 1 min. 6 Isolate the amplified DNA fragment of the correct size by agarose gel electrophoresis, digest and ligate with the appropriate vector.

Error prone PCR is able to produce large libraries of random mutants in a sequential fashion where deleterious mutations are accumulated in conjunction with beneficial ones. This can seriously limit the evolutionary process because of the high probability that the effects of the few beneficial mutations are lost due to the many deleterious ones. From a biological point of view, sequential random mutagenesis is equivalent to an asexual evolutionary process.

Recombination: DNA shuffling The breakthrough in directed evolution was made by Stemmer in 1994 with the introduction of DNA shuffling that allows the in vitro formation of recombinant genes from a set of parental ones (29, 30). DNA shuffling requires the availability of a pool of homologous genes that can be derived from a naturally occurring protein family or from a library of mutants produced by error prone PCR, as described in the previous paragraph. Error prone PCR can introduce a number of point mutations in a gradual fashion. It lacks the essential features of block changes typical of DNA shuffling that also enables the removal of deleterious mutations by back-crossing with parental or wild-type DNA (Figure 10). Block changes are introduced with the

Figure 10 Recombination of mutant or homologous genes by DNA shuffling.

Previous Page PROTEIN ENGINEERING FOR BIOSENSORS

fragmentation of the pool of genes using DNase I. The fragments are purified by agarose gel electrophoresis and reassembled into a full length gene by repeated cycles of PCR without added primers (Protocol 15). During this step the fragments prime each other on the homologous regions, resulting in recombination when fragments derived from one parental gene prime on another one, causing a template switch. The recombinant genes obtained with this procedure can then be used for other rounds of mutation and recombination.

Protocol 15

DNA shuffling Equipment and reagents See Protocol 5 and 14.

Method Step 1. DNAse I digestion 1 Dilute the genes or PCR products with the random mis-incorporations (Protocol 14) to 2-4 ng in 10 mM Tris-HCl (pH 7.4), 1 mM MgCl2. 2 Equilibrate the mixture at 15 °C for 5 min in a thermocycler. 3 AddO.SU of DNAse I. 4

Digest for 2 h at 15 °C.a

5

Stop the digestion by heating at 90 °C for 15 min.

6

Purify fragments of ca. 50 bp by 2% agarose gel electrophoresis.

Step 2. Fragment reassembly 7 Reassembly is achieved by Taq polymerase in the absence of primers. A 100 [d PCR mixture contains: 10-30 ng/|ol of fragments 0.2 mM each dNTPs 2.2 mM MgCl2 50 mM KC1 10 mM Tris-HCl (pH 9.0) 0.1% Triton X-100 2.5 U Taq polymerase. 8

Perform the PCR cycles (30-45) as follows:

96 °C for 3 min 50-55 °C for 1 min 72 °C for 1 min. 9 Incubate the mixture at 72 °C for 5 min.

227

GIANFRANCO GILARDI Protocol 15 continued

10 Reassembly of fragments of 100-200 bp size usually yield PCR products of the correct size; smaller fragments of 10-50 bp show some heterogeneity in the size of the PCR product, but this is normally rectified by restriction digest of the PCR product.

Step 3. Amplification of the reassembled fragments 11 A typical 100 |ol PCR reaction contains: 30 mmol of each primer 0.2 mM each dNTP 2.5UTaq/P/it (1:1) 1 x Taq buffer. 12 Perform the PCR cycles as follows. 13 Incubate at 96 °C for 2 min. 14 Perform 20 cycles as: 94 °C for 30 s 55 °C for 30 s 72 °C for 1 min. 15 Isolate the amplified DNA fragment of the correct size by agarose gel electrophoresis, digest and ligate with the appropriate vector. a

Time may need to be adjusted down to 10-20 min depending on the length of the target DNA.

Key features of DNA shuffling are: the requirement of considerable quantities of DNA templates with homologous regions able to prime each other; the presence of sequences of homology separated by regions of diversity; the block changes typical of sexual recombination brought about by template-switch or cross-over. These features have been subject of studies that led to modifications and improvements of original basic protocol. This paragraph reports only some examples of these new approaches. The Staggered Extension Protocol (StEP) has dramatically decreased the amount of the DNA template required by DNA shuffling (31). This method allows the template switching during the synthesis to yield chimeric genes without the requirement of the DNase I fragmentation step (Figure 11 (a)). The DNA templates are mixed with one or more primers and subjected to cycles of denaturation and short annealing/extension steps. The growing PCR products, or fragments, randomly prime to different templates and further extensions will contain template switching. The process is repeated until full length genes are obtained and purified on agarose gel electrophoresis. Parental-like genes are eliminated by digestion with Dpn I as described for the QuikChange method (see section on Quikchange® PCR method, Protocol 13).

228

PROTEIN ENGINEERING FOR BIOSENSORS

Figure 11 Procedures for the (a) StEP, (b) the ITCHY, and (c) the SHIPREC methods.

DNA shuffling generally relies on homologous gene recombination where template switching occurs at points of high sequence identity. Therefore, the DNA shuffling methods described so far can recombine only closely related sequences, that are more than 70% identical. Although many interesting target proteins show very similar functions and high three-dimensional structural homology, they often share only a very low sequence identity. In these cases it is convenient to refer to the Iteractive Truncation for the Creation of Hybrid Enzymes (ITCHY). This method allows the generation of N- and C-terminal fragment libraries of two genes by progressive truncation of the coding sequences with exonuclease III, followed by ligation of the products to make a single crossover hybrid library. This strategy yields cross-overs throughout all the coding region, as shown in Figure 11 (b) (32). Only a small number of cross-overs will connect the two parent genes at sites where the sequences align. Another method able to generate libraries of hybrid genes from distantly related sequences is the Sequence Homology Independent Protein Recombination (SHIPREC). A key feature of SHIPREC is that it ensures cross-overs at positions sharing a similar structural environment, maximizing the probability of obtaining functional proteins (33). The SHIPREC procedure starts with the fusion of two genes to give a heterodimer (Figure ll(c)). This is then digested by DNase I in the presence of Mn 2+ that favors double-strand breaks over nicks, producing an ensemble of fragments of random lengths. Fragments of length corresponding to that of either gene parent are isolated and treated with SI nuclease or T4 polymerase to produce blunt ends. The chimeric genes are first circularized by intra-molecular blunt end ligation, followed by linearization by restriction 229

GIANFRANCO GILARDI

digestion. This creates a library of chimeric DNA sequences where the gene that was at the 5' position in the dimer will now be at the 3' position and will donate the C-terminus of the hybrid protein. The cross-over will therefore be distributed over the entire length of the gene. This approach has been applied to produce soluble hybrids of the membrane associated human cytochrome P450 1A2 and the soluble haem domain of the bacterial cytochrome P450 BM3, that share only 16% sequence identity. The SHIPREC method successfully produced cross-overs mainly at structurally related sites along the sequences. The resulting folded and partially soluble hybrid P450s had cross-overs close to one of the termini, where there is no structural similarity. Simple cross-overs in the interior of the proteins were too disruptive to produce a folded and active protein.

3.3 Functional screening of the library A key requirement to proceed in the iterative cycles leading to the best recombinant protein is the availability of a screen or selection procedure that is sensitive to the properties target in the in vitro evolution process. This step is not trivial, as the vast majority of the mutations are deleterious and only very few will lead to the improved target property. The number of possible variants (V) of a protein that can be created by introducing M substitutions simultaneously over N amino acids is given by the formula: V = 19M[N!/(N-M)!M!]. This means that even for a small protein, when only 10 positions are targeted for 3 simultaneous changes of amino acids, there are 823,080 possible variants. The ability to identify one possible active variant among this large library is close to the limit of most screen or selection methods. Traditionally the term selection is reserved for the methods of searching a library based on an enzymatic function that confers a growth or survival advantage to the host organism. An example of selection is given by Stemmer in the first application of DNA shuffling (30). In this work Stemmer used a mixture of /Mactamase genes to carry out three cycles of DNA shuffling and two cycles of back-crossing with wild-type DNA. Escherichia coli XLl-Blue containing the plasmid expressing the mutagenized /Mactamases was grown on agar plates containing increasing amount of the antibiotic cefotaxime. Only the Escherichia coli expressing the /Mactamase with improved activity toward cefotaxime could grown on plates containing increasing amounts of the antibiotic. This allowed a 32,000-fold increase of the minimum inhibitory concentration from 0.02 (xg/ml typical of the wild-type to 640 (xg/ml typical of the evolved enzyme. Generally the use of a host organism as a means for biological selection is very efficient and it allows searches of libraries containing more than 10s variants. There is, however, a high risk that, under the selective pressure, the host organism itself develops alternative metabolic mechanism unrelated to the target activity. On the other hand, a screening procedure involves the construction of an arrayed protein library and the application of a rapid and high throughput 230

