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Biosensors & Bioelectronics 15 (2000) 273–282 www.elsevier.com / locate / bios Materials and techniques for

Biosensors & Bioelectronics 15 (2000) 273–282

Biosensors & Bioelectronics 15 (2000) 273–282 www.elsevier.com / locate / bios Materials and techniques for

www.elsevier.com/locate/bios

Materials and techniques for electrochemical biosensor design and construction

S. Zhang *, G. Wright, Y. Yang

Centre for Science and Technology in Medicine, WE Dunn Unit, Keele Uni ersity, Staffordshire ST5 5BG, UK

Received 2 November 1999; accepted 20 June 2000

Abstract

New developments in biosensor design are appearing at a high rate as these devices play increasingly important roles in daily life. This review aims to highlight recent developments in materials and techniques for electrochemical biosensor design and construction. Rapid growth in biomaterials, especially the availability and application of a vast range of polymers and copolymers associated with new sensing techniques have led to remarkable innovation in the design and construction of biosensors, significant improvements in sensor function and the emergence of new types of biosensor. Nevertheless, in vivo applications remain limited by functional deterioration due to surface fouling by biological components. However, new copolymers based upon biomembrane mimicry have been extensively investigated during the last two decades, raising hopes that the problems related to interactions between foreign surfaces and biological fluids and tissues may soon be solved. © 2000 Elsevier Science S.A. All rights reserved.

Keywords: Biomaterial; Biosensor; Immobilisation; Biological recognition; Copolymers; Biocompatibility

1. Introduction

In recent years, biosensors have been increasingly used for continuous monitoring of biological and syn- thetic processes and to aid our understanding of these processes. Typical applications include environmental monitoring and control, and chemical measurements in the agriculture, food and drug industries. Biosensors can also meet the need for continuous, real-time in vivo monitoring to replace the intermittent analytical tech- niques used in industrial and clinical chemistry (Fraser,

1997).

Consequently, biosensor development has become a highly active field and a review of currently available materials and fabrication techniques is opportune. In constructing this review, we have chosen to concentrate upon developments that have taken place during the last 5 years with reference to earlier publications that impinge upon the current state of knowledge. More than 1000 relevant publications have appeared since 1995, indicating the intense research activity devoted to this important subject.

* Corresponding author. Tel.: +44-1782-584300; fax: +44-1782-

583516.

The systematic description of a biosensor should include five features (Go¨pel and Schierbaum, 1991). These are (1) the detected, or measured parameter, (2) the working principle of the transducer, (3) the physical and chemical/biochemical model, (4) the application and (5) the technology and materials for sensor fabrica- tion. Many parameters have been suggested to charac- terise a biosensor. Some are commonly used to evaluate the functional properties and quality of the sensor, such as sensitivity, stability and response time; while other parameters are related to the application rather than to sensor function, for example the biocompatibility of sensors for clinical monitoring. When attempting to design a new biosensor, the first question to answer is ‘what parameter is the sensor to be used to detect?’ The first blood pO 2 electrode was introduced by Clark et al. (1953) and the first biosensor was constructed by applying an enzyme membrane onto this electrode by Clark and Lyons (1962). Since then, many biosensors have been developed to detect a wide range of biochemical parameters. Biosensors in- corporating enzymes have been developed to measure concentrations of carbohydrates (glucose, galactose and fructose), proteins (cholesterol and creatinine), amino acids (glutamate) and metabolites (lactate and urea) in

