Materials and Techniques for Electrochemical Biosensor Design And

8/14/2019 Materials and Techniques for Electrochemical Biosensor Design And

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Biosensors & Bioelectronics 15 (2000) 273282

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 Uni6ersity, 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

www.elsevier.com/locate/bios

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.

The systematic description of a biosensor should

include five features (Gopel 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 tosensor 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 pO2 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 measureconcentrations of carbohydrates (glucose, galactose and

fructose), proteins (cholesterol and creatinine), amino

acids (glutamate) and metabolites (lactate and urea) in* Corresponding author. Tel.: +44-1782-584300; fax: +44-1782-

583516.

0956-5663/00/$ - see front matter 2000 Elsevier Science S.A. All rights reserved.

PII: S 0 9 5 6 - 5 6 6 3 ( 0 0 ) 0 0 0 7 6 - 2


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S. Zhang et al. /Biosensors & Bioelectronics 15 (2000) 273282274

blood and other body fluids and tissues. It is evenpossible to measure the concentrations of neurotrans-mitter molecules by means of a neuronal biosensor andthe application of this technique to study the actions ofanaesthetics 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, thedevelopment of biosensors for the detection of DNAdamage and mutation (Healey et al., 1997; Palecek etal., 1998), and the identification of DNA sequences andhybridisation (Wang, 1998) offers considerable promisein several medical fields.

The operating principle of a biosensor tells us howthe biological process being monitored is converted andtransduced to obtain a detectable electrical, optical orother physical signal. Electrochemical electrodes havebeen used for pH monitoring for over 100 years

(Kohrausch, 1885) and the principle established in thistechnique provides the basis for the most widely usedelectrochemical biosensors. The electrochemical princi-ple is now well established and both chemical andmathematical models have been developed, includingboth two and three dimensions (Gopel and Schierbaum,1991). In the simplest applications, the electrochemicalreactions occur directly on the electrode surface, or inthe space between the electrodes, by the restoration ofredox balance between the target molecule, or ion, andthe 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 andHicks, 1967), sometimes combined with mediators, suchas ferrocene and its derivatives (Cass et al., 1984),cofactors, such as nicotinamide adenine dinucleotide(NADH+ and NADP) (Albery and Bartlett, 1984), orspecial indicators (Thorp, 1998). Non-electrochemicaltechniques including photochemistry (Ramsden, 1997),thermochernistry (Jones and Walsh, 1995), piezoelectricdetection 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 analysesprovide 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 possibleto estimate the sensitivity and signal-to-noise ratio of amembrane-covered, amperometric gas sensor (Linek etal., 1985). Furthermore, computer-assisted mathemati-cal modelling can be used to describe the very complexbiochemical 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 ofbiosensors.

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 techniquesis 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 (Cespedes andAlegret, 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

Cespedes and Alegret (1996). Each material retains its

original properties, while giving the composite distinctchemical, mechanical and physical properties that differ

from those exhibited by the individual components. A


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S. Zhang et al. /Biosensors & Bioelectronics 15 (2000) 273282 275

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 impuremetal working electrode with the catalyst and enzyme

adsorbed onto pyrolysed cobalttetramethoxyphenyl-

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 H2O2 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 ZieglerNatta 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 organometalliccomponents (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 at365 nm, 20 W cm2. 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, thesensors 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 andSefton (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 ofwater 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 systemand 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 (Lofas, 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, severalattempts 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 structurallythe 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 (p2-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 DNAprovides 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 leavingthe 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 materialsscience 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 bedetected 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 noblemetal (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 nowfrequently used to prepare electrodes for biosensors.

Turners 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 electrodesby 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 andBuck, 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 (B1 mm) 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 forcemicroscopy (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 functionalizedpolypyrrole 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-dopedpolypyrrole 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) palladiumcomplex. 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 forthis 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 avidinbiotin inter-

action (Hianik et al., 1997).

A layer-by-layer deposition technique may be used tooptimise 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 ingold film evaporated onto a supporting substrate. The

nanometer-scale proximity of the recognition compo-


8/10


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 multilayerscomposed 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 currentlyexpanding 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 ofDr Francis Walker.

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