Chemical sensors and biosensors for medical and biological applications ursula e spichiger keller (wiley VCH, 1998)

Spichiger-Keller Chemical Sensors and Biosensors for Medical and Biological Applications... When applying biochemical or chemical sensors, the analytical process is straight- line, sin

Ursula E Spichiger-Keller

Chemical Sensors

and Biosensors for

Medical and Biological Applications

Further titles of interest:

W Gopel, J Hesse, J N Zemel (eds.)

Volumes 1-9

ISBN 3-527-26538-4

H Baltes, W Gopel, J Hesse (eds.)

Sensors Update

Volumes 1-3

ISSN 1432-2404

Ursula E Spichiger-Keller

Chemical Sensors

Medical and Biological Applications

Weinheim - New York Chichester

Brisbane - Singapore Toronto

Prof Dr Ursula Spichiger-Keller

Zentrum fur Chemische Sensoren/Biosensoren

und bioAnalytische Chemie

Departement fur Pharmazie

Library of Congress Card No applied for

A catalogue record for this book is available from the British Library

Die Deutsche Bibliothek - CIP-Einheitsaufnahme

Spichiger-Keller, Ursula E.:

Chemical.sensors and biosensors for medical and biological applications /

Ursula E Spichiger-Keller - Weinheim ; Wiley-VCH, 1998

ISBN 3-527-28855-4

0 WILEY-VCH Verlag GmbH, D-69469 Weinheim (Federal Republic of Germany), 1998

Printed on acid-free and low chlorine paper

All rights reserved (including those of translation in other languages) No part of this book may be repro- duced in any form - by photoprinting, microfilm, or any other means - nor transmitted or translated into machine language without written permission from the publishers Registered names, trademarks, etc used

in this book, even when not specifically marked as such, are not to be considered unprotected by law Printing: strauss offsetdruck GmbH, D-69509 Morlenbach

Bookbinding: Wilh Osswald + Co., D-67433 Neustadt

Printed in the Federal Republic of Germany

care testing (POCT) in medicine An unexplored area is the use of chemical sensors in toxicology In order to cope with various fields of applications, the brand "the Lab in the Bag" was coined specifying the trend of further developments

Several comprehensive volumes on chemical sensors had been published However most of them are more focused on the development of the physical part, the transducers This volume intends to provide an overview on the variety of chemical sensors focusing on analytical- chemical aspects generally, and on biological applications specifically The field of chemical sensors could be depicted as a space which is spread by 3 coordinates: the biological or life sciences along one axis, physical-chemistry and chemistry along another, and mathematics and statistics along the third axis This %pace'' reflects the complexity of the field This volume tries

to take sufficient account of each axis and gives an overview of the field with special focus on the developments in the goup of Prof W Simon, Laboratory for Organic Chemistry, involving the habilitation thesis of the author, and on developments in the Centre for Chemical Sensors/Biosensors and bioAnalytical Chemistry at ETH Ziirich-Technopark Each chapter is devoted to a separate theme So the references have been inserted after each thematic block or

chapter, beginning with chapter 1 Each thematic block or section is closed by conclusions

In the first chapter, the question as to whether chemical sensors and biosensors have to be differenciated is discussed In the course of this chapter, chemical sensors are defined and related to particular areas in analytical chemistry A brief history of the field is given describing the development of chemical sensors This is followed by a discussion of market trends and

comments on possible future developments of the general situation in analytical laboratories

The second and third chapter sets out to give an overview on the chemical and physico- chemical principles underlying the preparation of chemical and biochemical sensors These chapters cope with the modelling of interactions, the investigation of interactions, and the basic theories underlying a reversible response which enables continuous monitoring An understanding of these principles is assumed in chapter five and six, where some sensors developed and tested by the author's own research group are presented In many cases, only a brief description is given, but this is compensated for by the provision of extensive references

A major subject of the author% research has been the investigation of the influence of the medium, the bulk of the sensing layer, incorporating the active compounds (chapter 4), and the development of the magnesium-selective electrode so that it can be routinely used in plasma and whole blood Major efforts were devoted to the synthesis of the magnesium-selective ionophore

VI Preface

ETH 5506 in order to make this ligand accessible as ETHT 5506 to industrial production

The seventh chapter discusses the problems of reliability and interpretability of results In all

fields of analytical chemistry, these are at least as important as the development of new methods and procedures Several sections focus on decision and discrimination problems analogous to analytical data treatment in medicine, in order to solve decision problems in general analytical chemistry The author's experience with quality control and discrimination analysis is referred

to

h the interests of completing this book, it has not been possible to go into great detail about the experimental conditions and fundamental explanations for all results presented However, many of these can be found in the references provided In selecting topics, I was governed by a

desire to cover those which fill a gap in the existing comprehensive volumes of other authors In addition, these topics provide insights into the actions of specific sensors, which illustrate their characteristics in detail, and which show the differences of basic concepts

I would like to dedicate this book first to the memory of the late Prof Wilhelm Simon in recognition of his outstanding contribution to the field It was in his laboratory that I realised that productive research is, among other things, the reflection of personal and scientific discipline, the unguarded exchange of ideas and daily critical discussions In writing the Habilitation thesis,

I missed his critical comments and suggestions, and his sometimes strange, but always stimulating ideas

Secondly, I dedicate this book to those students and colleagues who are new to the field of chemical sensors and who will, I hope, find it a useful reference work The appendices, in particular, are intended to be helpful for those involved in the development and in practical applications of chemical sensors The appendices, specifically appendix 9, contain much information not easily available elsewhere

I would especially like to thank my assistants and my doctoral students for their collaboration and support They contributed to the writing of this book in many ways, not least, through their

Wiesli, Remo Wild and Bruno Rusterholz; Gudrun Rumpf, Aiping Xu, Ruedi Eugster, Ulrich Schaller, Erika Haase, Ulrich Korell, Daniel Freiner, Mathias Nagele, Daniel Citterio, Jurg Muller, Caspar Demuth, Alphons Fakler, Wei Zhang, Michael Linnhoff, Thomas Roth I am also grateful to my teachers, my colleagues and the postdoctoral fellows who had been working with me in the group, Dres Maria Csosz, Maria Bochenska, Nik Chaniotakis, Kemin Wang, Honbing Li, Peter Holy, Eva Vaillo, Luzi Jenny, Stefan Rasonyi and Gerhard Mohr for their contributions My special thanks go to Dr Silvia Dingwall who checked my English professionally, and Dr Markus Rothmaier who formatted this manuskript

This work was supported by the Swiss National Science Foundation, by the Swiss Commission for Technology and Innovation, the Swiss Priority Programmes "Optique" and

"MIOS", by AVL LIST GmbH, 8020-Graz, Austria, and by Orion Research, Inc., Beverly,

MA 02129, USA

Ursula E Spichiger, August, 1997

Contents

Preface v

1 Introduction 1

1.1 Chemical Sensors as Alternative Analytical Tools 1

1.2 The Concept of Chemical and Biochemical Sensors 6

1.3 Recognition Processes and Sensor Technology: Milestones 10

1.4 Goals for Future Developments and Trends 13

1.4.1 Trends 13

1.4.2 Miniatuization Nanotechnology 16

1.4.3 In Vivo and In Situ Monitoring 21

1.4.4 The Analytical Laboratory in the 21SfCentury 25

References 27

2 Chemical and Biochemical Sensors 33

2.1 Classification Specification and Nomenclature of Chemical Sensors 33

2 2 Molecular Recognition Processes for Ions and Neutral Species 38

2.2.1 Introduction 38

2.2.2 Molecular Interactions: Tools and Calculations 41

2.2.3 Molecular Recognition of Ions 48

2.2.4 Hydrogen Bonds 56

2.2.5 Molecular Recognition of Enantiomers 58

2.2.6 Molecular Interactions within the Aqueous Medium 59

2.2.7 Catalysis by Enzymes, Enzyme Mimics and Host-Reactands 63

2.2.9 Multitopic Recognition of Immunological Systems 71

2.2.10 Conclusions and Considerations for Ligand Design 74

2.2.8 Catalytic Antibodies 70

References 76

VIII Contents

3 Controlling Sensor Reactions 83

3.1 Thermodynamically Controlled Sensor Reactions: Reversibility and Thermodynamic Equilibrium 83

3.1.1 The Chemical Potential and the Partition Equilibrium 83

3.1.2 The Recognition and Transduction Process 93

3.1.3 The Electrochemical Potential and the @otentiometric) Sensor Response 100

3.2 Thermodynamics of Nonequilibria: D f i s i o n and Steady-State 104

3.3 Rate Controlled Sensor Reactions: Mediated Enzyme Reactions 106

3.4 Nonthermodynamic Assumptions 114

3.4.1 Activity Versus Concentrations 114

3.4.2 Ionic Strength and Estimates of Activity Coefficients 118

3.4.3 Activity and Concentration of an Electrolyte: IFCC / TUPAC Definitions 121

3.4.4 The Osmotic Coefficient 124

3.4.5 Calibration, Standardization and Comparison with Definitive or Reference Procedures 127

3.4.6 The Liquid Junction Potential under Physiological Conditions 134

References 136

4 The Artificial Analyte-Selective Membrane Limitations Technological Precautions and Developments 139

4.1 Introduction 139

4.2 Types of Membranes and Membrane Models 140

4.2.1 The Biological Membrane 140

4.2.2 MicialMembranes 144

4.3 The Selectivity Coefficient 155

Contents IX

4.4 The Membrane Composition and the Membrane Medium 161

4.4.1 The Influence of the Permittivity and of Plasticizers 162

4.4.2 The Effect of Electron Pair Donor (EPD) and Acceptor (EPA) Properties of Solvents Solubilization Properties of the Membrane 169