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blood and other body fluids and tissues. It is even possible to measure the concentrations of neurotrans- mitter molecules by means of a neuronal biosensor and the application of this technique to study the actions of anaesthetics has been described by Tvarozek et al. (1998) and by Coon et al. (1997). A range of biologi- cally active molecules, including antibodies and anti- gens has also been measured using immuno-sensors (Van Regenmortel et al., 1998). Most recently, the development of biosensors for the detection of DNA damage and mutation (Healey et al., 1997; Palecek et al., 1998), and the identification of DNA sequences and hybridisation (Wang, 1998) offers considerable promise in several medical fields. The operating principle of a biosensor tells us how the biological process being monitored is converted and transduced to obtain a detectable electrical, optical or other physical signal. Electrochemical electrodes have been used for pH monitoring for over 100 years (Kohrausch, 1885) and the principle established in this technique provides the basis for the most widely used electrochemical biosensors. The electrochemical princi- ple is now well established and both chemical and mathematical models have been developed, including both two and three dimensions (Go¨pel and Schierbaum, 1991). In the simplest applications, the electrochemical reactions occur directly on the electrode surface, or in the space between the electrodes, by the restoration of redox balance between the target molecule, or ion, and the electrolyte (Clark et al., 1953). Field effect transis- tors (FETs) are more complex devices that can also be used in electrochemical biosensors (Bergveld, 1972). However, modern electrochemical biosensors incorpo- rate enzymes, such as glucose oxidase (Updike and Hicks, 1967), sometimes combined with mediators, such as ferrocene and its derivatives (Cass et al., 1984), cofactors, such as nicotinamide adenine dinucleotide (NADH + and NADP) (Albery and Bartlett, 1984), or special indicators (Thorp, 1998). Non-electrochemical techniques including photochemistry (Ramsden, 1997), thermochernistry (Jones and Walsh, 1995), piezoelectric detection and, quite recently, magnetic permeability (Kriz et al., 1996) have also been used to detect biolog- ical signals, but these are not covered in this review. Physical, chemical and mathematical analyses provide the theoretical basis for the design and con- struction of electrochemical biosensors. For example, by including electrode diameter and membrane thick- ness in one- and two-dimensional models, it is possible to estimate the sensitivity and signal-to-noise ratio of a membrane-covered, amperometric gas sensor (Linek et al., 1985). Furthermore, computer-assisted mathemati- cal modelling can be used to describe the very complex biochemical processes that occur in the boundary re- gion close to the sensor interface. This type of mod- elling is particularly valuable in the mass production of biosensors.

Biosensors have been successfully applied in many environmental and industrial situations. However, most clinical applications have, so far, been restricted to academic studies rather than to routine clinical moni- toring. The principal reason for this limitation is the poor biocompatibility of available materials which in- terferes with sensor function (Turner, 1997). The selection of materials and fabrication techniques is crucial for adequate sensor function and the perfor- mance of a biosensor often ultimately depends upon these factors rather than upon the other factors men- tioned above. Consequently, future developments in biosensor design will inevitably focus upon the technol- ogy of new materials, especially the new copolymers that promise to solve the biocompatibility problem and offer the prospect of more widespread use of biosensors in clinical monitoring.

2. Materials

Materials used in electrochemical biosensors are classified as: (1) materials for the electrode and support- ing substrate, (2) materials for the immobilisation of biological recognition elements (3), materials for the fabrication of the outer membrane and (4), biological elements, such as enzymes, antibodies, antigens, media- tors and cofactors.

2.1. Electrodes and supporting substrates

Metals and carbon are commonly used to prepare solid electrode systems and supporting substrates. Metals such as platinum, gold, silver and stainless steel have long been used for electrochemical electrodes due to their excellent electrical and mechanical properties. Carbon-based materials such as graphite, carbon black and carbon fibre are also used to construct the conduc- tive phase. These materials have a high chemical inert- ness and provide a wide range of anode working potentials with low electrical resistivity. They also have a very pure crystal structure that provides low residual currents and a high signal-to-noise ratio (Ce´spedes and Alegret, 1996). Weber (1989) suggested that carbon fibres could be valuable in sensor construction and he showed how a parallel array consisting of a large number of carbon fibres, separated by insulators, can be prepared to obtain a very high signal-to-noise ratio. More recently, a number of new mixed materials have appeared for the preparation of electrodes. A conducting composite formed by the combination of two, or more, dissimilar materials was introduced by Ce´spedes and Alegret (1996). Each material retains its original properties, while giving the composite distinct chemical, mechanical and physical properties that differ from those exhibited by the individual components. A