PROTEIN ENGINEERING FOR BIOSENSORS

Figure 12 Screening procedures developed for (a) Cytochrome P450cam (36), (b) Cytochrome P450 BM3 (37) and (c) more generally for NAD(P)H dependent oxidoreductases (13).

assay sufficiently sensitive and specific to enable the identification of positive variants. The development of the screening assay is usually the critical step in the directed evolution of a particular enzyme. Although in some cases visual screens based on color or halo-formation are rapid and straightforward, they are nonquantitative and often insensitive to small changes in the enzymatic properties. Preference is given to quantitative assays based on 96 (or more) well plates. This format not only is quantitative, but it is also amenable to automation using robotic arms, liquid handling systems and plate readers with automated data acquisition. There is a number of examples of screening procedures described in the literature (see reviews 34, 35). In keeping with this chapter's theme on cytochrome P450, this paragraph will give three examples of screening procedures developed for two members of this class of enzymes. Cytochrome P450s are haem-thiolate monoxygenases able to catalyze the insertion of one atom of oxygen from O2 into a wide variety of substrates with concomitant production of water, using the reducing equivalents of NAD(P)H normally provided through a redox chain involving one or two electron transfer proteins. They are involved in key reactions of detoxification of xenobiotics and biosynthesis of steroids, generating much interest in the bioanalytical area. Arnold and coworkers (36) developed mutants of P450 cam from Pseudomonas putida that are able to utilize the electrons and oxygen provided by hydrogen peroxide, eliminating the need for expensive cofactors and complex redox chains. P450 cam was evolved to acquire hydroxylating activity toward the un-natural substrate naphthalene. The screening procedure shown in Figure 12(a) relied in the co-expression of the horseradish peroxidase (HRP) mutant 231

GIANFRANCO GILARDI

Asn255Asp (HRP1A6). This mutant was previously obtained by directed evolution to allow expression of HRP in an active form in Escherichia coli. After 16 h growth on agar/TB plates, the P450 cam variants and HRP1A6 were replicated using a nitrocellulose membrane and transferred onto fresh agar plates containing 6 mM naphthalene and 10-30 mM hydrogen peroxide. The naphthols generated by the P450 cam variants were converted by HRP1A6 into fluorescent dimers and polymers that were detected by fluorescence digital imaging. Different naphthols obtained by diverse P450 cam variants of altered regio-specificity of hydroxylation are polymerized by HRP1A6 in products with different fluorescence spectra. These characteristics were used to select P450 cam variants tuned to different regio-specificity in catalysis. Variants with improved properties were subjected to in vitro recombination by StEP, yielding to several mutants with ~20 fold improvement in naphthalene hydroxylation activity over the wild-type. Another interesting monoxygenase is P450 BM3 from Bacillus megaterium. This enzyme contains the haem catalytic domain fused with the FMN/FAD reductase on the same polypeptidic chain. Wild-type P450 BM3 hydroxylates fatty acids of chain length between C12 and CIS. Arnold and co-workers engineered this enzyme to hydroxylate short chain alkanes such as octane (37). The screening procedure used an octane analogue, 8-pnpane, that upon terminal hydroxylation produces an unstable hemiacetal. This decomposes into the aldehyde and p-nitrophenolate that is detected by its yellow color at 410 nm (Figure 12(b)). Two round of error prone PCR and the screening of 2000-3000 colonies produced a variant with activity 2-3 fold higher than the wild-type. Both the screening procedures described so far are specific for the substrate under investigation, and in the case of octane the procedures are also dependent on the position of the hydroxylation. A general screening procedure developed using P450 BM3, but applicable to all NAD(P)H-dependent enzymes has been developed in our laboratory (13). The screening procedure relies on the conversion of the NADP+ resulting from enzymatic activity into a fluorescent product upon treatment with alkali (Figure 12(c)): for this reason, it has been named "alkali assay." The procedure can be applied to purified enzyme as well as to whole Escherichia coli cells expressing the target NAD(P)H-dependent enzyme. The wide applicability, the use of whole cells, the simplicity of procedure, the suitability for a microtiter plate format are very important properties for high throughput screening purposes. The alkali assay offers a powerful tool for important areas of applications such as screening for NAD(P)H-linked enzymatic activity of molecules of pharmacological and biotechnological interest and screening libraries of random mutants of NAD(P)H-dependent enzymes to allow design of new biocatalysts. The overall process is shown in Figure 13 and the procedure is described in Protocol 16.

232

PROTEIN ENGINEERING FOR BIOSENSORS

Protocol 16

High throughput screening for NAD(P)H-dependent activity Equipment and reagents Standard LB media are made using per liter 10 g of tryptone, 10 g of sodium chloride, 5g of yeast extract. Ampicillin stocks contain 100 mg/ml of ampicillin and are used to a final concentration of 100 (xg/ml and stored at — 20 °C when not in use. IPTG stocks contained 0.05M IPTG and are used to a final concentration of 0.5 mM and stored at — 20 °C when not in use. NADPH stocks contained 15 mM NADPH and are used to a final concentration of 1.5 mM and stored at — 20 °C when not in use. 9M NaOH buffer, 0.3M HC1 buffer, and 0.1M KH2PO4 and the pH of the latter was adjusted to eight using 10M KOH. The buffers are stored at room temperature. Microtiter plates and a fluorescence and/or absorbance microtiter plate reader are required.

Method 1

From a freshly grown (overnight at 37 °C) pick one Escherichia coli colony and transfer it into 5 ml of LB broth supplemented with ampicillin. Incubate overnight at 37 °C with vigorous shaking (230-250 rpm).

2 Dilute 1:100 the overnight culture in fresh LB supplemented with ampicillin, and incubate at 37 °C with vigorous shaking for 3-3.5 h. 3 Transfer 200 [d aliquots of the liquid culture in the well(s) of the microtiter plate and add IPTG to induce protein expression. 4 Incubate for 2 0 h a t 3 7 ° C with vigorous shaking. 5 Spin the cells at 4000 rpm for 10 min at 4 °C. 6 Remove the supernatant and resuspend the cells in 200 |ol 0.1M KH2PO4 pH 8. Check cell density by determining the absorbance at 600 nm using a microtiter plate reader after mixing the plate for 10 s. The optimal absorbance is ~0.3. If necessary, dilute the cells further in 0.1M KH2PO4 pH 8. 7 Add the substrates, including a positive and a negative control and mix the plate prior to incubating for 1 h at room temperature. If checking activity against a natural substrate the incubation length is 1 h. If checking activity against a nonnatural substrate, the incubation length is 2 h. 8 Add 1.5 mM NADPH and mix the plate prior to incubating for 1 h at room temperature. If checking activity against a natural substrate, the incubation length is 1 h. If checking activity against a non-natural substrate, the incubation length is 4 h. Cover the plates in foil because NADPH is photo-sensitive. 9 Stop the reaction by transferring 80 jol aliquot (mix first) to a new microtiter plate to which 80 |ol 0.3M HC1 was previously added and after mixing incubate at room temperature for 15 min. This step essentially lowers the pH to 1.0-2.5. 233

GIANFRANCO GILARDI Protocol 16 continued

10 Transfer 80 |ol aliquot to a new microtiter plate to which 270 jol 9M NaOH was previously added and after mixing, cover in foil and incubate in dark for 2.5 h. This step essentially increases the pH to 14.8. 11 After mixing, read the plate in the range from 340 to 550 nm and check for active mutants.