4.4.3 The Influence of the Aqueous Sample Environment 170

4.4.4 The Influence of the Surface Tension 172

4.4.5 The Effect of Lipophilic Anionic Sites 173

4.4.6 The Effect of the Ligand Concentration 176

4.5 Response Behavior Sensitivity and Detection Limit 179

4.6 Lifetime Lipophilicity and Immobilization 182

4.7 Interactions by the Biological Matrix and Precautions 183

4.7.1 Biocompatibility 183

4.7.2 Possible Mechanism of Protein Adsorption 185

4.7.3 Influence of Thrombocytes on Solvent Polymeric Membranes 188

4.7.4 The Donnan Potential 188

4.7.5 The Influence of Anticoagulants 191

References 193

5 Potentiometric Chemical Sensors and Biological Applications 199

5.1 Principles of Ion-Selective Electrodes 199

5.2 The Symmetric Potentiometric Cell 203

5.2.1 The Asymmetry of ISE Membranes and Reference'Electrodes 205

5.2.3 How About Human Whole Blood? 214

5.2.2 Analysis During Hemodialysis 211

5.3 The Magnesium-Selective Electrode 215

5.3.1 Characteristics of the Magnesium Ion 217

5.3.2 Analytical Techniques 217

5.3.3 Natural Carriers 222

5.3.4 Synthetic Carriers 225

5.3.5 Applications 237

5.3.6 Stop-Flow Analysis, the Continuous Flow System 239

5.3.7 Significance of Magnesium-Selective Assays 240

5.4 Microelectrodes for Intracellular Measurements 242

5.4.1 The Nitrite-Selective Microelectrode 245

X Contents

5.5 Miniaturized pH Probe for Intraluminal Monitoring of Gastric Juice 246

5.6 Chloride-Selective Measurements in Blood Serum and Urine 248

References 253

6 Optical Sensors Optodes

6.1 Introduction and Medical Applications 259

6.2 Sensors Based on Intrinsic Optical Effects of the Target Compound 260

6.2.1 Sensors Based on Inherent Optical Characteristics of a Specific 6.2.2 Sensors Based on Inherent Optical Characteristics of a Host Responding to Analyte Quantity with an Optical Effect 261

M y t e 260

6.3 Sensors Based on a Labeled Host Compound or a Labeled Competitive Analyte 269

6.4 Chemical Sensors Based on a Second Component: "Simon Optodes" 270

6.4.1 Chemical Principles of Operation 270

6.4.2 Optode Membranes for Cations 272

6.4.3 Optode Membranes for Anions 275

6.4.4 Optodes for Gases and Neutral Species 278

6.4.5 Principles of Reactions, Thermodynamic Equilibria and Response Functions 282

6.4.6 Medical Assays: Applications to Diluted Plasma 285

6.4.7 Analytical Performance Parameters 289

6.5 The Optical Transduction Process 299

6.5.1 Absorbance Measurements in Transmission Mode 299

6.5.2 Optical Transducing Elements Based on Multiple Internal Reflection(MIRE) 300

6.6 Trend to Miniaturized Integrated Optical Sensors (MIOS) 304

6.7 NZR-Absorbing Dyes 309

6.8 Conclusions: Electrodes versus Optodes Possibilities of Neutral Substrates 312

Contents XI

7 Data Validation and Interpretation 321

7.1 Introduction: What Does "Data" Mean? What Does "Information" Mean? 323

7.2 The Results of Analytical Tests: Random Numbers or Information Base? 323

7.2.1 Information Interpretation and Decision Making 323

7.2.2 What Does Information Mean? 325

7.2.3 The Bayesian Approach 330

7.2.4 General Validation of Clinical Tests and Analytical Results 331

7.2.5 ROC Analysis (Receiver Operating Characteristics) 334

7.2.6 The Likelihood Ratio 337

7.2.7 Multivariate and Clustering Procedure 339

7.3 Goals in Analytical and Clinical Chemistry 343

7.3.1 Analytical Errors and Biological Variation ; 344

7.3.3 Accuracy Assessment 352

7.3.4 Conclusions and Recommendations for Planning Diagnostic Tests 354

7.3.2 The Biological Scatterhg Range as the Dynamic Range 348

References 355

Appendices 359

Appendix 1: Appendix 2: Appendix 3: Appendix 4: Appendix 5: Appendix 6: Appendix 7: Appendix 8: Milestones in the Development of Chemical and Biochemical Sensors 359

Terminology for the Diagnostic Performance of a Test 361

Biological Setting Points for Electrolytes Allowable Analytical Errors for Electrolytes in Medical Assays Important Cations 367

363

365

Physicochemical Characteristics of the Five Biologically Structures and Physical Data of Plasticizers Nomenclature and Molecular Masses of Plasticizers Materials and Methods Used for Preparation of Ion-Selective 369

373

Electrodes and Synthesis of Hydroxy-Poly(viny1 chloride) 376

Appendix 9: Required Logarithmic Selectivity Coefficients for Ion-Selective

Electrodes 383 Appendix 10: Synthesis and Identity of the Ion-Selective Carriers ETH 7025

ETH 3832 and ETH 5506 used in this Work 387 Appendix 11: List of Structures and Selectivity Coefficients of Investigated

Magnesium-Selective Ligands 392 Appendix 12: IUPAC Units and Statistical Considerations 401

Index 405

Chemical Sensors and Biosensors for Medical and BiologicalApplications

Ursula E Spichiger-Keller copyright 0 WILEY-VCH Verlag GmbH, 1998

1 Introduction

1.1 Chemical Sensors as Alternative Analytical Tools

The technical potential of analytical chemistry has continued to grow over the past 30 years It has evolved from being a field little more scientific than alchemy to becoming an exact science with almost no limits to its applications

Analytical chemistry is the chemist's way of answering the question: "What's in it?" The

chemical components of a substance are determined through chemical analysis [ 11 The number

of chemical components which are identified by the analytical method depends on its resolution

or detection limit For example, with detection limits in the range 10-g-10-12 moles L-l, the

number of components in drinking water increases exponentially In practice, therefore,

analytical chemistry is mostly concerned with determining characteristic components of a

substance in order to answer specific questions and to yield specific information These

characteristic components are known as "analytes" or "laboratory parameters" when subjected to the analytical procedure and when listed in a report [2] In what follows, the originally chosen,

unchanged material is called the "specimen", whereas an appropriately representative portion of

a substance, which is fed to the analytical instrument after adequate pretreatment, is called the

"sample" [2] For each specimen there are certain typical chemical components or analytes about which the analytical chemist seeks chemical information

In analytical chemistry today, it appears that the most important decision is to select an appropriate, highly capable instrument, together with the necessary hard- and software, and to

.adapt the chemical procedure recommended by the standardization authorities Ideally, the

results flowing from the analyzer merely need to be collected and reported Although this may seem extremely direct, the time spent on collecting, transporting, and pretreating specimens causes a bottleneck in most analytical processes; in many cases this is not seriously addressed, and needs to be reduced Moreover, unstable analytes and analytes present at very low levels (ppb) are best analyzed on-site As a result, the laboratory has to move closer to the source of the

specimen, which means developing more user-friendly analytical instruments Along with innovations in analytical chemistry, social pressures from the environmental movement, and economic pressures arising from health care reform, have been responsible for many new

trends

Choice of the analytical instrument is important as it is the central element of the analytical

procedure and is routinely handled by analytical chemists and chemists The chemical and instrumental analysis is more likely to be limited by the chemistry of the specimen and its characteristic components than by the instrumental procedure For example the biological

matrix, specifically the protein content, has a crucial influence on the analytical procedure, owing

to interactions with reagents and adsorption on surfaces Special efforts are required to get rid of the effects of these interaction and to ensure that high-quality information is obtained by an appropriate analytical procedure The term "high-quality information" is not restricted to the uncertainty of results and aspects of quality assessment, but is primarily concerned with getting

Given this situation, an eclectic approach to chemical and instrumental specimen analysis seems most appropriate Used well, it should: (a) allow immediate on-site measurements; (b) eliminate matrix effects; (c) achieve high selectivity and user-friendly handling; (d) allow screening based on c e d e d cut-off limits; (e) expand the application area; (f) allow modification

of the methods or instruments A novel concept in analytical chemistry needs to be governed by

a strategy which involves and defines the necessary steps and procedures not the best possible

In addition, such a concept may not be oriented to increasing throughput, but, rather to increasing the efficacy of the analytical process (see below) Necessarily, such a concept must evolve not only from a profound and global insight into analytical processes, but also from a thorough understanding of the underlying chemical and physicochemical processes, and may be supplemented by chemometric approaches

1.1 Chemical Sensors as Alternative Analytical Tools 3

In conventional analytical chemistry, determining an analyte involves various steps (see also Figure 1-1 and [31):

1 Define the problem; 2 collect the specimen; 3 identify the specimen; 4 transport the

specimen to the laboratory; 5 select an appropriate method; 6 pretreat the specimen and prepare the sample; 7 perform the measurements; 8 compare with reference and quality control specimens; 9 calculate statistical parameters; 10 decide on the performance and reliability of the analysis; 11 transform data to give an interpretable value; and, 12 present the data

The complete procedure is challenging for the analytical chemist as it normally requires

considerable skill and a feeling for automation and robotics Often it is necessary to use several

different techniques and instruments in solving an analytical problem In between identifying the analyte and presenting the results, additional steps may be necessary, e.g., choosing and evaluating sophisticated additional separation steps or chromatographic columns, connecting specific detectors, specifying a flow-cell, or eliminating interfering solutes and solvents

Despite the skill involved in carrying out the complete analytical process, more and more analytical tests can now be carried out on-site rather than in a central laboratory Such front-line

analysis has ecological and economic advantages, such as:

- Eliminating the need to transport specimens, which is particularly problematic with unstable

- Reducing the effort required to identify the analyte, and to interpret and transmit the results

- Providing immediate answers to a problem

- Avoiding queues in the high-tech, central laboratory

- Stimulating thinking in terms of eflcucy, which may be defined either the number of true

positive results per total number of analyzed specimens or samples (positive efficacy) or the number of true negative results per total number of analyzed specimens or samples (negative

efficacy), in contrast to eficiency, which is the number of correctly allocated specimens per total number of analyzed samples, and throughput (total number of analyzed specimens per unit time) (see section 7.2.4 and Appendix 2)

analyteS

In order to tackle an analytical problem, the customer and analyst must agree on the information needed, and the specimen and sampling required to obtain it It is then up to the analyst to decide which procedure will be appropriate for dealing with the special properties of the specimen The two triangles in Figure 1-1 denote communication between the customer and the analytical laboratory which is catalyzed by the analytical chemist in the middle of the sandwich When applying biochemical or chemical sensors, the analytical process is straight- line, since sensor technology allows the analyte in a specimen to be quantified directly Thus, the analyst can avoid having to transport the specimen, pretreat it, and prepare a sample (at least steps 4 to 6 above) By using sensor technology, the steps in the first part of the data evaluation process can be reduced in principle to just one, namely, the interaction between the sensing element or sensor surface and the specimen However, in many cases pH- and/or ionic strength

buffering of the specimen (conditioning) is recommended in order to improve accuracy, which

means making use of a continuous flow system Instead of having to choose the chemical procedure and the detecting system, the required selectivity and detection limit have to be estimated, and the limiting operational conditions need to be considered when defining the

4 I Introduction

analytical problem The more selective the sensor, the more dedicated the system, and, as a result, the cheaper and easier it is to use If the technology applies a couple of poorly selective sensing elements arranged in a sensor array, it may be necessary to resort to sophisticated exterior data processing in order to interpret the measured values and to ensure that the sensing system was accurate Sensor technology can be particularly useful and preferable to well- established analytical procedures in testing situations where:

- Continuous or periodical testing by laypeople is necessary

- There is a shortage of skilled manpower and/or natural resources

- Working with chemical reagents is avoided

- The analytes are not stable and quick answers are required

- Front-line screening saves on resources for economic and/or ecological reasons

In order to improve the analytical methods involving sensor technology and the evaluation of

their results, it will be necessary, in the future, to find ways of: (a) ensuring adequate sensitivity

and accuracy; (b) validating the results reliably; (c) providing matrix independence and ruggedness of the analytical procedure; and (d) making the procedures user-friendly Technological advances will involve: optimizing the performance over time (long-term stability), combining sensor techniques, designing modular and multidimensional sensing systems, and facilitating specific applications Sensor technology is particularly appropriate in life sciences (biotechnology), in novel cultivation techniques, in the medical field, and in process monitoring, but there is considerable room for improving applications in these areas

Chemical and biochemical sensors have attracted considerable attention because they can provide information about the active molality of the free fraction of an analyte No other analytical techniques can do this It is likely that, with future sensor technology, elemental analysis will be refined or replaced by the speciation of specific chemical fractions This has already happened to some extent with a few inorganic and organic analytes Unfortunately, there

is still a tendency to evaluate total concentrations solely, especially in biology and medicine, although the activity of a metal ion is at least as relevant as the total concentration, and is, presumably, the most relevant fraction in toxicological studies It is essential that the correlations between sensor outputs and the toxicity of a species should be investigated in tests using animals

so that animal-free toxicology tests can be performed subsequently (see next section) There is also a growing need to determine active fractions for medical purposes, e.g., for electrolytes such as calcium and magnesium ions where the complexed fraction amounts to around 50% of the total concentration Standard techniques in general analytical chemistry have very limited ways of dealing with the problem of direct, selective detection of a defined fraction of an analyte

in the specimen or sample

Biosensors

Another very direct detecting system is the living organism In response to the Toxic Substances

Control Act (TSCA), the U.S Environmental Protection Agency (EPA) was charged with the

1.1 Chemical Sensors as Alternative Analytical Tools 5

responsibility of assessing the hazards particular chemicals posed for human health [4] For this purpose, whole-organism bioassays and physiological studies were used effectively in identifying potentially common modes of action, common analytical approaches, and in developing a knowledge base for an expert system designed to predict toxic mechanisms from the structure A variety of organisms have been used in testing the toxicity of xenobiotics

Among these are protoma, especially ciliates such as Tetruhymena pyrifomzis, and various fish,

in particular the rainbow trout The assessment of the $sh acute toxicity syndrome (FATS) has

been investigated through careful examination of the behavorial responses of trout, and associated variations in some commonly used diagnostic parameters which are correlated to the respiratory-cardiovascular toxic effects of xenobiotics dissolved in water [4]

Although using living organisms may seem a very simple and inexpensive technique, special care must be taken to ensure that the biochemical environment is controlled, usually by monitoring it electronically The reliability of biological monitoring is sometimes impaired by individual variations in the inbuilt repair mechanisms of damage associated with resistance to different agents However, the most relevant parameter in toxicological risk assessment is the lipophilicity of a xenobiotic and, therefore, the partition of the free species between water and the

living organism This suggests that at least some of the in vivo tests could be replaced by in vitro

tests using chemical sensors Some typical bulk membrane sensors, where the active component

is incorporated into an apolar solvent polymeric layer, respond preferably to the lipophilicity of a target compound (see section 3.1) Currently one of the most interesting questions is whether the

physicochemical activity of a xenobiotic correlates with its biological activity Analytical experiments may help to answer this question for both charged and uncharged species

A living organism is a complex and sophisticated biosensing system Some chemical senses

in animal species as well as in plants are so exquisitely developed that communication can take place through "biochemical" reactions Biosensor research has sought to mimic such natural processes in the laboratory by fixing and connecting isolated cells and organs to a transducing and/or detecting system, usually an electrochemical receiver and amplifier [5, 61 In.one such study, the latent potential of living "bioreceptors", in this case the olfactory organs or the antennules of Hawaiian crabs, were treated so as to create an intact neuronal chemoreceptor- based biosensor called a receptrode [7] This project involved confronting new aspects of

detection and data processing The various neurons of the antennular receptrodes generated

action potentials with different amplitudes The complex multiunit data were analyzed by

employing an amplitude sorting program similar to that used in clinical encephalography The amplitudes were associated with the selectivity of the olfactory organ, incorporating a multitude

of different receptors, whereas the frequency of the depolarization and voltage change corresponded to the intensity of the stimulus by volatile amines (e.g., trimethylamine oxide)

A chemoreceptor-based biosensor or receptrode like the one described above has some very

desirable characteristics, such as: high specificity, extremely low detection limit, large dynamic range, and very short response time [7] The major problem with using the antennules of Hawaiian crabs was they only had a short life-time of 48 h for the following reasons:

1 Autolytic processes destroy parts of the tissue

2 Neurons needed to be continually supplied with nutrients, electrolytes, and oxygen

In 1991, a very critical review on biosensors was published by G.A Rechnitz Ell] Since this time, several excellent overviews and books have appeared, the latest ones were edited by F.W.S Scheller et al [12], R.F Taylor and J.S Schultz [13] Ludi et al [14] have discussed possible applications of sensors in industry

Since both living organisms and isolated organs are selectively sensitive to agents and irritations, attempts have been made to develop artificial systems with comparable sensitivity In these, enzymes incorporated in "biosensors" have been mainly used to mimic the recognition process [12c, 15, 161 In 1991 Schultz defined biosensors as: "Raflnierte moderne Pendants zu den Kanarienvogeln in Kohlebergwerken, deren Verhalten Hauer und Steiger vor gefahrlichen Ansammlungen von Grubengas warnte, basieren auf pflanzlichen oder tierischen Molekiilbausteinen" (they are refmed modem equivalents to the caged canary used in coal mines

to warn miners of dangerous collections of methane (mine gas) and are based on vegetable or animal molecular building blocks)

Biosensors and chemical sensors differ in that they employ different recognition processes In biosensors, natural materials are coupled to physical transducers Excellent transducing elements are generally available, although the molecular recognition component is rarely satisfactory, owing to its short lifetime or the complexity of the signal In chemical sensors, the recognition component is, in some cases, a fully synthetic, specially tailored molecule The most successful chemical sensor involves incorporating valinomycin into a synthetic membrane Since valinomycin is essentially a natural peptide, it is open to debate as to whether this may be considered to be a fully synthetic recognition model

1.2 The Concept of Chemical and Biochemical Sensors

It is not easy to distinguish clearly between a sensor and a complex analytical system Integrated gas chromatographs, infrared and mass spectrometers may be called chemical sensors However, a chemical sensor is typically more versatile and cheaper than traditional instrumentation Some definitions of "chemical sensor" are given by ANSI, DIN, VDUVDE, ICE-Draft a.q [17] However confusing the range of definitions may be to the layperson, it is

quite clear to experts what is meant This is why only a rough and rather arbitrary definition is

given here [ 181

Janata stresses that a chemical sensor must provide "a real time insight into the chemical composition of the system'' and couple "recognition and amplification" with a resulting electrical signal One definition supported by the IUPAC commission in a provisional draft is [ 19a]:

1.2 The Concept of Chemical and Biochemical Sensors 7

Analytical chemical sensors are miniaturized transducers that selectively

and reversibly respond to chemical compounds or ions and yield electrical

signals which depend on the concentration

If this is interpreted strictly, reversibility must mean that successive concentration changes in both directions can be continuously monitored As a consequence, sensors integrating antibodies which are regenerated by a washout process cannot be considered as chemical sensors according

to this definition [20]

