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Biosensors & Bioelectronics 15 (2000) 273 – 282

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 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

1. Introduction The systematic description of a biosensor should


include five features (Göpel and Schierbaum, 1991).
In recent years, biosensors have been increasingly These are (1) the detected, or measured parameter, (2)
used for continuous monitoring of biological and syn- the working principle of the transducer, (3) the physical
thetic processes and to aid our understanding of these and chemical/biochemical model, (4) the application
processes. Typical applications include environmental and (5) the technology and materials for sensor fabrica-
monitoring and control, and chemical measurements in tion. Many parameters have been suggested to charac-
the agriculture, food and drug industries. Biosensors terise a biosensor. Some are commonly used to evaluate
can also meet the need for continuous, real-time in vivo the functional properties and quality of the sensor, such
monitoring to replace the intermittent analytical tech- as sensitivity, stability and response time; while other
niques used in industrial and clinical chemistry (Fraser, parameters are related to the application rather than to
1997). sensor function, for example the biocompatibility of
Consequently, biosensor development has become a sensors for clinical monitoring.
highly active field and a review of currently available When attempting to design a new biosensor, the first
materials and fabrication techniques is opportune. In question to answer is ‘what parameter is the sensor to
constructing this review, we have chosen to concentrate be used to detect?’ The first blood pO2 electrode was
upon developments that have taken place during the introduced by Clark et al. (1953) and the first biosensor
last 5 years with reference to earlier publications that was constructed by applying an enzyme membrane
impinge upon the current state of knowledge. More onto this electrode by Clark and Lyons (1962). Since
than 1000 relevant publications have appeared since then, many biosensors have been developed to detect a
1995, indicating the intense research activity devoted to wide range of biochemical parameters. Biosensors in-
this important subject. corporating enzymes have been developed to measure
concentrations of carbohydrates (glucose, galactose and
* Corresponding author. Tel.: +44-1782-584300; fax: +44-1782- fructose), proteins (cholesterol and creatinine), amino
583516. acids (glutamate) and metabolites (lactate and urea) in

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
274 S. Zhang et al. / Biosensors & Bioelectronics 15 (2000) 273–282

blood and other body fluids and tissues. It is even Biosensors have been successfully applied in many
possible to measure the concentrations of neurotrans- environmental and industrial situations. However, most
mitter molecules by means of a neuronal biosensor and clinical applications have, so far, been restricted to
the application of this technique to study the actions of academic studies rather than to routine clinical moni-
anaesthetics has been described by Tvarozek et al. toring. The principal reason for this limitation is the
(1998) and by Coon et al. (1997). A range of biologi- poor biocompatibility of available materials which in-
cally active molecules, including antibodies and anti- terferes with sensor function (Turner, 1997).
gens has also been measured using immuno-sensors The selection of materials and fabrication techniques
(Van Regenmortel et al., 1998). Most recently, the is crucial for adequate sensor function and the perfor-
development of biosensors for the detection of DNA mance of a biosensor often ultimately depends upon
damage and mutation (Healey et al., 1997; Palecek et these factors rather than upon the other factors men-
al., 1998), and the identification of DNA sequences and tioned above. Consequently, future developments in
hybridisation (Wang, 1998) offers considerable promise biosensor design will inevitably focus upon the technol-
in several medical fields. ogy of new materials, especially the new copolymers
The operating principle of a biosensor tells us how that promise to solve the biocompatibility problem and
the biological process being monitored is converted and offer the prospect of more widespread use of biosensors
transduced to obtain a detectable electrical, optical or in clinical monitoring.
other physical signal. Electrochemical electrodes have
been used for pH monitoring for over 100 years
(Kohrausch, 1885) and the principle established in this 2. Materials
technique provides the basis for the most widely used
electrochemical biosensors. The electrochemical princi- Materials used in electrochemical biosensors are
ple is now well established and both chemical and classified as: (1) materials for the electrode and support-
mathematical models have been developed, including
ing substrate, (2) materials for the immobilisation of
both two and three dimensions (Göpel and Schierbaum,
biological recognition elements (3), materials for the
1991). In the simplest applications, the electrochemical
fabrication of the outer membrane and (4), biological
reactions occur directly on the electrode surface, or in
elements, such as enzymes, antibodies, antigens, media-
the space between the electrodes, by the restoration of
tors and cofactors.
redox balance between the target molecule, or ion, and
the electrolyte (Clark et al., 1953). Field effect transis-
2.1. Electrodes and supporting substrates
tors (FETs) are more complex devices that can also be
used in electrochemical biosensors (Bergveld, 1972).
Metals and carbon are commonly used to prepare
However, modern electrochemical biosensors incorpo-
rate enzymes, such as glucose oxidase (Updike and solid electrode systems and supporting substrates.
Hicks, 1967), sometimes combined with mediators, such Metals such as platinum, gold, silver and stainless steel
as ferrocene and its derivatives (Cass et al., 1984), have long been used for electrochemical electrodes due
cofactors, such as nicotinamide adenine dinucleotide to their excellent electrical and mechanical properties.
(NADH+ and NADP) (Albery and Bartlett, 1984), or Carbon-based materials such as graphite, carbon black
special indicators (Thorp, 1998). Non-electrochemical and carbon fibre are also used to construct the conduc-
techniques including photochemistry (Ramsden, 1997), tive phase. These materials have a high chemical inert-
thermochernistry (Jones and Walsh, 1995), piezoelectric ness and provide a wide range of anode working
detection and, quite recently, magnetic permeability potentials with low electrical resistivity. They also have
(Kriz et al., 1996) have also been used to detect biolog- a very pure crystal structure that provides low residual
ical signals, but these are not covered in this review. currents and a high signal-to-noise ratio (Céspedes and
Physical, chemical and mathematical analyses Alegret, 1996). Weber (1989) suggested that carbon
provide the theoretical basis for the design and con- fibres could be valuable in sensor construction and he
struction of electrochemical biosensors. For example, showed how a parallel array consisting of a large
by including electrode diameter and membrane thick- number of carbon fibres, separated by insulators, can
ness in one- and two-dimensional models, it is possible be prepared to obtain a very high signal-to-noise ratio.
to estimate the sensitivity and signal-to-noise ratio of a More recently, a number of new mixed materials
membrane-covered, amperometric gas sensor (Linek et have appeared for the preparation of electrodes. A
al., 1985). Furthermore, computer-assisted mathemati- conducting composite formed by the combination of
cal modelling can be used to describe the very complex two, or more, dissimilar materials was introduced by
biochemical processes that occur in the boundary re- Céspedes and Alegret (1996). Each material retains its
gion close to the sensor interface. This type of mod- original properties, while giving the composite distinct
elling is particularly valuable in the mass production of chemical, mechanical and physical properties that differ
biosensors. from those exhibited by the individual components. A
S. Zhang et al. / Biosensors & Bioelectronics 15 (2000) 273–282 275