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carbon-polymer based composite is firstly prepared by dispersing powdered graphite in a polymer resin, such as epoxy, silicone, methacrylate, polyester or polyurethane. With the biological recognition element previously immobilised onto carbon particles, modifier, catalyst or mediator, the polymer composite is then mixed to form the integrated electrode unit. Using this method, Atanasov et al. (1996) prepared an impure metal working electrode with the catalyst and enzyme adsorbed onto pyrolysed cobalt–tetramethoxy–phenyl- porphyrin (CoTMPP). From this basic pressed matrix tablet, it is possible to manufacture numerous elec- trodes with identical functional properties in terms of sensitivity, linearity and lifetime. Organic electroconductive polymers have aroused considerable interest in recent years. These materials can be used to prepare electrodes, or to provide a substrate for the immobilisation of biological elements (see next section) simultaneously. Khan and Wernet (1996) fabricated a novel electrode by the use of a flexible conductive polymer film of polypyrrole doped with polyanions and a microporous layer of platinum black. Glucose sensors produced with this material provided a H 2 O 2 oxidation current at a lower applied potential than conventional sensors.

2.2. Materials used for the immobilisation of biological elements

Most of the materials traditionally used for this purpose are multifunctional agents such as glutaralde- hyde and hexamethyl diisocyanate, which form cross- links between biocatalytic species, or proteins. The process is known as coreticulation, since it creates complex matrices that make multienzyme immobilisa- tion possible. Alternatively, non-conductive polymers, such as polyacrylamide and polyphenol, can be used to entrap elements physically. Organic conductive polymers provide advantages, in- cluding the formation of an appropriate environment for enzyme immobilisation at the electrode and for its interaction with metallic and carbon conductors (Bartlett and Cooper, 1993; Trojanowicz and Krawczyk, 1995). Therefore, electrical communication between the redox centre and the electrode surface is more efficient. These polymers can be deposited electrolytically from solution onto a conducting support to provide a three- dimensional matrix for immobilised enzymes where reac- tants are converted to products. The polymers can be produced by a variety of chemical processes, including the Ziegler–Natta reaction for polyacetylene (Shirikawa, 1982; Naarmann and Theophilou, 1987), the creation of an electrolyte solution e.g. poly(p-phenylene) (Lenz et al., 1988), the coupling of organometallic components (polythiophene) (Kobayashi et al., 1984) and the oxidation of monomers (Ratcliffe, 1990).

Redox polymer hydrogels entrap oxidoreductases efficiently and transfer electrons from enzymatic oxida- tion/reduction reactions through the gel to the conduct- ing surface (Sirkar and Pishko, 1998). Gels based on networks of polyethylene glycol, diacrylate and vinyl- ferrocene can be formed by illuminating a solution containing the comonomers and the ultraviolet pho- toinitiator, 2,2 -dimethoxy-2-phenylacetophenone at 365 nm, 20 W cm 2 . The enzyme can then be loaded by dissolving it in this mixture followed by exposure to light. Latex particles also provide suitable substrates for the controlled attachment of biomolecules in the recog- nition of analytes. Studies on the formation of two-di- mensional latex assemblies covalently immobilised on conducting solid surfaces were performed by Slomkowski et al., (1996). Computer simulations illus- trated the general properties of the 2-D latex assemblies and a real example of the composite, polystyrene/acro- lein latex, on a quartz surface were presented. The immobilisation of human serum albumin and proteins from goat anti-HAS serum on 2-D latex assemblies was also discussed.

2.3. Membrane materials and biocompatibility

Biosensors are usually covered with a thin membrane that has several functions, including diffusion control, reduction of interference and mechanical protection of the sensing probe. Commercially available polymers, such as polyvinyl chloride (PVC), polyethylene, poly- methacrylate and polyurethane are commonly used for the preparation of these membranes due to their suit- able physical and chemical properties. Biosensors with polymer membranes have been successfully applied in many fields such as the monitoring of food production, environmental pollution and pathological specimens. However, when biosensors are placed in a biological environment, numerous factors operate to affect their performance, the most significant ones being sensor surface interactions with proteins and cells (Donald and Jeffrey, 1996). Therefore, although biosensors have great potential for real-time clinical monitoring, the sensors so far constructed lack functional stability after implantation and sensor lifetime is usually restricted to several hours, or days (Fischer, 1995). Thus, functional stability is profoundly affected by the biocompatibility of the biosensor materials that are in contact with the biological medium. Attempts to improve the biocompatibility of artificial surfaces by bonding anticoagulants have not been very successful. For example, the surface treatment of mem- branes with heparin sulphate is commonly used to improve haemocompatibility, but when Smith and Sefton (1993) analysed thrombin adsorption onto hep- arin treated polyvinyl alcohol and polyurethane, they