In another IUPAC paper, devices such as indicator tubes and test strips, which do not provide continuous signals, are considered to be dosimeters rather than sensors [21a] However, the IUPAC definition does not take into account the fact that the signal yield is closely related to the molality of the free analyte in the sample, which might differ from the concentration In addition to the very restricted definition, chemical sensors involve a broad spectrum of

trumducing process performed optically, gravimetrically, calorimetrically, or in various other

ways as shown in Figure 1-2 Remarkably, the transducing process, including coupling the chemical recognition element to the physical part of the sensor, may have a profound effect on chemical selectivity and analytical performance (see chapter 4) Those aspects were taken into account in a new draft by the IUPAC Commission on General Aspects of Analytical Chemistry This draft provides a more detailed and more general definition and a broad discussion of chemical sensors which states in the f i t phrase[l9b]:

A chemical sensor is a device that transforms chemical information, ranging

from the concentration of a specific sample component to total composition

analysis, into an analytically useful signal

At present, the final signal in a chemical sensor is still always electrical, but this may change with the development of optical computers

According to Figure 1-2, biosensors constitute a subgroup of chemical sensors where biological host molecules, such as natural or artificial antibodies, enzymes or receptors or their hybrids, are equivalent to synthetic ligands and are integrated into the chemical recognition process

Selectivity is related to specijkio Selectivity means that an interfering species responds with the same type of signal, e.g., with the same wavelength or working potential, but with an intensity different from that of the target analyte High selectivity means that the contribution of

an interfering species to the signal relative to the primary analyte is minimal, although the active molality of both covers the same range (see chapters 3 and 4) Specificity, on the other hand,

characterizes the unique property of a bioreceptor, e.g., an enzyme, which, in responding to a

specific target substrate, generates a specific product Therefore biosensors, in responding to that

specific substrate or product, generate a specific signal or signal change In the case where an

enzyme shows cross-reactivity to an interfering substrate, it is assumed that it produces a different product which results in a sensor response clearly different from that of the primary substrate (e.g., different wavelength, different working potential) In practice, only two classes of

8 I Introduction

THERMAL

ELECTRICAL MECHANICAL (MASS)

SPECIMEN

SIGNAL AND COMPUTING TRANSPUTING

Figure 1-2 General model for chemical sensors, differentiating between molecular recognition, uansduction and data processing

enzymes are used in sensor technology, namely oxidases and dehydrogenases, which produce products such as hydrogen peroxide or NADH (nicotinamide adenine dinucleotide, reduced form) which are detected in a wide range of biosensors Therefore the term "selectivity" has been used to describe the discriminative power of a biosensor in the same way as for other chemical sensors [22] Generally, the selectivity of a biosensor allows for a mixed response to both the target analyte and the interfering species Therefore, characterizing the selectivity coefficient for a typical application may be more relevant to the operation of such biosensors than relying on its Specificity [22]

Biosensors as a subgroup of chemical sensors are defined as operating with either high specificity or an exceptionally high natural selectivity, but with considerably restricted stability and lifetime in many cases As a consequence, the lifetime of the sensor has to be sacrified in favour of the natural selectivity or specificity

The quantity detected is always a measure of the active molality of the analyte, whose calibration is strongly correlated with quantities such as: the active molality of the interfering species, the pH and temperature of the sample, and the ionic strength and osmolality relevant for both charged and uncharged analytes (see chapter 3) For biotechnological as well as medical applications, the analyte activity delivers only the biologically relevant information when measured in the specimen directly, preferably by a "realtime" approach The most important features of chemical and biochemical sensors are shown in Table 1-1

Thermodynamic reversibility is important in ensuring continuous monitoring with chemical sensors Individual sensor reactions include: thermodynamically reversible reactions, steady- state reactions and non-reversible reactions in disposable sensors Thermodynamics is central in understanding the principles involved in operating the individual devices Chapter 3 is devoted to these reaction mechanisms

The key to the design of a chemical or biochemical sensor is the recognition process of an

organic or inorganic substrate by a receptor-molecule generating a host-guest product (see

1.2 The Concept of Chemical and Biochemical Sensors 9

Table 1-1 Features and benefits of chemical and biochemical sensors

reagent-poor operation

electronic processing and electronic control consumer friendliness, ensuring safety of the

consumption of the analyte

chapter 2) The sensing schemes of the molecular recognition element are based on bulk or

surface interactions, on mechanisms where the analyte is adsorbed or where it partitions between

the sample and the bulk phase (see section 2.1) The target analyte or substrate may be any organic or inorganic ion or any uncharged molecular species In order for a sensor to detect an analyte successfully in a complex sample matrix containing some analogously reacting species, high selectivity is required

Selectivity may be achieved by using various designs of optical as well as electrochemical sensors furnished with synthetic carriers (see section 2.2), enzymesJ23-251, or antibodies [26-

281, or by using sensors based on competitive binding [29] Enzymes are defined as reacting reversibly In fact, what really happens is that they reach a stable steady state, assuming a constant mass transfer of substrates and products In contrast, antigen-antibody reactions exhibiting high specificity are, in most cases, not reversible with a reasonable rate constant, owing to the exceptionally high affinity for their substrates associated with low detection limits

On reviewing the literature, it seems that an artificial recognition process can overcome the severe limitations of natural compounds [7,8]

One of the most outstanding recent developments has been the design of artificial enzymes or catalytic antibodies [30-321 The molecular recognition principles based on synthetic host compounds are more modest than those of artificial enzymes When modelling host-guest

10 1 Introduction

interactions (see section 2.2), the shape of the analyte has to match the site of the host species A

broad range of electrodes specified for various anions and cations are available, and are routinely used with reasonable analytical performance in diagnostic instruments, in clinical analyzers [33,

341, and in environmental analysis In clinical chemistry, highthroughput analyzers, preferably based on optical assays, produce 5000-15 000 results per hour Furthermore, the use of ion- selective electrodes (ISEs) in discrete analytical systems has increased throughput considerably

In the best case, the resident time of a sample in the ISE-module, which analyzes at least four parameters in series equivalent to four different typical ions, is 6 s

Although ligands and ligand c o c h l s are currently used worldwide, the approach developed

by Simon for recognizing and sensing ions is not mentioned in any of the volumes on sensors [35, 361 The design of ligands for molecular recognition has been extended to include the recognition of uncharged species, such as: humidity (H20) [37,38], ethanol [39,40], glucose [41], creatinine [42], gases such as C02 [43], HSO3- and SO2 [44], and NH3 [45, 461 It offers

exciting prospects for the optical translation and transduction of reversible host-guest interactions

The sensing system can, to a certain extent, be adapted and tailored to fit its applications The detection limit, the selectivity, and the dynamic range may be shifted by modifying the ligand or the optical transducer and the surrounding bulk medium according to chemical or physicochemical principles (see chapters 4-6) Applications in various fields as different as medical analysis and biotechnology have been undertaken successfully r47-491 A survey evaluating optical assays is given in [50]

Strong competition in the field of sensor technology over the past 10 years has led to an increase in the number of models available Nevertheless, only a few types of chemical and biochemical sensors appear to be viewed as reliable tools for analytical chemistry and to be used widely in this growing market sector Physical sensors, on the other hand, have become well-

established in a competitive market and are regularly used in different monitoring systems and

devices The concept of the chemical sensor is, however, not new A brief history focusing on the development of chemical sensors, especially on aspects of commercial use, will be presented

in the following section

1.3 Recognition Processes and Sensor Technology: Milestones

The technology of sensors and actuators has a long history Wilhelm von Siemens built one of the first sensors in 1860 He made use of the temperature dependence of a resistor made of copper wire to measure temperature [51] The fundamental principles behind physical sensors and transducers largely apply to chemical and biochemical sensors The history of the development of chemical sensors for medical applications is summarized in Appendix 1 [52,

531 The first really significant event from the commercial point of view occurred around 1932, when Arnold Beckman developed the modern glass electrode [54] In 1937, Kolthoff and Sanders [55] published a paper made use of solid-state electrodes, such as the silver halide and fluoride-selective electrodes (for an account of the development of solid-state and glass- electrodes, see Frant [54])

1.3 Recognition Processes and Sensor Technology: Milestones 1 1

The key feature of carrier-based chemical sensors involves the recognition of the analyte by using a ligand tailored for the purpose The sensing element is that critical part of the sensor where the primary transduction occurs and, as such, is vitally important in the operation of the whole sensor The basic concept of chemical sensors owes much to the investigations of Moore and Pressman into the effects of naturally occurring neutral antibiotics on biological membrane systems in 1964-1965 [56] Valinomycin (Figure 1-3) was reported to change the permeability

of cells for potassium by a factor of 4 x 104 Two years later, the highly selective and reversible complexing properties for alkali metal ions were described by Stefanac and Simon [57] In the meantime, Ross in the United States and Simon in Switzerland had both applied for a patent covering the K+-selective electrode; the patent application of Simon was accorded priority [58]

Simon was certainly the first to introduce the class of chemical sensors based on neutral curriers Subsequently, in 1970, Frant and Ross described how the valinomycin K+-selective electrode was first employed in serum measurements [59] Orion received a licence under the Simon patent and developed a prototype electrolyte serum analyzer for NASA's Space Shuttle and, subsequently, the first commercialized sodiudpotassium analyzer SS-30 for whole blood Ironically, spin-offs from the Orion project led to the business becoming commercially successful In 1972, another clinical analyzer using a valinomycin-based sensor, namely the

In 1967, the term "ionophore" was coined by Pressman et al [61] In the same year the structure of the first macrotetrolide-ion complex was elucidated (see Figures 1-3, and Appendix

1) [62] The ionophores were, typically, lipid-soluble peptides with a relative molecular mass <