carbon-polymer based composite is firstly prepared by Redox polymer hydrogels entrap oxidoreductases
dispersing powdered graphite in a polymer resin, such efficiently and transfer electrons from enzymatic oxida-
as epoxy, silicone, methacrylate, polyester or tion/reduction reactions through the gel to the conduct-
polyurethane. With the biological recognition element ing surface (Sirkar and Pishko, 1998). Gels based on
previously immobilised onto carbon particles, modifier, networks of polyethylene glycol, diacrylate and vinyl-
catalyst or mediator, the polymer composite is then ferrocene can be formed by illuminating a solution
mixed to form the integrated electrode unit. Using this containing the comonomers and the ultraviolet pho-
method, Atanasov et al. (1996) prepared an impure toinitiator, 2,2%-dimethoxy-2-phenylacetophenone at
metal working electrode with the catalyst and enzyme 365 nm, 20 W cm − 2. The enzyme can then be loaded
adsorbed onto pyrolysed cobalt – tetramethoxy – phenyl- by dissolving it in this mixture followed by exposure to
porphyrin (CoTMPP). From this basic pressed matrix light.
tablet, it is possible to manufacture numerous elec- Latex particles also provide suitable substrates for
trodes with identical functional properties in terms of the controlled attachment of biomolecules in the recog-
sensitivity, linearity and lifetime. nition of analytes. Studies on the formation of two-di-
Organic electroconductive polymers have aroused mensional latex assemblies covalently immobilised on
considerable interest in recent years. These materials conducting solid surfaces were performed by
can be used to prepare electrodes, or to provide a Slomkowski et al., (1996). Computer simulations illus-
substrate for the immobilisation of biological elements trated the general properties of the 2-D latex assemblies
(see next section) simultaneously. Khan and Wernet and a real example of the composite, polystyrene/acro-
(1996) fabricated a novel electrode by the use of a lein latex, on a quartz surface were presented. The
flexible conductive polymer film of polypyrrole doped immobilisation of human serum albumin and proteins
with polyanions and a microporous layer of platinum from goat anti-HAS serum on 2-D latex assemblies was
black. Glucose sensors produced with this material also discussed.
provided a H2O2 oxidation current at a lower applied
potential than conventional sensors. 2.3. Membrane materials and biocompatibility