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observed that, whereas the rate of adsorption of thrombin was reduced by heparin coating, the final volume of thrombin adsorbed remained similar. An- other problem is that the heparin tends to leach from the membrane surface into the surrounding medium. Materials with hydrogel-like properties are generally considered to favour biocompatibility. Water associates with the water-soluble polymers and the presence of water around the polymer hinders protein adsorption due to the energetically unfavourable displacement of water by protein and compression of the polymer upon the approach of protein. These factors have been de- scribed in terms of steric repulsion, van der Waals attraction, and hydrophobic interactive free energies by Jeon et al. (1991) and by Jeon and Andrade (1991). Surfaces grafted with water-soluble polymers have been developed using a number of techniques, including end-grafting (Shoichet et al., 1994) and in situ poly- merisation by photo- or wet- chemistry, and by radio frequency glow discharge deposition (Lopez et al., 1992). An alternative to surface grafting is the adsorp- tion of amphiphilic molecules onto hydrophobic poly- mers. Amphiphilic molecules can rearrange themselves on a surface in an attempt to maximise packing density and can be covalently immobilised on the surface to create a permanent adsorption layer (Nojiri et al., 1992). Polyethylene glycol (PEG) has been used exten- sively to modify surfaces, so that protein adsorption and platelet interactions with the foreign surface are reduced (Amiji and Park, 1994). The natural cell membrane is a self-assembled system and the extra-cellular matrix is a nano-structured sys- tem. Based upon these observations, amphiphilic, self- assembled multilayers and nano-structured surface systems have been exploited in the production of lipo- somes modified with PEG. These surfaces suffer from significantly less protein adsorption and immune clear- ance mechanisms are reduced (Woodle and Lasic, 1992). Chemical adsorption has been used to produce self-assembled monolayers on metals and ceramics. Alkanes terminated with thiols form densely packed monolayers, with the alkane chain oriented outwardly from the substrate surface. Dimilla et al., (1994) re- ported that protein adsorption could be virtually elimi- nated by alkane thiol terminated with oligoethylene glycol. An optical biosensor chemically adsorbed with a monolayer of 16-mercaptohexadecan-1-ol has been pro- duced to measure protein interactions with gold coated surfaces (Lofa˚s, 1995). Surface modification of polymers has led to modest improvements in biocompatibility, but it is still not satisfactory for long-term in vivo applications, so there is an urgent need to design and develop new biocom- patible materials. During the last two decades, several attempts have been made to do this by synthesising new phospholipid copolymers based upon the principle of

biological membrane mimicry. These copolymers have been successfully used as drug carriers as well as biosen- sor membranes. The basic philosophy behind this idea is due to Zwaal et al., (1977), who described the com- plex relationships between cell membrane structure and blood coagulation. In vitro coagulation tests demon- strated that the inner surfaces of the plasma membrane of erythrocytes and platelets are highly procoagulant, but the outer surfaces are inactive. Liposomes having the same phospholipid composition as the outer sur- faces of erythrocyte and platelet membranes were also inactive and did not reduce the time for recalcified plasma to clot. The simplest common feature of these non-reactive cellular and model membranes is the high content of electrically neutral phospholipids with phosphoryl- choline head groups (Zwaal, 1978). Consequently, the synthetic copolymer, poly(MPC-co-BMA) which con- tains head groups of 2-methacryloyloxyethyl phospho- rylcholine (MPC) copolymerised with n-butyl methacrylate (BMA), also exhibits surface properties that are favourable for haemocompatibility. The sur- faces are extremely hydrophilic and they contain large volumes of water (Ishihara et al., 1990). During the construction of the membrane by liquid evaporation, the whole phospholipid molecule, which includes two fatty acid chains, undergoes significant rotation to min- imise interface energy and this results in the orientation of the phosphorylcholine head group towards the side of the membrane that is exposed to air. The MPC moiety also has a strong affinity for natural phospho- lipid molecules in plasma, so a well organised natural lipid layer, biomembrane-like structure, forms on this surface during its exposure to plasma (Kojima et al., 1991; Zhang et al., 1998). Protein adsorption is signifi- cantly reduced on poly(MPC-co-BMA) surfaces com- pared with other medical polymers (Ueda et al., 1990; Zhang et al., 1998) and the in vitro and in vivo perfor- mance of biosensors is significantly improved when poly(MPC-co-BMA) is coated onto the sensor surface (Chen et al., 1993; Zhang et al., 1996a,b). Zhang et al. (1998) suggested that these membranes might simulate natural membranes functionally as well as structurally the natural phospholipids may be continuously replen- ished in a continuous process of erosion and repair.