Functional characterization of the mutants Once the presence of the desired mutation has been confirmed by DNA sequencing, the protein can be expressed and purified. The yields should reach levels of at least 10 mg of pure protein per litre of bacterial culture to allow reasonable applications in the biosensor area. In the first analysis, the functional performance of the mutant should be checked with a number of complementary physical-chemical methods. The detailed functional characterization of the mutants aims at establishing what effect, direct or indirect, the mutation has on the function of the protein. Although X-ray crystallography and NMR spectroscopy are the methods of choice for a detailed analysis of the effect of the mutation on the structure and its consequences on function, the application of these techniques is not always quick and straightforward. Information on stability and folding can be obtained from circular dichroism, fluorescence spectroscopy, and calorimetry. Depending on the presence of cofactors or prosthetic groups, UV-vis and EPR spectroscopy can also provide useful information on the active site of enzymes. Stopped flow spectroscopy can also provide a detailed description of the enzyme kinetics. As a whole these investigations should confirm that the desired function has been achieved without significant perturbation of the other properties. This allows the establishment of general principles for the design in iterative cycles as shown in Figure 2. This task is not trivial because the effect of a mutation on the overall three-dimensional scaffold of the protein is not always obvious.

Other aspects of protein engineering This chapter has so far covered the most widely used methods for engineering proteins. There are many other aspects that cannot be adequately covered in the space limits of this chapter, but I would like to briefly address some of them in this paragraph, pointing the reader toward the cited literature. Often, after the expression of mutants constructed by molecular biology methods, some level of chemical modification of the protein may be required. This is the case, for example, for the site-specific labeling of engineered unique cysteines as shown in Figure 1. Protocol 17 describes how to label the unique cysteine 337 of the S37C mutant of the maltose binding protein with the thiol-reactive fluorophore LANBD.

234

PROTEIN ENGINEERING FOR BIOSENSORS

Figure 13 Screening process for the identification of active NAD(P)H-dependent oxidoreductases in a library of random mutants.

Protocol 17

Fluoresence labelling of the S37C mutant of the maltose binding protein This method describes the labelling of the unique cysteine 337 of the S37C mutant of the maltose binding protein with the thiol-reactive fluorophore IANBD. Only minor changes on parameters such as length of incubation with the fluorophore may be needed to adapt this protocol to other mutants or other thiol-specific fluorophores. The final binding curve obtained from this experiment is shown in Figure 1.

235

GIANFRANCO GILARDI Protocol 17 continued

Equipment and reagents The mutant S337C is constructed, expressed, and purified as described in (1, 2). Dithiothreitol (DTT) is supplied by Sigma; prepare a stock solution 0.85 mg/ml. The source of the fluorophore IANBD (N-((2-(iodoacetoxy)ethyl)-N-methyl)-amino-7-nitrobenz-2-oxa-l,3-diazole) is Molecular Probes Inc. The IANBD stock solution is 0.9 mg/ml in dimethylformamide (DMF). The maltose is supplied by Sigma and the stock solution: 10 mM maltose in 10 mM potassium phosphate buffer, pH 7.0. Fluorescence emission measurements are made at room temperature on a standard fluorimeter, for example a Perkin-Elmer LS 50.

Method 1 Determine the concentration of the pure S337C mutant. This can be done spectrophotometrically using an s28o for mbp of 78,570 M^cm" 1 . 2 Work out the volumes so that 2 ml of protein are chosen to be labeled. 3 Incubate the S337C on ice for 30 min with five fold excess of dithiothreitol. This step is necessary to ensure that the cysteine is reduced and free to react with IANBD in the following step. We have seen a tendency of S337C to form dimers via intermolecular disulfide bonds. From the concentration of the protein, work out the volume of DTT to be taken from the stock solution. Typically this is around 20 |ol when a pure fraction of S337C is used directly after purification on a DEAE column as described in (2). 4 Add the appropriate volume of the IANBD stock solution to reach a 10-fold excess in relation to the protein. Usually this is around 100 |ol; larger volumes are not recommended because the IANBD is dissolved in DMF and this may affect the protein. Keep the mixture on ice for 30 min. This time is very crucial for efficient labeling of the protein and may need to be adjusted. Reproducible results are only obtained by control over the time and the temperature of this step. 5 After the reaction, promptly separate the excess free fluorophore by gel filtration on a Sephadex G25 column equilibrated with ice-cold 10 mM potassium phosphate buffer, pH 7.0. Perform this step in a cold room. 6 Collect 1 ml fractions, carefully keeping them on ice. 7 Measure the absorbance at 280 nm and 478 nm and plot an elution profile. The first peak that eluted in the void volume is the labeled S337C (called S337C-NBD), the second is the free fluorophore. 8 Calculate the molar ratio of fluorophore-to-protein (Cf/Cp) using the formula: C

f/ C p = (Anaxf

X

e280p)/(-A280 X emaxf -Amaxf X e 2 gof),

where the Cf is the molar concentration of fluorophore, Cp is the molar concentration of protein, Amaxf is the value of the absorbance maximum of the fluorophore (478 nm for IANBD, g2gop is the molar extinction coefficient of the

236

PROTEIN ENGINEERING FOR BIOSENSORS

Protocol 17 continued

protein at 280 nm, A280 is the total absorbance of the sample at 280 nm, gmaxf and fi2gof are the molar extinction coefficient of the fluorophore at the absorbance maximum and at 280 nm, respectively. In the case of IANBD bound to S337C, the relevant constants are: g280 S337C = 78,570/M cm; g280 S337C-NBD = 80,315/M cm; g478 IANBD = 26,000/M cm; g280 IANBD = 1745/M cm. 9 Select the fractions with Cf / Cp = 1 and pool them. These will be used for the fluorescence titrations with the ligand, maltose. 10 Adjust the concentration of S337C-NBD to be 1 |oM using 10 mM potassium phosphate buffer, pH 7.0. This is needed to allow the fitting to the model described by the hyperbolic binding curve described below. 11 Collect the emission spectra scanning from 500 to 600 nm upon excitation at 480 nm. An emission peak for the fluorophore will be observed at 530 nm. Store this first spectrum obtained in the absence of maltose. 12 Add aliquots of the 10 mM maltose stock solution in the same buffer by using a high-precision Hamilton syringe. Typical additions are 10 aliquots of 1 jol each, followed by 5 x 2 (0,1 and 3 x 10 |ol Collect and store the emission spectra at each step, but direct readings of emission at 530 nm are also adequate. 13 Plot the reading of the fluorescence emission at 530 nm against the concentration of maltose added in the cuvette. Fit the data to the equation: AFobs = AFmax[Mal]/(KD + [Mai]), where AF obs is the observed change in fluorescence intensity, AFmax is the maximum attainable change in fluorescence intensity, [Mai] is the molar concentration of maltose and Ku is the dissociation constant. A typical Ku for the S337C-NBD for maltose is 62 ± 0.2 |oM Figure 1. Proteins can also be created from first principle by solid state synthesis (38, 39). The four helix bundle fold is a popular target for the de novo design of model cytochrome and a range of binding proteins because of the relative simplicity of their intra-chain hydrogen bonding pattern and the well-understood hydrophobic effect that drives their folding. On the other hand, the progress in creating E-structures has been slower due to the complex long-range inter-chain patterns of hydrogen bonding that characterize this type of folding (40). A variety of programs are now available in guiding the rational de novo design of proteins, allowing to predict the folding of a particular sequence. An alternative approach to the de novo rational design of proteins is the combinatorial synthesis. Here libraries of peptides and proteins are generated either by chemical solid phase synthesis or by combinatorial synthesis of genes that are then expressed by Escherichia coli. For example, Haehnel and co-workers have developed a method for the combinatorial synthesis of four helix bundles attached to a cellulose membrane via a cleavable linker and assembled to a cyclic

237

GIANFRANCO GILARDI

peptide (41). The immobilized peptides can be easily screened for metal binding and redox activity for exploitation in electrochemical devices (42). Libraries of peptides folding into helical bundles can also be obtained with DNA methods using, for example, a binary pattern of polar and non-polar amino acids, in which the characteristic of the residue is specified but the exact side chain is varied randomly. This can be achieved by constructing a library of synthetic genes containing patterns of degenerate NAN codons (in which N denotes any base), where polar amino acids are required (lysine, histidine, glutamic acid, glutamine, aspartic acid, asparagine), and NTN where apolar amino acids are specified (methionine, leucine, isoleucine, valine, phenylalanine). This approach is successfully used by Hecht and co-workers for the creation of peptides with peroxidase activity (43). A whole range of possibilities are available for tagging proteins to facilitate their purification or immobilization or to obtain combined activities. Suitable plasmids allow to clone the gene of the target protein next to strings of typically 4-6 histidines (His-tags) or biotin-acceptor peptides, designed to be either at the N- or C-terminus of the protein. This has been used for a wide range of applications, for example, the immobilization of peroxidases (44, 45). On a larger scale, gene fusion is another method emerging in the bio-analytical area. Two examples of fusions are the green fluorescent protein and the maltose binding protein. These can provide useful systems for detecting gene expression or for immobilization on solid supports. The molecular Lego developed in our laboratory uses gene fusion to link electron transfer and catalytic modules of redox proteins and enzymes to build artificial electron transfer chain for amperometric devices (11, 12). Protein engineering has also contributed to achieve the controlled immobilization of protein on surfaces. This is a very important achievement for the rational design of sensing surfaces. Cysteine residues are very interesting targets for this purpose owing to their low occurrence in natural proteins and to the specific reactivity of the thiol of the side chain, in particular toward fluorescence and redox labels as well as gold surfaces. Hill and co-workers attached mutants of P450cam to gold surfaces via a unique, surface exposed cysteine (46). Scanning tunneling microscopy has proven to be an invaluable technique to study the coverage of proteins on atomically flat gold (47). The same approach has been used for other proteins such as azurin (48, 49).