2000 Some of them, such as those in the valinomycin class, had in common molecular masses

of 500-1500 and a curious alternation of D- and L- configurations of the participating

aminoacids as well as a lack of ionizable groups The structure of the K+-valinomycin complex

was elucidated in 1969 by Pinkerton [63] In contrast, the neutral ionophores with lower molecular mass were classified as "carriers" Today, the selectivity of valinomycin for potassium ions still seems striking, and compares favorably with the properties of other ligands developed later

After testing other naturally occurring antibiotics (macrotetrolides) with remarkable selectivities, Pedersen [66,67] , Lehn [68,69], and Cram [70] began to study synthetic ligands (crown compounds, synthetic macrocyclic polyethers, macrohetero-bicyclic ligands, cyclophanes and others) Cram [71] uses the term host for the synthetic compounds that are the

counterparts of acceptor sites in biological chemistry, and the term guest for compounds that are

the counterparts of substrates or inhibitors in the acceptor sites, according to Kyba [72] Pedersen, Cram, and Lehn were awarded the Nobel prize for these achievements in 1987

In the early development of the industrial electrode, organic ion exchangers were used in a solid configuration Moody, Oke, and Thomas showed that incorporating the ligands into a plasticized PVC membrane prevents the membrane components from becoming fully hydrated and allows the active components to be sufficiently mobile [73] This technique enabled ion- selective electrodes to become not just practically, but also commercially feasible The influence

of ion-selective complexing agents on the ion selectivity of liquid membranes was discussed theoretically by Eisenman's school [74,75], by Covington [76], by Pungors' group [77] and by Sandblom [78] and Orme [79] and later by Buck [80] and by Wuhrmann, Morf and Simon [81,

12 1 Introduction

Figure 1-3 Constitution of valinomycin, as presented by [MI, and the macrotetrolide antibiotics

monactin and nonacth [65] The ligand-cation complexes are positively charged The alkali- or ammonium ions are complexed by 5-8 polar oxygen atoms The conformation of the complex

is characterized by an outer nonpolar shell and the polar groups oriented towards the center

atoms

821 Electrically neutral and electrically charged ligands were strongly debated Eventually, charged ligands were shown not to work in nonpolar phases The working priniciple and the functional distinction between charged and neutral ligands was accepted empirically rather than being rigurously defined and investigated The complex formation was described for the interface between an aqueous phase and a relatively nonpolar membrane phase, where the selective transport of cations was to be expected

More than 12 years later, the first optical potassium test, based on dry reagent chemistry was

evaluated by the author, and commercialized subsequently by the Ames Division The evaluation of silicone rubber membranes for the valinomycin electrode (see Figure 1-3) has led

to extensive collaboration between the groups of E Pungor and W Simon, within the context of

a friendship which has not prevented occasional decisive discussions of fundamental issues

[831

At the same time, the concept of the biosensor was proposed by L.C Clark Jr et al in 1962

[84] They measured pH, pC02, and PO;? for intravascular continuous monitoring (see section

1.4.3) Also in 1962, Enson, Briscoe, Polanyi, and Cournand [85] introduced intravascular

reflection oximetry Bergman [86] described the first oxygen fluorosensor in 1968, which was introduced into medicine by Lubbers and Opitz in 1975 [87, 881 At first it was thought that, unlike electrochemical sensors, optical sensors would not require a reference element Satisfactory results were obtained by normalizing the optical signal of the analyte to a second

reference wavelength, which involved evaluating relative intensity changes Enson et al [86]

proposed the use of the isosbestic point as a reference

1.4 Goals for Future Developments and Trends 13

Considerable progress in developing medical sensors has been made during the past few years [89-911 In nearly all cases of optode design sensitive to the chemical properties of the analyte, the optical detection principle is based on either quenching of luminescence by oxygen

or on a change in luminescence intensity due to pH alteration Pulse oximetry, as well as some other new approaches, are exceptions [12, 921 Different from other ions, pH is the unique parameter where the ion activity is measured and reported Providing there is reliable calibration,

pH assays are relatively unproblematic since no assumptions are necessary for calculating the concentration

For p02 measurements, the sensors are considerably more rugged than those used in the evaluation of enzymatically generated oxygen or quenching compounds such as S 0 2 , halides,

etc [91,92] Thus, assays of most low-concentration analytes based on fluorescence quenching

as well as pH evaluations demonstrate that these systems are too unreliable for direct monitoring

in blood or in any biological sample An element for selective recognition of the analyte, acting

as a selective filter to take account of interfering entities, would ensure greater reliability

An important landmark was the introduction by D.W Lubbers et al [92] of immobilized indicators into the development of optical sensors for continuous monitoring in biological fluids They introduced the term "optrode" by analogy with "electrode" The term "optrode", however,

is etymologically incorrect; optode [93] would be more appropriate

Enumeration of all the scientists involved in developing combinations of sensing elements and transducers is beyond the scope of this study Appendix 1 indicates some of the historic landmarks, whereas Table 1-3 shows some of the components and combinations that are possible in constructing chemical sensors and biosensors Many of these have not yet been

tested in real sensor configurations Apart from the development of the recognition process, the transducers, actuators, or amplifiers have been improved considerably In summary, the development of physical transducers is much more advanced than is that of the chemical recognition components as used in sensors [94] At the Optical Meeting in The Hague 1991, the lack of innovation and development in this area was deplored

1.4 Goals for Future Developments and Trends

1.4.1 Trends

One of the catchier titles in the program of the 1991 Pittsburgh Conference was that of I.J Higgins et al.: Biosensors: Philosopher's Stone or Fool's Gold? The authors presented

statistical data and forecasts regarding the sensor market

Table 1-2 shows an extract from various reports on sales in the sensor market and their

forecasts for the future [17,95-981 The first row shows figures taken from a report by Prognos

AG (Basel, Switzerland) [17] In 1988, the world market for sensors, physical and chemical, amounted to US$ 24.1 billion Regional shares in the market reflect the strength of the sensor industries there: US$ 5.2 billion in both the United States and Western Europe, and US$ 3.0 billion in Japan [ 171 A wide variety of application-specific demands means that there is a fairly

14 I lntroducrion

Table 1-2 Overview of current sales in US$ X lo6 and forecasts of the sensor and biosensor market respecting different fields of applications (different references)

24100

1200

13.6 3.1 1.9

by 2004 *

1380 (9-1 1%)

* Projected in 1994 dollars (sales, contract research and development)

heterogeneous market for measuring parameters According to the report cited, chemical parameters constituted only US$ 1.2 million of the total world market for sensors of US$ 24.1

billion in 1988 The report predicts an annual growth rate of 5 1 0 % for the next ten years

According to its forecasts, the United States will be the leading vendor and, maybe also consumer in the chemical sensor market by the year 2000 (US$ 14.5 billion), ahead of Western Europe (US$ 13.5 billion) and Japan (US$ 10 billion) This means that, relatively, Japan will

have the highest growth rate It is, however, to be expected that the regional market shares will change

Another report [95] estimated that the growth of the United States biosensor market would

be from only US$ 3.1 million in 1989 reaching US$69 million by 1993 This report foresaw

the application with the fastest growth during this time to be the monitoring and control of

1.4 Goals for Future Developments and Trends 15

process industries It suggested that the largest market segment in 1993 would be in health care (US$29.4 million), and that it would consist mainly of single-analyte instruments

Depending on the source, the forecasts (Frost and Sullivan, Desjardin, Battelle, Market Intelligence Research) differ considerably Whereas the figures for chemical sensors by Prognos

AG include biosensors, the report by Taylor [96] clearly separates the two classes, so that the

worldwide sales figures for chemical sensors and biosensors in 1994 have to be added together

in order to compare them with the Prognos figures for 1988 Rather surprisingly, sales of chemical sensors and biosensors actually went down during the 6 years between 1988 and 1994, which might indicate some "cleaning" process in view of unsuccessful developments in chemical sensor technology Notwithstanding this decline, a sharp upturn in the market, which may be entering the "take-off stage" [95], is predicted in both reports [95] and [96] By way of

comparison, in vitro diagnostics had amarket of US$ 9 billion worldwide in 1992 [97] 5% of

this market consisted of consumer testing (US$ 450 million), 20% of which was dedicated to

decentralized testing Medical in vivo monitoring has, on the other hand, declined as it has met

with ethical and legal barriers, and is now limited to a few applications

Table 1-3 Possible components of a biosensor / chemical sensor characterized by selective molecular recognition and solubilization of target analytes

synthetic ionophores

synthetic carriers

supramolecular structures, clusters

solid layers: metals

- metal oxides, crystals

- polymers, conducting polymers

natural organic and inorganic molecules

micelles, reversed micelles

16 1 Introduction

Commission (DG XII) of the European Union [98] published an interesting report on

science and technology in Europe in 1991, in which it was estimated that the total world market

for sensors in 1990 was approximately 10 billion ECU (US$ 8.3 billion) Western Europe had

about half the world market for sensors in engineering, whereas Japan was the leading user of sensors in robotics, and the United States was the major user of sensors for electronic applications in vehicles

European competitiveness in the hightechnology market declined in the 1980s so a financial framework program for supporting the technical potential of European industries was introduced in 1989 It included sensor technology For four years (1989-1992), some 500 million ECU (approx US$ 416 million) was budgeted for the whole programme Large

investments in research and development and highly qualified researchers in Europe were expected to increase competiveness There are several reasons for the comparatively modest performance of Europe, which include: an unbalanced and fragmented distribution of human and financial resources, the lack of mobility of the workforce, little coordination of research efforts, and the lack of a large integrated home market