2.4. Biological elements

Improvements in interface design have frequently been directed at the incorporation of active molecules, including enzymes such as glucose oxidase (Clark and Lyons, 1962) and lactate oxidase (Vadgama et al., 1986), mediators, such as Ferrocene ( 2 -bis-cyclopenta- dienyl iron) and its derivatives (Cass et al., 1984), cofactors based on nicotinamide adenine dinucleotide (NADH + and NADP + (Jaegfeldt et al., 1981), cata-

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lysts (Cosnier et al., 1997), antibodies and antigens (Umezawa et al., 1980). Studies on the use of biosensors for gene detection are relatively recent and still uncommon. Deoxyribonu- cleic acid (DNA) has recently been suggested as a biological recognition element for such biosensors (Vadgama and Crump, 1992; Mulchandani and Bassi, 1995). The unique nucleotide base structure of DNA provides the basis of the technique which allows single- stranded DNA (ssDNA) to be used to identify other ssDNA molecules with the complementary bases (Zhai et al., 1997). Therefore, nucleic acid hybridisation is the underlying operating principle of DNA biosensors. During the last decade, there have been many advances in DNA biosensor technology and most work has focused on electrochemical, piezoelectric and optical transducers. Attempts to develop an electrochemical DNA biosensor have been made by several groups (Millan et al., 1994; Marrazza et al., 1999). In these sensors, an ssDNA strand is covalently bound to the surface of an electrode. Hybridisation of the immobilised sequence with its dissolved complement forms the double strand that can be detected using a DNA-specific redox-active metal/polypyridine complex. Damaged segments of DNA can also be detected by measuring changes in the redox signals of base residues in DNA immobilised on carbon electrodes. Covalently closed circular DNA can be attached to an electrode surface to obtain a sensor that detects a single break in the DNA sugar-phos- phate backbone, or for the detection of agents leaving the DNA backbone such as hydroxyl radicals, ionising radiation or nucleases (Palecek et al., 1998). DNA sensing protocols, based on different modes of nucleic acid interaction have been reviewed by Wang et al. (1997). The review describes recent efforts to couple nucleic acid recognition layers to electrochemical transducers. Peptide nucleic acids (PNAs) have been found to exhibit unique and efficient hybridisation properties that may offer significant advantages for sequence-spe- cific recognition compared to their DNA counterparts. The advantages include higher sensitivity and specific- ity, faster hybridisation at room temperature and mini- mal dependence upon ionic strength. The use of PNA incorporated with a Co(phen)(3)(3+ ) redox indicator on a carbon electrode for the detection of sequence specific DNA has been discussed by Wang et al.

(1996).

3. Techniques for the preparation of biosensors

Design and construction technology and materials science are intimately linked in biosensor development. Therefore, discussions of biosensor design and fabrica-

tion should always involve the selection of materials. An electrochemical biosensor usually consists of a transducer such as a pair of electrodes or FET, an interface layer incorporating the biological recognition molecules and a protective coating. Sensor design, including materials, size and shape and methods of construction, are largely dependent upon the principle of operation of the transducer, the parameters to be detected and the working environment. Traditional electrode systems for measurements of the concen- trations of ions in liquids and dissolved gas partial pressures contain only a working electrode (usually a noble metal wire) and an electrically stable reference electrode, such as Ag/AgCl, though a counter electrode is sometimes included. A simple electrical, or chemical, modification may sometimes improve specific electrode properties. For example, repeated potential cycling of 0.3 mm diameter carbon rods in 0.1 M potass- ium hexacyanferrate improved the stability of glu- cose sensors for up to 6.5 days (Jaffari and Pickup,

1996).