6 Concluding remarks Proteins and enzymes are undoubtedly an attractive target for the analytical biotechnology area. Their dimensions are intrinsically in the nanoscale, they are formed by precise scaffolds folded to perform specific and often coupled functions such as binding, stereo-specific catalysis, pumping, and self-assembling. All these functions can be detected by an array of different biophysical methods that have made much progress allowing to reach the level of single molecule detection. This chapter has shown how the field of protein engineering 238

PROTEIN ENGINEERING FOR BIOSENSORS

has refined many different strategies to specifically tune or even create de novo the biological element to perform a wide range of desired functions imposed by the experimentalist. We are no longer limited by the properties or the availability of natural proteins. There is a very bright future for protein engineering in nanobiotechnology.

Acknowledgments I would like to acknowledge all the members of my group, past and present. Particular thanks to Professors Gerard Canters and Tony Cass for introducing me to the fields of protein engineering and biosensors.

References 1. Gilardi, G., Zhou, L. Q.., Hibbert, L, and Cass, A. E. G. (1994). Anal. Chem., 66, 3840-7. 2. Gilardi, G., Mei, G., Rosato, N., Agro, A. F., and Cass, A. E. G. (1997). Protein Eng., 10, 479-86. 3. Brune, M., Hunter, J. L., Corrie, J. E. T., and Webb, M. R. (1994). Biochemistry, 33, 8262-71. 4. Marvin, J. S. and Hellinga, H. W. (1998). J. Am. Chem. Soc., 120, 7-11. 5. Renard, M., Belkadi, L., Hugo, N., England, P., Altschuh, D., and Bedouelle, H. (2002). J. Molec. Bio!., 318, 429-42. 6. Schonbrun, J., Wedemeyer, W. J., and Baker, D. (2002). Curr. Opin. Struct. Bio!., 12, 348-54. 7. Risen, S. C. G. and Thornton, J. M. (2002). Curr. Opin. Struct. Bio!., 12, 374-82. 8. Hardin, C., Pogorelov, T. V., and Luthey-Schulten, Z. (2002). Curr. Opin. Struct. Bio!., 12, 176-81. 9. Guex, N. and Peitsch, M. C. (1997). Electrophoresis, 18, 2714-23. 10. Macarthur, M. W., Laskowski, R. A., and Thornton, J. M. (1994). Curr. Opin. Struct. Bio!., 4, 731-7. 11. Sadeghi, S. J., Meharenna, Y. T., Fantuzzi, A., Valetti, R, and Gilardi, G. (2000). Faraday Discuss., 116 135-53. 12. Gilardi, G., Meharenna, Y. T., Tsotsou, G. E., Sadeghi, S. J., Fairhead, M., and Giannini, S. (2002). Biosens. Bioelectron., 17, 133-45. 13. Tsotsou, G. E., Cass, A. E. G., and Gilardi, G. (2002). Biosens. Bioelectron., 17, 119-31. 14. Corpet, F. (1988). Nucleic Adds Res., 16, 10881-90. 15. Ravichandran, K. G., Boddupalli, S. S., Hasemann, C. A., Peterson, J. A., and Deisenhofer, J. (1993). Science, 261, 731-6. 16. Li, H. Y. and Poulos, T. L. (1997). Nature Struct. Bio!., 4, 140-6. 17. Williams, P. A., Cosme, J., Sridhar, V., Johnson, E. F., and McRee, D. E. (2000). Mo!ec. Cell, 5, 121-31. 18. Connolly, M. L. (1983). Science, 221, 709-13. 19. Nicholls, A. and Honig, B. (1991). J. Comput. Chem., 12, 435-45. 20. Kunkel, T. A. (1985). Proc. Not!. Acad. Sci. USA, 82, 488-92. 21. Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987). Meth. Enzymol, 154, 367-82. 22. Taylor, J. W., Ott, J., and Eckstein, F. (1985). Nucleic Acids Res., 13, 8765-85. 23. Nakamaye, K. and Eckstein, F. (1986). Nucleic Acids Res., 14, 9679. 24. Higuchi, R., Krummel, B., and Saiki, R. K. (1988). Nucleic Acids Res., 16, 7351-67. 25. Sarkar, G. and Sommer, S. S. (1990). Biotechniques, 8, 404-7. 26. Derbyshire, K. M., Salvo, J. J., and Grindley, N. D. F. (1986). Gene, 46, 145-52. 27. Leung, D. W., Chen, and Geddel, D. V. (1989). Technique, 1, 11-15.

239

GIANFRANCO GILARDI 28. Cadwell, R. C. and Joyce, G. J. (1995). Mutagenic PCR. In PCR primer: A laboratory manual (ed. C. W. Dieffenbach and G. S. Dveksler, pp. 583-9, CSHL Press, New York. 29. Stemmer, W. P. C. (1994). Proc. Natl. Acad. Sd. USA, 91, 10747-51. 30. Stemmer, W. P. C. (1994). Nature, 370, 389-91. 31. Zhao, H. M., Giver, L, Shao, Z. X., Affholter, J. A., and Arnold, F. H. (1998). Nature Biotechnol, 16, 258-61. 32. Ostermeier, M., Shim, J. H., and Benkovic, S. J. (1999). Nature Biotechnol, 17, 1205-9. 33. Sieber, V., Martinez, C. A., and Arnold, F. H. (2001). Nature Biotechnol, 19, 456-60. 34. Petrounia, I. P. and Arnold, F. H. (2000). Curr. Opin. Biotechnol, 11, 325-30. 35. Cirino, P. C. and Arnold, F. H. (2002). Curr. Opin. Chem. Biol, 6, 130-5. 36. Joo, H., Lin, Z. L, and Arnold, F. H. (1999). Nature, 399, 670-3. 37. Farinas, E. T., Schwaneberg, U., Glieder, A., and Arnold, F. H. (2001). Adv. Synth. Catal, 343, 601-6. 38. Wilken, J. and Kent, S. B. H. (1998). Curr. Opin. Biotechnol, 9, 412-26. 39. Borgia, J. A. and Fields, G. B. (2000). Trend. Biotechnol, 18, 243-51. 40. DeGrado, W. F., Summa, C. M., Pavone, V., Nastri, F., and Lombardi, A. (1999). Annu. Rev. Biochem., 68, 79-819. 41. Rau, H. K, Dejonge, N., and Haehnel, W. (2000). Angewandte Chemie-International Edition, 39, 250-3. 42. Willner, L, Heleg-Shabtai, V., Katz, E., Rau, H. K., and Haehnel, W. (1999). J. Am. Chem. Soc., 121, 6455-68. 43. Moffet, D. A., Certain, L. K., Smith, A. J., Kessel, A. J., Beckwith, K. A., and Hecht, M. H. (2000). J. Am. Chem. Soc., 122, 7612-13. 44. Presnova, G., Grigorenko, V., Egorov, A., Ruzgas, T., Lindgren, A., Gorton, L, and Borchers, T. (2000). Faraday Discuss., 116 281-89. 45. Zhang, J. K. and Cass, A. E. G. (2000). Anal Chim. Acta, 408, 241-7. 46. Lo, K. K. W., Wong, L. L, and Hill, A. O. (1999). FEBS Lett., 451, 342-6. 47. Davis, J. J., Hill, H. A. O., and Bond, A. M. (2000). Coord. Chem. Rev., 200, 411-42. 48. Chi, Q. J., Zhang, J. D., Friis, E. P., Andersen, J. E. T., and Ulstrup, J. (1999). Electrochem. Commun., 1, 91-6. 49. Chi, Q.. J., Zhang, J. D., Nielsen, J. U., Friis, E. P., Chorkendorff, L, Canters, G. W., Andersen, J. E. T., and Ulstrup, J. (2000). J. Am. Chem. Soc., 122, 4047-55.