The thrust of financial support has been toward the mass production of sensors and also toward miniaturization However, unlike the glucose testing market with an expected global turnover of US$2 billion (US$2 X log), only a few other applications will have a comparable sales volume [96] Comparatively, another type of sensor, e.g., for ethanol or ammonia

monitoring could also be produced on a smaller scale, supposing a frequent demand in various fields of applications, e.g., in bioprocess control, medical monitoring, and other specialized areas In bioprocess control, voltammetric oxygen probes, pH probes and unselective redox

sensors are currently commercially available In the future, an ammonia-selective sensor is likely

to have a wide range of applications

Table 1-3 shows some possible sensor components In view of the large number of these proposed over roughly the past 20 years, only those combinations and principles which look likely to result in effective analytical devices are listed In the future, the design of sensor systems will involve combining those sensors with the most attractive features in order to solve

a specific analytical problem The client is unlikely to suggest the type of sensor to be used, but will be more inclined to specify the analytical parameters (detection limit, dynamic range, lifetime, etc.) the system should provide Sensing systems must be constructed so that they are flexible and versatile enough to integrate various types of effective physical and chemical sensors working with standardized units or modules

1.4.2 Miniaturization, Nanotechnology

Miniature analytical instruments and devices have several advantages since they allow:

- on-site analysis, analysis in security areas

- extremely small amounts of substances to be rapidly analyzed, thus complying with recent

- smaller analytical devices to be produced which are more suitable for in vivo measurements

trends in combinatorial chemistry, peptide synthesis, and DNA-fragment analysis

since they are less likely to impair the functions and structures of living organisms

1.4 Goals for Future Developments and Trends 17

There are two ways in which progress is being made in miniaturizing analytical devices so as

to produce more versatile and smaller instruments First, through the development of microstructured, miniaturized systems; and secondly through the development of dedicated systems Microstructured chips for capillary electrophoresis and gas chromatography [99 1001

are examples of the first group (see Figure 1-4) They are especially attractive for the analysis of pico-, femto-, and attomol- quantities of peptides and DNA-fragments In the second group belong: the ion mobility spectrometer [ 1011, hyphenated techniques [ 1021, and the accelerated development of chemical sensors, sensor arrays, and microsensors A further goal of miniaturization might be to produce an instrument combining or hyphenating miniaturized single elements based on different working principles without sacrifying their versatility These developments here look promising The terms "mini", "micro", and "nano" are often confused

A "ministructure" means a structure in the "milli" range (dimensions of, e.g., to c 10-2 m

or 10-3 to c 10-2 cm3) Structures which are 1000-fold smaller are microstructures or

microsensors Microelectrodes have tip diameters in the range of c 10 pm down to 50 nm and

reach, in some cases, the nunoelectrode size The name "microsensor" means that the size of the

active sensing area is in the micro range, whereas the sensing device itself is much larger and designed so that it can be handled easily There is a history behind the name

"ultramicroelectrode" [ 1031 Ultramicroelectrodes are usually larger than microelectrodes

Currently they are in the process of being down-scaled to the nano-range for in vivo

the resolution of a light microscope is 10" m, implying that working with nanostructures

requires special tools for visualizing the local activity

Miniaturization of an analytical device or a sensor does not have to mean making it less rugged On the contrary, miniaturization may result in a more rugged device, such as the voltammetrically operated ultramicroelectrode Furthermore, other specific features may be improved by miniaturization Thus, the total information yield may be greater despite the fact that the dimensions have been reduced by several orders of magnitude The merging of sensing elements with electronic devices for transduction and readout has led to new capabilities, but has also imposed some constraints (see below) on the sensor system In the case of biomedical sensors, increasing the information gathering capability per unit volume is often the motivation for integrating electronics in the system Integration also results in some very promising properties, not only because the resulting system is more versatile, but also because it allows: drifts to be corrected; fitting and calculation software to be implemented for the measurement based on nonlinear calibration functions; and interferences to be compensated by using sensing arrays

Miniaturization is particularly appropriate for sensors that improve their performance with

smaller sensor and sensing layer areas, and with reduced distances from the working electrode

to the reference and counter electrode This holds for sensors based on electrokinetic techniques concerned with determining the rate of electron transfer at the working electrode which is of special interest when associated with chemical regenerating electrode reactions, and mass

transport For these electrodes, sensitivity refers to the decreasing surface area exposed to the

target analyte h the best case, mass transport is purposely restricted to diffusion in transient

to

18 I Introduction

Figure 1-4 The miniaturized gas chromatograph M200; left hand side: capillary column; right

hand side, upper case: gas sampling chip; right hand side, lower case: conductivity detector chip (with permission [loo])

techniques, and to forced convection and diffusion in steady-state techniques [ 1031 For electrokinetic techniques, the radius r is the lateral diffusion area of amperometric and voltammetric ring or disk electrodes Since the double-layer capacitance of the electrode cell and the total current are proportional to the surface area of the electrode -r* the cell response time

and the ohmic drop both decrease with the reduced surface area, whereas the diffusion and reaction rate vary with lh2 Given this situation, voltammetric ultramicroelectrodes have the

following advantages: when detecting catecholamines in stimulated brain tissue [ 1061, they enable a high scan rate and a response speed in the ms range For the same reason,

ultramicroelectrodes are often used in spectroelectrochemistry [105]

Potentiometric sensors, on the other hand, are basically affected by the size of the active area However the size of the exposed ion-selective surface area is supposed to limit the ion-exchange current, reducing the selectivity and the sensitivity of the electrode [107] Those parameters seem

to be correlated to the stability constants and detection limits of the ligands and electrodes Similar to the sensors discussed before, potentiometric sensors show a diffusion-limited response, where the response time increases with 8 depending on the thickness of the static Nernstian diffusion layer d at the sensor surface The Nernstian diffusion layer decreases, not only with increasing flow or convection, but also with decreasing diameter and detection volume

on the miniaturized electrode tip

A further dimension is also important in this discussion of sensors, namely, the thickness of the sensing layer This relates to the working principle of a sensor Miniaturization is most appropriate for sensors that are not sensitive to the thickness of the sensing layer In optical transmission sensors, however, the optical path length is equal to the thickness of the optode

membrane In optical bulk membrane technology (see section 6.2), the optical sensing layer

equilibrates with the target analyte in the sample phase Therefore, the thickness of the sensing layer determines not only the speed of equilibration, but also the optical path length So reducing the thickness of the sensing layer also reduces the sensitivity As a consequence of this

1.4 Goals for Future Developments and Trends 19

equilibration process, the concentration of the target analyte in the sample phase is reduced by a

contribution equivalent to -Ac and an uptake of +Ac by the membrane phase This size of

uptake is smaller than 1%0 for membrane volumes < 10-9 L incorporating less than 10-1 mol

L-' ligand in contact with an amount of analyte in the sample of more than lo4 mol referred to the whole dynamic range of the optode membrane However, at low amounts of analyte c lo4 mol, it might be possible that the optical signal involves the consumption of the target analyte (see section 6.4) Moreover, the extraction steady-state, associated with the stability constant of the ion ligand complex, affects and modulates the detection limit and may induce a shift to very low detection limits < 10-9 m o m when operating under continuous-flow conditions Inevitably,

if this process takes place, the response in this range will be slower compared with a higher concentration range To avoid having to use thick membranes, associated with slow equilibration, it was decided tQ investigate novel deposition techniques and optical detection technologies (see sections 6.5 and 6.6)

Another way to distinguish sensors is to classify them according to how they are produced

A microsensor is a device requiring microfabrication technology Microfabrication is used by the semiconductor (electronics) industry in the manufacture of integrated circuits (ICs) As Bergveld showed in 1970 [log], design and packaging are important in the development of electrochemical "minisensors" They are even more crucial in manufacturing micro- and nanosensors (sensors where the active sensing areas are small, but the size of the chip is still in the macroscopic range) Currently, these miniaturized electrodes, down-scaled to planar sensors cannot compete with the down-scaled microelectrodes for physiological applications (see section

5.4)

An alternative is to have the chip accommodating not just one sensing field, but also related functions such as a light source, a detector, or a whole sensor array This, plus the necessary

electronics, is known as an integrated chemical sensor For semiconductor devices, it is

necessary to encapsulate the electronics, which makes it difficult for the specimen to make contact with the sensitive transducer area This problem was solved by using a gate The ChemFET (chemically sensitive field-effect transistor) is an excellent example of a microsensor which has suffered several developmental setbacks, owing to a lack of understanding of the basic operational principles and constraints, due to insufficient theoretical analysis of the operation [ 181 However, one of the main problems with semiconductor ion-selective sensors (ion-selective field-effect transistors, ISFET) arose because it was assumed that they could be produced without a reference half-cell Not only due to the missing reference electrode, the ISFETs showed a relevantly drifting potential which was referred to the ill defined boundary where the ion conductivity is transformed into electron conductivity For more detail, the reader

is referred to [18] and [log-1 111 Recently, some new solutions have been proposed [log, 1 lo] One recent investigation covers the fundamentals of hysteresis, long-term drift, slope, and selectivity of ISFETs [ 11 11

Whereas the encapsulation of solid-state microsensors has been dramatically improved in recent years, the ChemFET response continues to demonstrate a significant long-term drift This

is a major obstacle in cases where a stable in situ calibration is essential, e.g., for implantable sensors This is due partly to the fact that the reference half-cell has a reduced capacity (volume) and, also, that the sensor field responds to uncharged interfering species such as gases, e.g., carbon dioxide and oxygen Furthermore, photochemical polymerization of the sensing layer does not allow the inclusion of the necessary amount of additives, apart from a limited selection

20 I Introduction

cartridge label sample entry

well gasket

fluid channel cartridge cover

sample entry

wall

calibrant pouch

Packaging, Sampling and Testing

i-STAT @

Cartridge

puncture barb cartridge base air bladder

Figure 1-5 i-STAT@ cartridge incorporating electrochemical sensors and biosensors (with

permission [ 1 121

of the prepolymer for some electrodes Tailoring the composition of the sensing layer to its

application, however, is one of the main factors in determining its performance (see chapters 4,