2.2. Materials used for the immobilisation of biological Biosensors are usually covered with a thin membrane
elements that has several functions, including diffusion control,
reduction of interference and mechanical protection of
Most of the materials traditionally used for this the sensing probe. Commercially available polymers,
purpose are multifunctional agents such as glutaralde- such as polyvinyl chloride (PVC), polyethylene, poly-
hyde and hexamethyl diisocyanate, which form cross- methacrylate and polyurethane are commonly used for
links between biocatalytic species, or proteins. The the preparation of these membranes due to their suit-
process is known as coreticulation, since it creates able physical and chemical properties. Biosensors with
complex matrices that make multienzyme immobilisa- polymer membranes have been successfully applied in
tion possible. Alternatively, non-conductive polymers, many fields such as the monitoring of food production,
such as polyacrylamide and polyphenol, can be used to environmental pollution and pathological specimens.
entrap elements physically. However, when biosensors are placed in a biological
Organic conductive polymers provide advantages, in- environment, numerous factors operate to affect their
cluding the formation of an appropriate environment for performance, the most significant ones being sensor
enzyme immobilisation at the electrode and for its surface interactions with proteins and cells (Donald and
interaction with metallic and carbon conductors Jeffrey, 1996). Therefore, although biosensors have
(Bartlett and Cooper, 1993; Trojanowicz and Krawczyk, great potential for real-time clinical monitoring, the
1995). Therefore, electrical communication between the sensors so far constructed lack functional stability after
redox centre and the electrode surface is more efficient. implantation and sensor lifetime is usually restricted to
These polymers can be deposited electrolytically from several hours, or days (Fischer, 1995). Thus, functional
solution onto a conducting support to provide a three- stability is profoundly affected by the biocompatibility
dimensional matrix for immobilised enzymes where reac- of the biosensor materials that are in contact with the
tants are converted to products. The polymers can be biological medium.
produced by a variety of chemical processes, including Attempts to improve the biocompatibility of artificial
the Ziegler–Natta reaction for polyacetylene surfaces by bonding anticoagulants have not been very
(Shirikawa, 1982; Naarmann and Theophilou, 1987), the successful. For example, the surface treatment of mem-
creation of an electrolyte solution e.g. poly(p-phenylene) branes with heparin sulphate is commonly used to
(Lenz et al., 1988), the coupling of organometallic improve haemocompatibility, but when Smith and
components (polythiophene) (Kobayashi et al., 1984) Sefton (1993) analysed thrombin adsorption onto hep-
and the oxidation of monomers (Ratcliffe, 1990). arin treated polyvinyl alcohol and polyurethane, they
276 S. Zhang et al. / Biosensors & Bioelectronics 15 (2000) 273–282