240

INDEX

Index Terms

Links

A ABD-F

114

124

ABD-N

112

114

determination of zinc concentration

123

Accelrys web site

189

acetone-soluble photoresist

158

acetylcholine

167

acousto-optical tunable filters acrylic resin

8

43 186

acryloylfluorescein

136

A.C.S. reagent grade buffer salts

117

active dyes

44

affinity sensors

10

agarose gel electrophoresis

210

AgCl-coated Ag wire

170

alamethicin

165

Albery, W. J.

87

alkyd resin

43

aluminium oxide, A12O3

42

53

amperometric enzyme electrodes

70

analytical protein engineering

228

143

ammonium ion

types of

58

110

acrylodan

amine amplification

123

122

ABD-T

esterase

119

89

69 187

This page has been reformatted by Knovel to provide easier navigation.

Index Terms anionic interferants anisotropy calibration of theory of determination of metal ions based on antibiotics, preparation of most commonly used antibody-antigen binding reaction

Links 9 124 111 220 91

apoCA

123

apocarbonic anhydrase

117

apolar amino acids

238

apoprotein, preparation of

116

aqueous microsensor suspension

139

Arnold, F. H.

231

aryl sulfonamides

111

affinity chromatography column ascorbate Ashworth, D.

116 9 87

asolectin

167

aspartic acids

142

Atanasov, P.

88

automated immobilization

44

azurin

123

238

B Bacha, S. Bacillus megaterium

87 199

232

haem domain of CYP102, and flavodoxin from Desulfovibrio vulgaris, ribbon diagram of complex between back-crossing

200 230

Bannon, T.

87

Bartlett, P.N.

59

BAS 100 Battaglini, F.

80

87

89

170 87

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

BCECF-AM (2′,7′-bis (2-carboxyethyl)-5(and, 6)carboxyfluorescein acetoxymethyl ester)

149

Beer-Lambert law

90

benzoin dimethyl ether

44

benzophenone

44

benzoquinones

44

Bergel, A.

87

Bicine binding agent Bioanalytical Instruments

116

150

121

43 170

BioDot Inc.

48

Biojet Quanti3000™

48

biological oxygen demand (BOD), quick estimation of

54

biological selection

230

bioluminescence

100

bioluminescent marine bacteria

100

biomimetic or molecular imprinting

54

biomimetic sensor

55

Bio-Rad Chelex-100

118

Bio-Rad metal-free pipet tips

118

biorecognition process and transduction mechanism, kinetics of biosensing

60 109

biosensors amperometric and potentiometric arrays design of made with polycationic redox polymers potentiometric and conductimetric Biosym/MSI

7 132 97 9 60 188

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

Biotech Validation Suite for Protein Structures

188

biotin-acceptor peptides

238

black lipid membrane (BLM)technique

154

literature

165

Blum, L. J.

97

bovine serum albumin (BSA)

34

Briggs, G. E.

61

105

buffers that contain metal ions such as Ca or Mg, preparation of

119

C CA

117

Ca(II)

111

119

calcein acetoxymethyl ester (calcein-AM)

148

Calvo, E. J.

87

Cambiaso, A.

87

carbamates

52

carbonic anhydrase

111

carbon paste (CP)

43

carbon paste electrodes (CPEs)

43

carbon-α three-dimensional models for the human CYP2D6 Caruana, D. J.

191 19

Cd(II)

111

cell array fabrication

148

encoding

147

viability

148

assay with BCECF-AM

150

test via pH sensitive nanospheres

150

cellulose acetate

46

ceramic

42

150

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

channel-forming peptides

165

chelating resin

118

chemiluminescence

101

chemiluminescent choline biosensor

107

Chen, Y.

87

chimeric DNAsequences, library of functional screening

230

chimeric genes

229

chip fabrication and pretreatment

157

chlorophenol sensor

107

cholera toxin

168

cholesterol

51

esterase

8

oxidase

8

choline oxidase

8

cholinesterase

53

clad, refractive indices of CnBr coupling

28 111

cobalt

43

Coche-Guerente, L.

88

Colicin N

174

collagen

101

177

102

colloidal silica

125

combinatorial cassette mutagenesis

224

combinatorial chemistry

153

combinatorial genetics

153

combinatorial synthesis

237

commercial coffee creamer

125

complementary DNA

202

composite electrodes, advantage of

10

131

Co(II)

membranes, enzyme immobilization on

180

2

This page has been reformatted by Knovel to provide easier navigation.

Index Terms Comtat, M.

Links 87

concentration change in, and net flux, schematic diagram showing relationship gradient conductive inks Connolly algorithm surface

83 64 43 193 199

contamination metal ions, preparation of solutions free of convection and biosensors

118 64

cosmids see plasmids Coulet, P. R. Coulter counter-like current modulations

97 164

coupled chemical reaction

68

coupled diffusion/reaction problem solving

74

Cramer, M. L.

109

Cranfield University

42

Crank-Nicholson (CN) method

85

creatinine

51

crosslinkers

4

crosslinking

1

75

Cu and other ions, determination using reagentless approach

123

Cu(II)

109

111

Cuvettes

127

CYP102

193

197

199

cysteine

238

337

234

cysteinyl residues

123

cysteinyl thiol

124

cystic fibrosis

167

This page has been reformatted by Knovel to provide easier navigation.

Index Terms cytochrome, human (CYP) P450

Links 92

231

enzymes

189

230

sequence alignment

190

232

D Danilowicz, C.

87

dansylamide (DNSA)

116

dansylaziridine

124

Darwinian evolution

224

Daunert, S.

147

dehybridization

145

dehydrogenases

101

Delfino, L. DelPhi

87 197

200

see also Insight II Deng, J. Q,

87

Denhardt's buffer

34

deoxynucleotides (dNTPs)

202

analogs

201

Desprez, V.

88

Desulfovibrio vulgaris

199

flavodoxin from

200

see also Bacillus megaterium dextran sulphate

38

dialysis

51

Diard, J. P.

88

Diaz, L.

87

2,4-dichlorophenoxyacetic acid

52

dielectric inks

43

116

diffusion and biosensors

64

limited first-order Langmuir model

36

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

digital simulation, use in electrochemistry

82

4-(4-(dimethyllaminopherylazo) benzoic acid) (DABCYL) dioleoyl phosphatidyl choline (DOPC)

145 174

1,2-diphytanoyl-sn-glycero-3phosphocholine (DphytPC)

167

180

dipicolinic acid (pyridine-2,6-dicarboxylic acid; DPA)

115

dipole interactions

20

directed evolution

187

protocols error prone PCR DNA shuffling

222

225 227

high throughput screening for NAD(P)H-dependent activity QuikChange® PCR mutagenesis steps of iterative cycles of

233 223 224

disposable biosensors

44

dithioerythritol (DTE)

124

DMF

114

DNA array fabrication detection

119

26 144

double stranded see dsDNA hybridization on solid surfaces

19

nonhomogeneous heat denatured sheared

34

polymerase

199

probe, non-fluorescent

144

sensor technology shuffling in directed evolution DNA-DNA interactions

203

213

54 227 144

DNAses

20

drier

43

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

Driscoll, B. J.

87

dsDNA

22

200

202

exponential growth as function of number of PCR cycles

205

DuPont Ludox

125

–

201

DUTPase(dut )

E Eadie-Hofstee plots

63

electrocatalytic activity of redox hydrogels and “wiring” of enzymes electrochemical-based detection of hybridization

2 26

electrochemical cell, two-electrode with Ag/AgCI counter electrode and gold working electrode, schematic view

175

electrochemical systems method, digital simulation of electrochemiluminescence (ECL)

86 97

fiber-optic biosensor based on

107

measurements, flow system for

100

101

electrons conducting redox polymers in biosensors

1

diffusivity of

2

transfer between enzyme and polymer redox centers

4

electrophoretic vesicle positioning in two different configurations

164

electroreduced chemicals

1

electrostatic interactions

20

electrostatic potentials electroxidized biochemicals, examples of

196 1

This page has been reformatted by Knovel to provide easier navigation.