However, with the expanding demands for more complex measurements, the rapid development of ma- terials science and the emergence of micro- and nano- process technology, indirect electrochemical methods in simple biosensors to monitor enzyme activity have gradually been replaced by more direct, but more com- plex processes. Methods for the preparation of electrochemical elec- trodes are well established. Some of these techniques are used to prepare the conductive supporting sub- strate, while others are employed to achieve an efficient electrical communication between the chemical reaction site and the electrode surface, high levels of integra- tion, sensor miniaturisation, measurement stability, se- lectivity, accuracy and precision. In addition, the technique used to immobilise the biological recognition components of the sensor can affect biosensor perfor- mance significantly.

3.1. Electrode fabrication

The electrode supporting substrate can be a noble metal (gold or platinum), carbon rod or paste, or an organic conducting salt or polymer. Techniques used for the production of conductive supporting substrates can be roughly classified as: (1) printing, (2) deposition, (3) polymerisation, (4) plasma induced polymerisation, (5) photolithography and (6) nano-technology. (1) Screen-printing is one of the thick-film techniques that have been widely used in industry for mass pro- duction. Paste material is printed onto a matrix di- rectly through a mask-net with a designed pattern. The technique of carbon, or graphite, screenprinting is now frequently used to prepare electrodes for biosensors. Turner’s group recently improved this technique by

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using solvent resistant materials, heat stabilised polyester sheet, carbon base tracks and an epoxy-based polymer (Kroger and Turner, 1997). These electrodes have no problems with solvent induced baseline shift and are therefore suitable for work in water-miscible organic solvents. Sensor arrays for the detection of more than one parameter by different sensing techniques, or to assem- ble a package of sensors to measure the same parame- ter, have potential practical applications. For this purpose, the screen printing technique has been used to prepare a seven-channel electrode for simultaneous am- perometric and potentiometric measurements. The ar- ray contains 14 gold working and counter electrodes and one Ag/AgCl reference electrode (Silber et al., 1996a). This sensor can be used to analyse blood and serum electrolytes and metabolites. Khan (1996a) introduced a technique to produce a printable biosensor. A printable paste is prepared by mixing glucose oxidase adsorbed organic charge trans- fer complex crystals with a binder and a solvent. This paste is printed onto a matrix cavity and dried under vacuum. A thin layer of gelatin is then cast on the electrode. The developed sensor provides a huge response current with minimum interference from oxy- gen and an extended linear range up to 100 mM glucose. Techniques (2), (3), (4) and (5) are thick- /thin-film techniques that are used in biosensors to form mono- or multi-layers of conducting film onto a supporting substrate in order to obtain a direct electrical commu- nication between the chemical/biochemical reaction site and the supporting surface. Factors affecting electron transfer from biological molecules to electrode surfaces have been reviewed by Cardosi and Turner (1991). (2) Traditional chemical, or electrochemical, deposi- tion methods can be used to deposit an electroconduc- tive film on a supporting substrate. The deposited film can be metal such as platinum, catalytic material such as TiO, or a metal complex. Biological elements can also be simultaneously coupled during the film deposi- tion process. Lorenzo et al. (1998) discussed the analyt- ical strategies for various electrodeposited films. (3) Polymerisation takes place due to the condensa- tion of small molecules in monomers, or by free radical creation and reaction by rearranging the bonds within each monomer. Free radicals are produced when the double bond is broken by initiation activated by heat, light, or electro-chemicals. Electrical conductivity can be achieved by the introduction of metal powder into the monomer before polymerisation, or through elec- trons that are not conjugated in the monomer. A new technique has been developed to generate poly- methylene blue-modified thick-film on gold electrodes by electropolymerisation to form an eletrocatalytically active conducting layer that is in intimate and stable