5 and 6) Therefore, the possibilities for creating sensors incorporating novel sensing layers are rather limited Despite these drawbacks, a whole set of analytical devices which can be used for testing near patients and in emergencies are currently available in the form of disposable sensors [112] (seeFigures 1-5 and 1-6)

In the biomedical environment, the protection of the electronics is especially problematic and

has limited the commercial success of ChemFETs [ 11 11 Microelectronic components are very

sensitive to temperature, humidity, pressure changes, and chemicals Additionally, integrated devices lack modularity The components of the sensor system which are incorporated on-chip

are fixed by the mask set and must be fabricated at the same time (see Figure 1-5) Any change

in the design requires an entirely new mask, and often a new process flow as well Whenever undefined or poorly understood transduction mechanisms are employed in microsensor designs, there are bound to be development delays and fabrication problems and the reliability of the application will be reduced

One of the most urgent goals for the near future is to combine the large-scale hardware technology of physical transducers and laser optics with the "softer" approaches of sciences such

1.4 Goals for Future Developments and Trends 21

as chemistry and materials science, in order to find the most suitable combinations for solving specific problems One product that involves combining miniaturization with integrated planar

sensors and sensor arrays is the electronic nose, which recently came onto the market Such

transducing elements make use either of the quartz crystal microbalance (QCM) [113] or,

alternatively, of various surface acoustic wave (SAW) transducers where different piezoelectric resonator materials, e.g., a quartz foil or YZ lithium niobate, is stimulated by two electrodes and works as an electromechanical transducer of oscillations with a defined frequency in theMHz range [114, 1151 Another type of sensing device for the electronic nose relies on electrochemical cells covered with a metal oxide layer [ 1 151 The heart of the electronic nose is a more-or-less analyte-selective polymer layer cast onto the oscillator Currently, the sensing principle is preferably based on partition equilibria of the analyte between the sample phase and polymer layer, and does not make use of known host-guest chemistry (see section 2.2 and chapter 3) The selectivity of some platforms could and should be improved In the electronic nose, the two trends in analytical chemistry discussed previously, miniaturization and integrated

design, are combined in one instrument; the electronic nose is a dedicated system making use of

miniaturized sensor arrays Similar systems are being created for solution chemistry

Frequently, nanodevices display new, unexpected properties The role of chemists in the

nano-range may seem less clear than their role in creating chemically selective tools However,

insights into chemical reactions and equilibria at the level of single molecules, and has led to insights into partitioning and interactions between single molecules At the macroscopic level, chemical reactions and equilibria are seen to involve a majority of molecules, so that descriptions

at this level are dealing with average molecular behavior Nanotechnology allows these hypothetical mechanisms to be studied at the molecular level, so that the behavior of single molecules in different states in, say, a solution can be distinguished For example, the permittivity and ionic strength of a contacting solution close to a surface may differ considerably from that of a bulk, and are affected by polarization phenomena [116] The complexing sites of a

polypodal ligand are not equivalent and may become saturated progressively, which means that

nanoscaled sensing elements may behave differently and need to be viewed more stochastically The availability of OH groups in different homologous pure alcohols will vary depending upon the lipophilicity of the alcohol, the free energy of hydrophobic interactions, and the density of intermolecular hydrogen bridges When the nanoscaled sensing element is exposed to the active

OH groups, the reactivity of homologous alcohols to the host molecule varies due to these effects, as well as to the extent of self-organization and the influence of electronic inductive effects Chemical microscopy makes use of miniaturized optical devices, such as chemically selective nanofiber tips and NSOM (near-field scanning optical microscopy) fiber tips [117], as

well as electrochemical techniques These techniques will result in some novel physicochemical

information and initiate a new area in chemistry and education which will be more adapted to chemical analysis on the micro- and nanoscale than traditional approaches

In summary, miniaturized analytical sensors and devices have great potential and are likely to have considerable impact on the development of analytical chemistry The dream of a whole laboratory installed in a glove-box may become reality in due course

22 I Introduction

1.4.3 In Vivo and In S i h Monitoring

A rule of the thumb in life-saving says: humans can survive 3 weeks without eating, 3 days without drinking, but only 3 minutes without breathing During a polio epidemic in 1952, two Danish anaesthetists, Bjorn Ibsen and Poul Astrup, reported on more than 100 patients artificially ventilated by volunteers squeezing oxygen bags They noticed the high carbon dioxide concentration in the victims [118] Astrup developed the first model of an equilibration method for measuring pH and pC02 in order to diagnose respiratory alkalosis or acidosis Severinghaus's electrode also contributed to this work ['I 191 Polio epidemics also provided motivation for the development of the C 0 2 and 0 2 electrodes in the United States In 1958, Clark presented the results of continuously monitoring oxygen partial pressure and pH with sensors mounted directly in the extracorporeal blood circuit that is used for perfusion of open- heart surgery patients [120] As a consequence of the commercial development and availability

of stable amplifiers and recorders, satisfactory systems for the rapid and accurate measurement

of blood pH, pC02, and p 0 2 were developed Improvements in the electrode systems meant that intraarterial oxygen partial pressure could be monitored continuously through an implanted catheter [84] Clark mentioned that heparinization was not necessary if the "microcatheter" was less than 3 - 4 feet in length In the same paper, he describes a new design of an enzyme electrode

Clark predicted:

By withdrawing blood through microcatheters, continuous recording of blood

composition for many hours, even days, is possible, using only about 10cc of

blood per hr Continued development of electrode systems may extend their

usefulness to the measurement of blood ions, sugar, and urea and finally result

in instruments with which analyses can be performed with a minimum of reagents and with but little delay

Another purely physical optical sensor, the pulse oximeter, measures the oxygen saturation transcutaneously, and has been applied to the monitoring of critically ill patients The method involves a pulsed diode spectrophotometer which detects the internal total reflectance at multiple

wavelengths [85, 121-1231 According to Severinghaus [118], Karl von Vierodt carried out the

first measurements of optical density in 1870 by monitoring changes in his hand His results were rediscovered in 1932 But it was not until the early 1970s that real progress was made by

T h o Aoyagi, Tokyo, and Akio Yamanishi of the Minolta company Pulse oximeters are now commercially available and have been used routinely for over 15 years in intensive care units (ICU) despite having many limitations, such as severe interference from HbF However, the device is appropriate for the long-term monitoring of a constant oxygen supply and uptake, and this is how it is mostly used today Where do we now stand? According to the US National Committee for Clinical Laboratory Standards (NCCLS, C27):

blood gas has more immediacy and potential impact on patient care than any

other laboratory determination

1.4 Goals for Future Developments and Trendr 23

The American Association of Clinical Chemistry (AACC) expanded upon this, clearly seeing

an essential link between the blood gases and the electrolytes Na+, K+, ionized Ca2+:

The measurement is essential for diagnosing and monitoring electrolyte disorders

In 1992 some devices for evaluating pH, pC02, and pO2 based on opticalfibers were marketed by BTI, Oximetric, and Puritan Bennett In these devices, optical fibers are introduced

by catheters over an interface adapted so as to calibrate and recalibrate the sensor For in vivo monitoring, optical methods have now replaced electrodes Optical methods rely on the fact that oxygen quenches the luminescence of characteristic dyes and that pH changes bring about a shift

in the emission spectrum Optical pH measurements were developed by Petersen et al., who described a sensitivity (resolution) of the optical fiber sensor of 0.01 pH unit [124] The sensors

were used in blood and demonstrated in vivo in 1975 [92] The probe did not, unfortunately, meet the in vivo biocompatibility and reliability requirements for a one-time use of up to 72 h In

1984 J.I Peterson and G.G Vurek concluded [89]:

Electrode development, although of intense interest for many years, has not

lived up to expectations for wide applicability and reliability in vivo Fiber optic

sensors are still too new to be of proven value in most applications

Long-term monitoring of electrolytes during hemodialysis is necessary since the side-effects

are aggravated the longer hemodialysis continues Continuous monitoring in an extracorporeal second circulation failed at the first attempt, owing to the instability of the calibrated setting points (see section 5.1) 1125-1281 These problems were partly solved by taking into consideration the asymmetry of the ion-selective membranes induced by blood plasma, and by varying the membrane composition

The concerted European action devoted to in vivo monitoring of blood glucose concentrations

and concentrations of several key metabolites during intensive care ended in 1996 A total of 36 European analytical centers were involved, working on a broad spectrum of projects [ 1291 Unfortunately, BIOMED 2, the new proposal, did not receive support from the European Union In view of the length of time it has taken to develop sensors (roughly 40 years), a 3-year period for this type of project is too short History shows the strong impact that the commercial availability of novel electronic systems and biocompatible materials may have on the realization

of projects At the same time, history also shows that the most significant progress is made when the scientists concerned are closely involved in clinical research, and are aware of see the

medical problems which arise For this reason, a concerted action on in vivo monitoring should

involve the participation of medical personnel and medical centers, especially as biocompatibility problems tend to be poorly understood by purely technical scientists In conclusion, the

following points seriously restrict the viability of long-term monitoring in vivo:

- the sterility of the device and its aseptic handling

- the maintenance of the calibrated setting points, which is partly related to the biocompatibility

of devices and materials

- the toxicity of components

- the cost-benefits of a patient having to carry an additional catheter, which is stressful

24 I Introduction

Implanted sensors are immediately entrapped by proteins as part of a reaction in the stressed tissue compartment, with subsequent diffusional problems This means that results in general do not correlate with determinations in whole blood