Index Terms enzyme electrodes

Links 4

basic electrochemistry

63

behavior model

66

8

immobilization on collagen membranes

102

on preactivated polyamide membranes

102

kinetics

60

data analysis

62

equilibrium and steady state

60

membrane|electrode problem, case diagram for

80

overcoating with membrane

8

wiring redox polymer

4

enzyme-based sensors enzyme-containing redox hydrogels enzyme-labeled microspheres enzyme-mediator kinetics

134 1 141 89

enzyme/pH array, fabrication of

137

enzymes covalent immobilization

142

with high chemical and thermal stability

222

immobilization

103

with glutaraldehyde

142

by physical absorption

141

loading of IDA sepharose beads sensors, disposable enzyme-substrate (ES) complex

104 41 61

kinetics

89

epoxy resin

43

Eppendorf Microfuge tubes

118

EPR spectroscopy

234

Epstein, J. R.

131

error prone PCR

224

in directed evolution

74

226

225

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

Erythrosin B

127

Escherichia

232

Escherichia coli

174

XL1-Blue

230

codon usage by

200

237

206

Escherichia.coli dam

+

–

Escherichia coli, dut ung

221 –

201

ETH 1001

173

ethanol

119

ethanolamine

114

ethidium homodimer-1 (EthD-1)

148

ExacTech excitation light introduced into fiber

42 132

external encoding in fluorescence based fiber-optic biosensors

140

F Feldberg, S.W.

82

ferrocene/ferricinium ion couple

41

ferrocenes

44

enzymatic electrochemistry mediated by fiber optic biosensors advantages bio-, chemi-, and electrochemiluminescence for

42 132 151 97

enzymatic

136

fluorescence-based

131

light-emitting reactions

100

preparation of sensing layer immobilization techniques

101

protocols chemical modification of HRP with His-tag

104

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

fiber optic biosensors (Cont.) enzyme immobilization on collagen membranes 102 enzyme loading of IDA sepharose beads

104

preparation of sensing layer with photocross-linkable polymer (PVA-SbQ)

104

sensing tip of

98

setup

98

fiber-optic based arrays of cells

147

fiber-optic microarray with molecular beacon (MB) functionalised microspheres fiber polishing

147 133

Fick first law

64

second law

66

Ficoll Fierke, C.

34 119

filter hybridization studies firefly

84

34 38 100

fish freshness estimation

52

Fitzgerald, C. A.

87

flavodoxin

199

Fleming, M. S.

131

flow-cell detection system

91

Flow Injection Analysis (FIA)

98

measurements in a batch system

98

in a flow system

98

fluorescein

126

136

145

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

fluorescence intensity of apo-CA

120

lifetime

113

sensing

110

measurements and imaging instrumentation used for

134

microscopy

109

polarization immunoassay

109

fluorescence anisotropy of apo-CA plus ABD-N as a function of free Zn(II) concentration

121

of apo-N67C-ABD-T as a function of the concentrations of Cu(II), Zn(II), Cd(II), Ni(II) and Co(II)

123

determination of metal ions by

109

microscopy

110

fluorescence based fiber optic biosensors

137

141

protocols acryloylfluorescein, preparation of

137

amine amplification with PEI

143

cell viability: assay with BCECF-AM

150

test via pH sensitive nanospheres

150

chemical etching of germania-doped imaging fiber bundle

139

enzyme immobilization with glutaraldehyde

142

by physical absorption

141

external encoding

140

fabrication of enzyme/pH array

137

of fiber-optic based living array of cells

148

internal encoding

140

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

fluorescence based fiber optic biosensors (Cont.) PAN-enzyme sensors

135

periodate oxidation

143

preparation of molecular beacon-modified microspheres

146

silanization/functionalization of fiber tip

135

ssDNA probe labelled microspheres, preparation of

145

fluorescent aryl sulfonamide

123

for zinc(II) determination

114

fluorescent calcein

149

fluorescent dyes

145

fluorescent sulfonamide

123

fluorophores

127

147

flux diagrams for membraneenzymeelectrode, simplified

70

of different kinetic processes for amperometric membrane| enzyme|electrode

70

“flyers”, identification of

127

formaldehyde

25

formamide

36

Förster transfer

123

Fox, R. O., Jr.

166

free amine groups on a microsphere surface

142

free radical polymerization

136

free zinc concentration

119

functional characterization of mutants

234

functional screening

38

2224

fused quartz

127

fused silica core

131

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

G Gajovic, N.

88

gel filtration

116

gene fusion

238

generalized biosensor, schematic diagram of

90

Genies, E.

87

Georgiev, T.

88

germania-doped imaging fiber bundle, chemical etching of

139

germania doped silica cladding

138

G-factor

127

giant unilamellar vesicles

160

formation of Gilardi, G. Gillespie, D. Giovangrandi, L

157

188

161

185 25 153

glass fiber

42

glass fiber-optic bundle

97

glucoamylase activity

52

production, control of

52

glucose blood, pocket meter electrooxidation monitors, personal

52 51 3 42

oxidase

5

sensors

46

glutamic acids

142

glycogen

125

glycols glycoproteins

107

23 103

This page has been reformatted by Knovel to provide easier navigation.

Index Terms glyoxal (ethanedial)

Links 25

gold electrode tethered lipid bilayers

168 179

surfaces

168

Gooding, J. J.

87

graphite screen-printed electrodes

54

Grattarola, M.

87

gravimetric sensors

60

green fluorescent protein

87

Gros, P.

87

Guiseppi-Elie, A.

177

238

238

Greer, J. C.

Gryczynski

174

110 88

H Haehnel, W.

237

haem-thiolate monoxygenases

189

Haldane, J. B. S.

61

Hall, E. A. H.

87

Hamamatsu R928 PMT

127

Hanes plots

63

Hanke, W.

167

heavy metals

117

Heller, A. HEPES sodium sulfate

1 121 116

Hibbert, D. B.

88

Higgins, I. J.

42

Higuchi, R.

202

Hill, H. A. O.

238

Hindle, A.

231

87

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

4-6 histidines (His-tags)

103

holoprotein

116

hopscotch method horseradish peroxidase (HRP)

8 104

mutant Asn255Asp (HRP1A6)

231

Huang, T.

238

86

with His-tag, chemical modification of

HPLC grade water

177

10

125 88

human cytochrome see cytochrome human diseases, point mutation Human Genome project human serum albumin antigen-antibody complex

54 144 92

Hunt, J. B.

115

hybrid lipid layer description

170

hybridization base mismatch

33

of DNA on nitrocellulose membranes

27

kinetics at solid surface

31

mass transport

34

nonspecific adsorption

34

at oligonucleotide sensitive electrodes

19

process of

38

of redox hydrogel bound DNA sequence

11

sensors

10

sequence discrimination

37

target, double stranded

38

temperature

31

hydrated silica matrices

7

hydrogen bonds

20

electrode

41

peroxide

10

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

hydrophobic interactions

20

hydroxides

44

I IANBD

185

thiol-reactive fluorophore

234

imaging fiber bundle

132

iminodiacetate residues

118

136

immobilization of “His-tag” enzymes

103

of large proteins

142

of peroxidases

238

via polymer photodeposition

136

impedance modules

170

spectral analysis

169

spectroscopy

153

basics of

168

data

172

measuring technique and electrochemical cell

170

of tethered lipid membranes

168

ink jet printing technique

44

48

inks typically used for screen-printed biosensors Insight II

45 188

193

196

internal encoding in fluorescence based fiber-optic biosensors

140

in vitro DNA synthesis

201

in vitro evolution process

230

in vivo glucose sensors

10

in vivo selection steps

201

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

ion channels activity electrical recordings of

153

in tethered lipid membranes on gold electrodes

154

self-integrating ionic strength

32

ionophore

173

ion selective electrodes

109

38

Iteractive Truncation for Creation of Hybrid Enzymes (ITCHY)

229

J Jobst, G.

88

Joslin, T.

86

K Kalia, Y. N. Kawski

87 124

kidney dialysis

51

kinematic viscosity

65

kinetic modeling of biosensors

59

applications

89

list of symbols

92

of enzyme electrodes, summary of literature on

87

immunosensors

91

optical and photometric biosensors

90

potentiometric enzyme electrodes

90

kinetic models case diagrams

79

deriving complete

78

experimental verification of This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

kinetic modeling of biosensors (Cont.) approximate analytical simplifying assumptions

81 70

Kirtikara, K.