contact with the electrode surface. This process allows for a reduced applied potential of only 200 mV, the avoidance of inference from co-oxidisable species and the minimisation of electrode fouling (Silber et al., 1996b). By depositing a thin electropolymerised film of poly(1,3-diaminobenzene), electrochemical interference from ascorbate, urate, acetaminophen and other oxi- disable species can be greatly diminished (Madaras and Buck, 1996). Sirkar and Pishko (1998) reported that a photo-initiated free-radical polymerised redox hydrogel polymer entrapped enzymes efficiently and increased the transfer of electrons from enzyme oxidation/reduc- tion reactions through the gel to the electrode surface. (4) Plasma-induced polymerisation is performed un- der high vacuum. The principle is to introduce func- tional groups onto the substrate surface and then ‘polymerisable’ gas plasma is coated onto this surface to form a layer of film. Plasma-polymerised film, gen- erated in a glow discharge, or plasma in a vapour phase, may offer a new alternative for biosensor inter- face design. The advantage of this technique is that it can produce an extremely thin ( 1 m) film that adheres firmly to substrates. Furthermore, the film is pin-hole free and both mechanically and chemically stable, and it allows a large amount of biological material to be loaded onto the surface (Muguruma and Karube, 1999). (5) Photolithography techniques have long been used in the semiconductor industry to produce integrated chips. Light passes through a photo-mask and is cast upon a photodegraded material surface to form a pat- tern. Nussbaum et al. (1997) used this technique to fabricate micro-lens arrays for sensors. The manufac- ture of integrate transducer arrays for measurements of a single parameter, or for several different parameters, is now possible by means of photolithography and plasma technology. These techniques have been used to increase the dynamic range and sensitivity of urea sensors (Steinschaden et al., 1997). (6) Nano-techniques have recently appeared with the maturation of modern technologies such as surface probe microscopy and lithography, atomic force mi- croscopy (AFM), AFM lithography and lateral force microscopy (LFM). A brief historical overview was given by Skladal and Macholan (1997), in which recent developments in miniaturisation, microfabrication, nanotechnology, immuno-sensors and gene-sensors are discussed. Sugimura and Nakagiri (1997) produced patterns with resolutions in the nanometer range based upon photo- and AFM- lithography with an orgaosi- lane monolayer resist. The combination of techniques mentioned above leads to multilayer structures that may well prove to be useful in the development of new types of biosensor. Bilayer polymer coatings consisting of polypyrrole acid poly(o-phenylenediamine) on a supporting substrate

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may improve selectivity and reduced inference from electroactive species like uric and ascorbic acids that are often present in biological samples (Vidal et al., 1999). Kranz et al., (1998) proposed a multilayer architecture to predefine electron-transfer pathways, integrate redox mediators, immobilise enzymes and restrict diffusional access by interfering compounds. A multilayer wafer can also be formed by depositing a thin functionalized polypyrrole film on the supporting surface and then covalently bonding a redox dye of polymerised quinoidic species to prevent electrode fouling. The top of this layer is coated with polypyrrole with entrapped tyroinase. Electrons transfer from the quinone to the electrode surface via the immobilised redox dye. Other new techniques have been suggested that could be useful in the design and construction of new biosen- sors. The most promising of these may be the forma- tion of a direct electrochemical communication between the active enzyme site and the electrode surface using a biocatalyst with a very low molecular weight, such as microperoxidase MP-11, immobilised on a thio-mono- layer. In this case, the distance between enzyme and electrode surface is greatly reduced in comparison with earlier constructions and this modification significantly increases the strength of the output signal (Lotzbeyer et al., 1996). Khan (1996b) reported a stable organic charge-trans- fer-complex (CTC) electrode for the direct oxidation of flavoproteins. To construct the CTC electrode, tetrathi- afulvalene-tetracyanoquinodimethane is grown at the surface of an electroconductive polyanion-doped polypyrrole film in such a way that it makes a tree- shaped crystal structure, standing vertically on the sur- face. By immobilising a glucose enzyme on the CTC electrode, direct electron transfer is achieved between the active enzyme and the crystal electrode and this leads to remarkably improved sensor performance. An electrically conductive and mechanically flexible composite polymer was prepared to construct a glucose sensor by Yamato et al. (1997). Using this technique, fine palladium particles are dispersed in polypyrrole/ sulfated poly(beta-hydroxyethers) by thermally decom- posing the bis(dibenzylideneacetone)–palladium complex. With Ag/AgCl as a reference electrode, a conventional platinum electrode responded to glucose at a working potential of 650 mV, whereas the new electrode responded at 400 mV. Elrhazi et al., (1997) used fibrinogen film to provide a porous, non-reactive layer over a carbon paste elec- trode to control the mass transfer rate of diffusing species and Muller et al. (1997) introduced the tech- nique of pulsed LASER deposition (PLD) with opti- mised LASER parameters and reaction atmosphere to obtain more efficient enzyme activities than the conven- tional platinum film electrode produced by argon sputtering.