Some companies have left the field Others have continued [130, 1311, and yet others have shifted towards investigating noninvasive techniques [ 132, 133 3 The electrochemical measurements of pH and p 0 2 are now the only "physicochemical" sensors used in biotechnology since the Severinghaus electrode is no longer used to determine the pCO2 as its response behavior is too slow In comparison, optical sensors are likely to have an increasing impact since they show many promising features associated with the development of new dyes [134] and chromogenic ligands (see section 6.2), and with a growing market of various small-

sized light sources A new domain of analysis has recently emerged in medicine called "point- of-care testing" (POCT) POCT allows clinicians to quickly assess their patients in emergency

units and on-site POCT is practiced under the assumption "faster is better" However, rapid

testing should not turn out to provide inaccurate results Which means that all the features found

in traditional instruments must be provided also in a small portable instrument based on miniaturized disposable cartridges shown in Figure 1-6 [135] The AVL OFTI 1 calibrates itself

Figure 1-6 AVL OFTI 1 cartridge incorporating optode membranes for the analysis of pH- and blood gas (p02, 602) in whole blood Additionally 10 calculated parameters are provided (with permission [ 1353)

1.4 Goals for Future Developments and Trends 25

and consumes typically only 80 ~1 of the specimen Novel developments are based on

experiences made with in vivo and ex vivo monitoring and have led to successful application of

some of the techniques in this field to on-site determinations and front-line analysis [136]

Novel approaches are likely to affect other fields in analytical chemistry, such as environmental chemistry, toxicological studies, process monitoring, and the analysis of seawater [ 1371

1.4.4 The Analytical Laboratory in the 21s' Century (Conclusions)

Any perspective on the future must remain incomplete; such a perspective can only be based on

a global view

Analysis in the Centre

Worldwide, a few giant companies will dominate the market for the enormously expensive and technologically highly developed hyphenated instruments such as ICP-MS, highresolution quadrupole MS sector instruments, and some N M R techniques However most instruments wiU undergo some degree of miniaturization, and, in many cases, distribution of the specimen, sample fractionation, and pretreatment wdl need much more space than the analytical instrument itself Space-filling robotic systems for specimen and sample distribution, literally speaking, are totally uneconomic; they are slow, space-filling, and inefficient Either these robotic systems have to be provided with additional skills, e.g., sensors for screening, or they have to be miniaturized or replaced by more intelligent solutions

In the clinical laboratones of major hospitals, automation is particularly advanced, and routine chemical and hematological analyses are virtually centralized Some analyzers can perform up to

36 analytical tests on plasma samples simultaneously and provide up to 15 0o0 analytical values

within 1 h under the supervision of only a few staff Such systems integrate ion-selective

electrodes, continuously monitoring the free molality of the most frequently analyzed electrolytes Such integrated systems are also being developed by national reference laboratories, and require capital investments of US$ 2-7 million They will be supplied by a few global companies, with national offices taking care of servicing

Generally speaking, economic trends will lead to the growth of opportunities for

economically competitive reference and consulting laboratories worldwide, and to the deporture

of some smaller laboratories from the large-scale routine treating queues of specimens The reference laboratories will run under the supervision of only a few staff However, individuals will still be required to ensure quality, since currently no quality control system can interpret reports, manage the sources of errors, or make constructive suggestions on corrections

Reference laboratories will fulfil many functions other than merely producing results, including

establishing a network of reference laboratories, for data excharige, and quality assurance worldwide These laboratories will be completely free of daily analytical batch services

Under these conditions, large-scale, rapid, and safe transports of specimens submitted to the central laboratories must be organized

26 I Introduction

Analysis in the Periphery

The laboratory in the periphery is supposed to work much closer to clients Presumably, they could provide services to the front-line analysis sites and also perform front-line analysis At the very least, they will be responsible for adapting screening methods using dedicated systems For analyses involving very small amounts of substances or a high risk of contamination, radiation, etc., glove-box-like analytical sites will be equipped with miniaturized analytical instrumentation, and with disposable and continuous monitoring sensing systems However with on-site analysis comes the need to supervise a number of nonlaboratory personnel In medical programmes, multiple techniques for initial trainings and subsequent follow-ups were exploited Video presentations keep the lessons consistent In addition, some laboratories have assigned point-of- care coordinators

Analytical methods fulfilling special medical requirements are, increasingly, having more direct medical impact, and some analytical services will move from the central laboratory to the bedside or periphery (ICU, ECU) These methods have an immediate impact on the therapy being carried out, such as screening for acute myocardial infarction (AMI) Similar to the situation of polio epidemic in 1952, the necessary tests will be developed and made available to

the medical community relying on early pioneers in point-of-care testing (POCT) which have accumulated a wealth of experience with such analytical techniques

In environmental technology, there is an increasing demand for continuous monitoring of

landfill sites and drinking water supplies Here, the main requirements are not necessarily selectivity and high sensitivity but rather longevity, appropriate detection of chemical classes of compounds, and the quantitation of a cut-off or limiting concentration The evaluation of free analyte concentrations, e.g., of lead ions, in contrast to total concentrations, may be controversial, owing to a lack of consensus about legal limits, even if the free concentration is in equilibrium

with the total concentration Discussions about what is to be analyzed and what is to be reported

will continue, and new solutions and a degree of consensus will be necessary Consideration of the free dissolved species rather than total concentrations may lead to new ways of viewing the toxicity of chemical compounds

For on-site screening, more and more chemical sensors will be evaluated and requested Ensuring the mobility and versatility of the analysis involves producing integrated sensing devices and cordless transmission of signals or results The sensing element itself will be incorporated in a multidimensional and multifunctional robust modular system where it can be replaced easily and safely [136] Chemical sensing elements will either have to ensure long-term stability, extended life-times, and reversibility or, alternatively, they will be offered as cheap, low-waste, disposable elements which can still perform at a suitably high level with respect to reliability and accuracy! The state-of-the-art would currently allow production sites to monitor

some highrisk effluents such as cyanide or heavy metals in situ and immediately

1.4 Goals for Future Developments and Trends 27

Analysis in and during Production

In biotechnology and food technology, real-time, continuous monitoring of bioprocesses saves resources, time and money Selectivity may be less important since the detected species is known and is the predominant or limiting product; dynamic range, sensitivity, and detection limits may be more relevant parameters The cut-off limits where metabolic processes are limited by the concentration of substrates or products are defined very precisely In this situation, the chemical sensor is specifically indicated and has a considerable potential since the design of dedicated systems is the major strength of these applications A large variety of sensors have actually been evaluated, to determine dissolved oxygen, carbon dioxide, inorganic cations and anions such as nitrite, neutral compounds such as alcohols, ammonia and amines, organic

charged compounds, enantiomers, and enantiomeric excess (see chapter 2, 5 and 6) Those who are concerned only with the final product of a process are often unaware of the potential of dedicated systems and their contributions to process design [136] Alternatively, there is no sensor which cannot be realised by one or other of the various well-known introduced concepts,

and, even an aseptic preparation or sterilization is feasible There are no fundamental barriers to

the application of chemical sensors in the future

References

[I] Encyclopedia Americana, 1975; Meyers Enzyklopadisches Lexikon, Mannheim: Bibliographisches Institut

AG, 1972 Analyse: "Auflosung", chemische Analyse: Die Auftrennung von Stoffgemischen in ihre

Einzelkomponenten und deren anschliessende Identifizierung

121 International Federation of Clinical Chemistry (IFCC), J Clin Chem Clin Biochem., 1979

[3] Willard, H.H., Merritt, L.L.M., Dean, J.A., Settle, F.A., Instrumental Methods of Analysis; Belmont: CA,

Wadsworth, Inc., 1988

[4] Several authors in: Karcher, W., Devillers, J., Practical Applications of Quantitative Structure-Activity Relationships (QSAR) in Environmental Chemistry and Toxicology ; EURO COURSES, Chemical and Environmental Science Series, Vol 1; Dordrecht: Kluwer Academic Publ., 1990

[5] a) Lo, C.-M., Keese, C.R., Giaever, I., Exp Cell Res., 1993,204, 102 b) Giaever, I, Keese, C.R., Nature, 1993,

366,590

[6] Nicolini, C., Lanzi, M., Accossato, P., Fanigliulo, A., Mattioli, F., Martelli, A., Biosensors & Bioelectronics,

1995.10.723

171 Buch, R.M., Barker, T.Q., Rechnitz, G.A., Anal Chim Acta, 1991,243, 157

[8] Lundstrbm, I., Gustafsson, A., bdman, S., Karlsson, J.O.G., Andersson, R.G.G., Grundstrom, N., Sundgre, H.,

Elwing, H., Sensors and Actuators, 1990, BI, 533

[9] Sugimoto, K, Teeter, J.H., Chemical Senses, 1991,16, 109

[lo] 24th Japanese Symposium on Taste and Smell (Jasts XXIV) held at Tsu Region Plaza, Tsu, Japan,

November 8-9,1990, Chemical Senses, 1991,16, 181

[ l l ] Rechnitz, G.A., Electroanalysis, 1991.3 73

[I21 a) Scheller, F.W., Schubert, F., Fedrowitz, J., Frontiers in Biosensorics I and I I ; Basel: Birkhiuser Verlag,

1997 b) Scheller, F.W., Hintsche, R., Pfeiffer, D., Schubert, F., Riedel, K., Kindervater, R., Sensors and

Actuators B, 1991,4, 197 c) Scheller, F., Schubert, F., F'feiffer, D., Hintsche, R., Dransfeld, I., Renneberg, R., Wollenberger, U., Riedel, K., Pavlova, M., Kiihn, M., MUller, H.G., Tan, P., Hofmann, W., Moritz, W.,

Analyst, 1991,114, 653

[13] Taylor, R.F., Schultz, J.S., Chemical and Biological Sensors; Bristol IOP Publ Comp., 1996