88

KM significance for biosensor applications

63

Kong, J. L

87

Krishnan, P.

88

Krull

31

Kunkel method

200

Kunkel, T. A.

201

Kurganov, B. L.

87

L Labbe, P.

88

labelling enzyme

10

lactate

51

lactate oxidase

107

6

Langmuir model

36

Langmuir-type isotherm

36

light-emitting bi-enzyme system of marine bacteria

101

Lineweaver-Burk plot

62

lipid bilayer selective antibody binding to SLIC in

180

for single-channel experiments, highly insulating, tethered

179

tethered via surface-attached proteins description

177

impedance spectroscopy data

179

liquid core fiber-optic

98

Liu, H. Y.

87

live-cell array fabrication

146

This page has been reformatted by Knovel to provide easier navigation.

Index Terms LIVE/DEAD Viability/Cytotoxicity Kit

Links 148

luminescence and fiber-optic biosensors, enzyme immobilization on membranes Immunodyne ABC type from Pall-Gelman on Ultra Bind type membranes

103 103

luminescence sensors analytes involving H2O2 detection

107

dehydrogenases as auxiliary enzymes analytes using

105

determinations with

105

oxidases as auxiliary enzymes

107

luminol chemiluminescence luminometer Luria-Bertani (LB) broth

107 97 218

Lyons, C. H.

87

Lyons, M. E. G.

87

lyophilization (freeze-drying)

114

M Magner, E.

87

Maines, A.

87

7-(5-maleimidyl)-pentylaminobenz-2oxa-1,3-diazole-4-sulfonamide) Maliwal, B. maltose maltose binding protein (MBP) mutant S337C

123 109 52 238 185

manganese

43

Marchesiello, M.

87

marine bacteria Martens, N.

234

101 87

This page has been reformatted by Knovel to provide easier navigation.

Index Terms mass transport Mayer, M.

Links 63 153

Mears, D. J.

88

mediated enzyme electrode

41

mediator-based sensors Medisense Inc.

1 46

megaprimer

212

melittin

165

178

membrane |enzyme|electrode configuration

89

flux diagram for

70

flux equations, solution of

73

problem solving

74

Menten, M. L. β-mercaptoethanol

60 124

metabolites measured with screen-printed sensors metal ions

51 5

contaminants, removal from solutions and buffers contamination, avoidance of

119 117

and fluorescence anisotropy, protocols ABD-N, synthesis of

115

anisotropy, measurement and calibration of apoprotein, preparation of

128 116

contamination metal ions, preparation of solutions free of

118

reagentless approach, determination of Cu and others using a

124

zinc concentration determination with ABD-N

122

This page has been reformatted by Knovel to provide easier navigation.

Index Terms Mg(II) Michaelis constants in conventional enzyme kinetics

Links 111 8 73

Michaelis, L.

60

Michaelis-Menten enzyme kinetics

60

one-dimensional case diagram for

79

equation, linear plots derived from

63

micromembranes, reproducible microspheres sensors

ssDNA probe labelled

145

migration and biosensors MINEQL

79

139 146

array platform preparation

75

8

molecular beacon-modified

microwells

91

132 138 64 121

modelling kinetic

69

purpose and practice of

59

molecular beacons

145

molecular diversity

224

molecular evolution, natural

222

molecular probes

148

monochromatic laser sources

125

monoclonal antibody Sp3E9

178

monolayer of thioalkanes

168

MOPS

121

Moser, I.

88

Mullard Prize

42

multi-analyte arrays

136

sensing

132

multipurpose biosensor

52

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

mutagenesis

185

mutagenic oligonucleotide

200

primers, design of

205

N NAD(P)H

100

231

dependent activity in directed evolution, high throughput screening for dependent enzymes Nafion

233 232 46

NAN codons nanocomposite enzyme electrodes nearest neighbour interactions Neher, E.

238 2 22 154

Nernst diffusion layer

73

equation

90

Neykov, A.

88

Ni(II)

111

nicotinamide adenine dinucleotide (NADH) oxidation at poly(aniline) electrodes

81

see also NAD(P)H Nigretto, J. M.

87

NIH 3T3 mouse fibroblast cells

148

nitrilotriacetic acid

121

177

26

42

nitrocellulose NMR spectroscopy

234

N-succinimidyl esters of carboxytetramethylrhodamine (TAMRA) functionality

140 135

This page has been reformatted by Knovel to provide easier navigation.

Index Terms NTN

Links 238

nucleic analogs

20

nucleotide duplex, structure of

20

numerical methods, explicit

82

numerical simulation methods

82

O oligonucleotide probes attachment to gold by chemisorption

27

in base pairs, length of

23

deposition onto a polymer coated electrode non-covalent or physisorption

28 27

protocols attachment of ligonucleotides

28

CnBr activated coupling to an edge plan graphite electrode

29

determination of solution concentration of single stranded oligonucleotide and assessment of purity of oligonucleotide

26

reactive electrophoretic deposition

30

oligonucleotides arrays

207 144

chemical attachment

28

devices

21

hybrids, stability of

36

primers

212

224

strands, hybridization of two complementary

21

This page has been reformatted by Knovel to provide easier navigation.

Index Terms oligonucleotide sensitive electrodes

Links 21

determination of meltingtemperature

32

function of

20

hybridization conditions

30

efficiency and sensitivity

21

OmpF

168

n i lipid bilayers

177

trans-membrane protein

174

“one-shot” biosensor optical fiber based DNA sensors optical sensors optical transduction mechanisms

42

44

131

133

144 60

104

131

optical waveguide

97

organic solvents

43

organophosphate pesticides

52

orthogonal collocation method

86

Os-complex mediator

52

osmium

175

4

oxidation polymerization

44

oxygen electrode

41

P P450 see cytochrome PAN see poly (acrylamide-co-Nacryloxysuccinimide) patch clamp on chip, concept of

154

patch pipet

154

PCR without added primers

227

amplification procedures

144

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

PCR without added primers (Cont.) experiment, schematic diagram of

213

megaprimer method

212

methods

200

site-directed mutagenesis

202

thermal cycles

205

penicillin

137

Penicillium chrysogenum

137

peptide nucleic acids (PNA)

20

32

peptides folding into helical bundles

238

periodate oxidation fluorescence-based fiber-optic biosensors

143

peroxidases

10

peroxides

44

Perrin equation

112

pesticides

52

Pfeiffer, D.

87

PfuTurbo DNA polymerase pH range

114

126

221 36

phenanthroline

115

phenolic resin

43

Phloxine B

127

phospholipid (POPC)

171

174

phosphorothioate method see Kunkel method Photinus pyralis

100

photocross-linkable polymer (PVA-SbQ), preparation of the sensing layer with, fibre-PLB see planar lipid bilayers photoluminescence techniques

109

photomultiplier tube (PMT)

127

of luminometer

97

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

photosensitive agents

44

planar bilayers on chip

157

165

planar lipid bilayers (PLB)

153

156

formation of

157

planar patch clamp

153

principle of

165

155

protocols dry cleaning and activation of silicon chips

158

fabrication of silicon chips

157

fabrication of Sylgard® pads

165

giant unilamellar vesicles, preparation of

161

PLBs, formation on silicon chip

162

surface modification of silicon chips

160

wet cleaning and activation of silicon chips

159

planar silicon microchips

154

plasmids and cosmids, most commonly used

221

Plasmodium falciparum

178

PLB see planar lipid bilayers Fletcher, D.

86

Poisson-Boltzmann algorithm

197

polar amino acids

238

poly(4-vinylpyridine) (PVP)

3

poly (acrylamide-co-N-acryloxysuccinimide) (PAN) 135 enzyme sensors

135

gel immobilization

135

polymer-based pH sensor

136

polyamide, synthetic-like derivatized

101

polycarbonate

42

polychlorinated biphenyls

52

poly(dT)

23

poly-ethyleneimine (PEI)

142

poly-L-lysine

160

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

polymer microspheres

142

photodeposition

136

polymerase chain reaction see PCR polynomial curve fitting poly(N-vinylimidazole) (PVI)

86 3

polyurethane resin

43

poly(vinylalcohol)

101

bearing styrylpyridinium groups (PVA-SbQ) polyvinylpyrrolidone

104

107

34

POPC see phospholipid Pratt, K. F. E. primers

87

89

207

printing process

48

probe attachment of

25

length and orientation

22

oligonucleotides hybridization kinetics

36

structure and dynamics

22

progesterone

51

protein-DNA interactions

144

protein engineering for biosensors

185

187

189

191

234 protocols calculation of a map of electrostatic potentials on a protein surface creation of three-dimensional models

197 191

fluoresence labelling of the S37C mutant of the maltose binding protein

235

This page has been reformatted by Knovel to provide easier navigation.