3.2. Immobilisation methods

Considerable progress has been made in the develop- ment of new methods of immobilising biological recog- nition elements onto sensor surfaces. The use of self-assembled mono- and multi-layers (SAMs) is in- creasing rapidly in various fields of research, and this applies especially to the construction of biosensors. SAMs can be used as interface layers upon which almost all types of biological components, including proteins, enzymes, antibodies and their receptors, and even nucleotides for DNA recognition, can be loaded (Wink et al., 1997). The use of biomembranes as recog- nition elements was introduced by the pioneering work of Mueller et al., (1962). Lipid membranes provide a relatively blocompatible surface and mass diffusion based sensors constructed with lipid membranes have fast response rates and high sensitivities. However, there was no practical use of lipid films until the development of physically stable bilayer lipid mem- branes (BLMs). BLMs can be formed on metal sur- faces, conductive polymer supports, or agar plates. The technique usually proceeds by two steps. First, the support substrate is coated with the lipid layer by immersing it in a lipid solution and then placed in an electrolyte solution to create the self-assembled lipid bilayer. The incorporation of biological recognition molecules into lipid layers and their immobilisation on BLMs depend upon the degree of access to reactive sites on the molecules. The various techniques used for this purpose have been described by Nikolelis et al.

(1999). Hianik et al. (1998) recently described the tech- nique used to construct an immunosensor based on a self-assembled BLM supported on a metal surface. Avidin modified monoclonal antibodies, originating from the E2/G2 clone (AMab), and matched antigens were incorporated in the membrane. Amperometric biosensors for glucose and urea measurement have also been produced by the immobilisation of glucose oxidase and urease into BLMs through the avidin–biotin inter- action (Hianik et al., 1997).

A layer-by-layer deposition technique may be used to

optimise enzyme loading in bienzyme systems (Chen et al., 1998). With up to ten monomolecular layers con- taining avidin, biotin residues and choline oxidase (ChOx), and two superficial layers containing choline

esterase, the sensor exhibits an increased response to acetylcholine.

A technique to prepare gold electrodes with nanome-

ter-sized openings for the immobilisation of biological recognition elements, such as antibodies, has been de- scribed by Padeste et al., (1996). Latex spheres are used as a masking material to create 60 nm diameter holes in gold film evaporated onto a supporting substrate. The nanometer-scale proximity of the recognition compo-

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nents to the conducting surface may facilitate the devel- opment of biosensors without mediators. A multi-enzyme sandwich comprised of layers of separately immobilised enzymes was prepared by Sorochinskii and Kurganov (1996). Factors controlling the concentration of enzyme, enzyme kinetics, and the permeability and thickness of the coating components had been evaluated previously. Enzyme multilayers composed of avidin, biotin-labelled glucose oxidase and ascorbate oxidase are considered to be resistant to ascorbate interference (Anzai et al., 1995). By the use of avidin to crosslink and immobilise the glucose oxidase, an electroreduction current may be measured at the extremely low potential of 100 mV (Vreeke and Rocca,

1996).

4. Conclusions

Biosensor development and production are currently expanding due to the recent application of several new techniques, including some derived from physical chem- istry, biochemistry, thick- and thin-film physics, materi- als science and electronics. The necessary expertise and facilities are sophisticated and expensive. Contrary to expectations expressed in market surveys conducted during the early 1990s, the biosensor market has not expanded as predicted, mainly due to unanticipated technological problems. This is especially true for po- tential applications in clinical real-time monitoring and diagnosis in vivo, which may remain at the experimen- tal stage for several more years. Before biosensors can make significant contributions in these fields, further developments will be needed to improve the long- term performance of biosensors in complex biological envi- ronments. This will inevitably involve the use of new biocompatible materials.

Acknowledgements

The authors gratefully acknowledge the financial as- sistance of Action Research and the valuable advice of Dr Francis Walker.

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