197

Index Terms

Links

protein engineering for biosensors (Cont.) molecular graphics, display and basic calculations on protein structures protein sequence alignment

194 189

proteins controlled immobilization on surfaces

238

modeling and calculations on protein structures

188

physical adsorption

141

rational design of, de novo

237

Pseudomonas putida PVC

231 42

pWhitescript™ plasmid

221

Pyrococcus furious

202

Q quaternary ammonium salts

33

Quickchange™ PCR method

201

221

228

mutagenesis in directed evolution

223

schematic procedure for

222

R Ramachandran plots

188

Raman band of water

125

scatter

125

127

signal excitation in UV 125 random collision between two complementary strands random mutagenesis, error prone PCR Randriamahazaka, H.

20 224 87

This page has been reformatted by Knovel to provide easier navigation.

Index Terms rational design flow chart showing experimental stages

Links 199 187

rational protein engineering

187

Rayleigh scatter

127

reaction schemes for CNBr activated coupling and electrophoretic deposition on electrodes “reagent” approaches

29 111

“reagentless” anisotropy-based material ion determination approach determination of Cu and others by using a

113 112 124

reassociation

33

reciprocal expressions, advantages of using

73

recombinant DNA technology

185

recombination

224

DNA shuffling

38

226

redox hydrogel based electrochemical biosensors

1

containing immobilized enzymes, cross-linked

75

redox polymer-enzyme system microscopic homogeneity and salt effects in

6

optimal compositions

6

redox polymers

1

crosslinked

2

and their electrochemistry

3

and enzymes

7

redox proteins

199

Rhodamine-based dyes

140

238

This page has been reformatted by Knovel to provide easier navigation.

Index Terms Rhodes, R. K. Richards, F. M.

Links 87 166

RNAses

20

RNA sequence

11

rotating disc electrode

65

rotation speed

65

rotational correlation time

68

113

S Sakmann, B.

154

SAMCO “Pasteur” pipets

118

Sansom, M. S.

167

saturated calomel electrode (SCE)

4

Schauer, C. L.

131

Scheller, F. W.

88

Scheller

41

Schlue, W. R.

167

Schulmeister, T.

87

screen-printed biosensors, applications

51

in clinical diagnosis

51

in environmental monitoring

52

in food analysis bioprocess control

52

and their performances

53

screen-printed lactate sensor

52

screen-printing apparatus

46

for biosensors diagram of preparation

41 48

protocol for screen-printed glucose electrode, preparation of

50

process

46

technology

42

This page has been reformatted by Knovel to provide easier navigation.

Index Terms segmental mobility

Links 1

semipermeable membranes

46

sensing element

44

sensor examples, specific

7

measuring turnover rate of hydrolytic and other non-redox enzymes made with diffusional mediators “self-encoding”

8 9 140

Sequence Homology Independent Protein Recombination (SHIPREC)

229

sequential chemisorption reactions of polymers

8

Sheppard, N. F.

88

Shults, M. C.

87

silanization/functionalization of fiber tip

135

silanized oxide surfaces

168

silica core chemically etched

138

silicon chip for planar patch clamp measurements, schematic diagram of

156

single analyte sensing

132

134

single stranded DNA see ssDNA single stranded oligonucleotides attached to surface, schematic of

24

complementary, equilibrium constant for hybridization process for two synthetic

21 54

single-channel conductance

167

recording of alamethicin pore, chip-based

166

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

SiO2

157

site directed mutagenesis

199

PCR method protocols hot start PCR

207

megaprimer PCR

213

purification of PCR products from agarose gels

211

submarine agarose gel electrophoresis of DNA

209

protocols isolation of plasmid from bacterial host

218

ligation of DNA insert in a vector

216

restriction digest of DNA

215

transformation of plasmid in bacterial host schematic diagram for site-directed mutants

217 200 187

sodium chloride

32

Solartron 1296

170

sol-gel method

7

solid state synthesis, proteins created by Somasundrum, M. sorbitol determination

237 88 106

Sorochinskii, V. V.

87

Southern blots

27

soybean peroxidase (SBP)

10

Sp3E9 spacers, amino-based Spectrosil Speigelman, S. SR 850

35

179 23 127 26 170

This page has been reformatted by Knovel to provide easier navigation.

Index Terms ssDNA (single stranded DNA)

Links 22

artificially synthesized

145

native

144

template

202

Staggered Extension Protocol (StEP)

228

Stanford Research Systems

170

starch

52

steady-state enzyme kinetics

60

Stemmer, W. P. C.

226

200

232

230

StEP see Staggered Extension Protocol steric hindrance to hybridization

22

steroids

231

Stitzel, S. E.

131

Stora, T.

153

Stratagene

221

substrate electrooxidation or electroreduction matrix

2 42

sucrose

52

sulfite

53

sulfonamide, affinity constant of

112

sulfonyl halide

114

supported lipid monolayers

168

Suprasil

127

surface concentration of single stranded oligonucleotides

22 23

surface plasmon resonance (SPR) technique

91

SWISS-MODEL, three-dimensional model built by Swiss-PdbViewer

188 188

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

Sylgard® pad

157

synthetic ligand-gated channel (SLIC)

168

178

180

T T4 DNA polymerase

200

Tan, T. C.

87

Tanticharoen, M.

88

Taq polymerase

224

Tatsuma, T.

87

TCEP (tris-(2-carboxyethyl)phosphine, hydrochloride)

124

Tebbutt, P.

87

Terrettaz, S.

153

Terrific Broth (TB)

218

tethered lipid bilayers

168

description

174

impedence spectroscopy data

176

protocols Ca2+ -sensitive hybrid lipid layer electrode, preparation of

172

OmpF-containing lipid bilayer tethered on a gold electrode, preparation of

175

proteolipid bilayer tethered by SLIC ot gold electrode, formation of tetradecanethiol tetraethylammonium T-format optical configuration

178 171 33 110

Therias, S.

88

thermal curing

43

thermal polymerization

44

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

Thermococcus litoralis

202

Thermus aquaticus

202

thioalkane

171

thiol-specific fluorophores

185

Thompson, R.

109

174

three-dimensional model of target protein, construction of

188

three-electrode system sensor

48

Toh, C. S.

59

Tongta, A.

88

toxicity screen of antiseptics and antioxidants transducer

147 89

transition metal ions

9

Turner

42

two photon excitation

110

two-electrode configuration sensor

48

electrochemical cell, schematic view of Tyrrell, C. H.

171 87

U ultraviolet (UV) light

136

curing process

44

inducible immobilization

43

Updike, S. J.

87

uracil-containing DNA

201 –

uracil N-deglycosidase (ung ) urate

234

201 9

Urban, G.

88

urea

51

uric acid

51

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

V Vadgama, P.

87

van der Waals interactions

20

Verreschi, G.

87

vesicles

161

see also giant unilamellar vesicles Vibrio fischeri

100

Vibrio harveyi

100

Vogel, H.

153

W Walt, D. R.

131

Warsinke, A.

88

Watanabe, T.

87

Watson-Crick hydrogen bonding

21

“wavelength-ratiometric” probes

110

Weber, G.

110

web sites for protein engineering and PCR applications, some useful Wilkins, E. “wired” redox enzymes, substrates of Wollaston prism

206 88 7 110

World Congress on Biosensors First

41

Wu, H. H.

88

X xenobiotics

231

XL-1 Blue

221

X-ray crystallography

234

structure for target protein, three-dimensional

193

This page has been reformatted by Knovel to provide easier navigation.

Index Terms

Links

Z Zeng, H. H.

109

Zhang, X. E.

41

Zhu, K.

88

zinc

43

buffers

121

from carbonic anhydrase (CA), removal of

115

chelator

115

concentration, determination with ABD-N

122

determination using reagent approach

119

ion buffer recipe

122

Zn(II) schematic of determination

112

116

111 120

This page has been reformatted by Knovel to provide easier navigation.