observed that, whereas the rate of adsorption of biological membrane mimicry. These copolymers have
thrombin was reduced by heparin coating, the final been successfully used as drug carriers as well as biosen-
volume of thrombin adsorbed remained similar. An- sor membranes. The basic philosophy behind this idea
other problem is that the heparin tends to leach from is due to Zwaal et al., (1977), who described the com-
the membrane surface into the surrounding medium. plex relationships between cell membrane structure and
Materials with hydrogel-like properties are generally blood coagulation. In vitro coagulation tests demon-
considered to favour biocompatibility. Water associates strated that the inner surfaces of the plasma membrane
with the water-soluble polymers and the presence of of erythrocytes and platelets are highly procoagulant,
water around the polymer hinders protein adsorption but the outer surfaces are inactive. Liposomes having
due to the energetically unfavourable displacement of the same phospholipid composition as the outer sur-
water by protein and compression of the polymer upon faces of erythrocyte and platelet membranes were also
the approach of protein. These factors have been de- inactive and did not reduce the time for recalcified
scribed in terms of steric repulsion, van der Waals plasma to clot.
attraction, and hydrophobic interactive free energies by The simplest common feature of these non-reactive
Jeon et al. (1991) and by Jeon and Andrade (1991). cellular and model membranes is the high content of
Surfaces grafted with water-soluble polymers have been electrically neutral phospholipids with phosphoryl-
developed using a number of techniques, including choline head groups (Zwaal, 1978). Consequently, the
end-grafting (Shoichet et al., 1994) and in situ poly- synthetic copolymer, poly(MPC-co-BMA) which con-
merisation by photo- or wet- chemistry, and by radio tains head groups of 2-methacryloyloxyethyl phospho-
frequency glow discharge deposition (Lopez et al., rylcholine (MPC) copolymerised with n-butyl
1992). An alternative to surface grafting is the adsorp- methacrylate (BMA), also exhibits surface properties
tion of amphiphilic molecules onto hydrophobic poly- that are favourable for haemocompatibility. The sur-
mers. Amphiphilic molecules can rearrange themselves faces are extremely hydrophilic and they contain large
on a surface in an attempt to maximise packing density volumes of water (Ishihara et al., 1990). During the
and can be covalently immobilised on the surface to construction of the membrane by liquid evaporation,
create a permanent adsorption layer (Nojiri et al., the whole phospholipid molecule, which includes two
1992). Polyethylene glycol (PEG) has been used exten- fatty acid chains, undergoes significant rotation to min-
sively to modify surfaces, so that protein adsorption imise interface energy and this results in the orientation
and platelet interactions with the foreign surface are of the phosphorylcholine head group towards the side
reduced (Amiji and Park, 1994). of the membrane that is exposed to air. The MPC
The natural cell membrane is a self-assembled system moiety also has a strong affinity for natural phospho-
and the extra-cellular matrix is a nano-structured sys- lipid molecules in plasma, so a well organised natural
tem. Based upon these observations, amphiphilic, self- lipid layer, biomembrane-like structure, forms on this
assembled multilayers and nano-structured surface surface during its exposure to plasma (Kojima et al.,
systems have been exploited in the production of lipo- 1991; Zhang et al., 1998). Protein adsorption is signifi-
somes modified with PEG. These surfaces suffer from cantly reduced on poly(MPC-co-BMA) surfaces com-
significantly less protein adsorption and immune clear- pared with other medical polymers (Ueda et al., 1990;
ance mechanisms are reduced (Woodle and Lasic, Zhang et al., 1998) and the in vitro and in vivo perfor-
1992). Chemical adsorption has been used to produce mance of biosensors is significantly improved when
self-assembled monolayers on metals and ceramics. poly(MPC-co-BMA) is coated onto the sensor surface
Alkanes terminated with thiols form densely packed (Chen et al., 1993; Zhang et al., 1996a,b). Zhang et al.
monolayers, with the alkane chain oriented outwardly (1998) suggested that these membranes might simulate
from the substrate surface. Dimilla et al., (1994) re- natural membranes functionally as well as structurally
ported that protein adsorption could be virtually elimi- the natural phospholipids may be continuously replen-
nated by alkane thiol terminated with oligoethylene ished in a continuous process of erosion and repair.
glycol. An optical biosensor chemically adsorbed with a
monolayer of 16-mercaptohexadecan-1-ol has been pro- 2.4. Biological elements
duced to measure protein interactions with gold coated
surfaces (Lofås, 1995). Improvements in interface design have frequently
Surface modification of polymers has led to modest been directed at the incorporation of active molecules,
improvements in biocompatibility, but it is still not including enzymes such as glucose oxidase (Clark and
satisfactory for long-term in vivo applications, so there Lyons, 1962) and lactate oxidase (Vadgama et al.,
is an urgent need to design and develop new biocom- 1986), mediators, such as Ferrocene (h 2-bis-cyclopenta-
patible materials. During the last two decades, several dienyl iron) and its derivatives (Cass et al., 1984),
attempts have been made to do this by synthesising new cofactors based on nicotinamide adenine dinucleotide
phospholipid copolymers based upon the principle of (NADH+ and NADP+ (Jaegfeldt et al., 1981), cata-
S. Zhang et al. / Biosensors & Bioelectronics 15 (2000) 273–282 277

lysts (Cosnier et al., 1997), antibodies and antigens tion should always involve the selection of materials.
(Umezawa et al., 1980). An electrochemical biosensor usually consists of a
Studies on the use of biosensors for gene detection transducer such as a pair of electrodes or FET, an
are relatively recent and still uncommon. Deoxyribonu- interface layer incorporating the biological recognition
cleic acid (DNA) has recently been suggested as a molecules and a protective coating. Sensor design,
biological recognition element for such biosensors including materials, size and shape and methods of
(Vadgama and Crump, 1992; Mulchandani and Bassi, construction, are largely dependent upon the principle
1995). The unique nucleotide base structure of DNA of operation of the transducer, the parameters to be
provides the basis of the technique which allows single- detected and the working environment. Traditional
stranded DNA (ssDNA) to be used to identify other electrode systems for measurements of the concen-
ssDNA molecules with the complementary bases (Zhai trations of ions in liquids and dissolved gas partial
et al., 1997). Therefore, nucleic acid hybridisation is pressures contain only a working electrode (usually a
the underlying operating principle of DNA biosensors. noble metal wire) and an electrically stable reference
During the last decade, there have been many advances electrode, such as Ag/AgCl, though a counter electrode
in DNA biosensor technology and most work has is sometimes included. A simple electrical, or chemical,
focused on electrochemical, piezoelectric and optical modification may sometimes improve specific electrode
transducers. properties. For example, repeated potential cycling
Attempts to develop an electrochemical DNA of 0.3 mm diameter carbon rods in 0.1 M potass-
biosensor have been made by several groups (Millan et ium hexacyanferrate improved the stability of glu-
al., 1994; Marrazza et al., 1999). In these sensors, an cose sensors for up to 6.5 days (Jaffari and Pickup,
ssDNA strand is covalently bound to the surface of an 1996).
electrode. Hybridisation of the immobilised sequence However, with the expanding demands for more
with its dissolved complement forms the double strand complex measurements, the rapid development of ma-
that can be detected using a DNA-specific redox-active terials science and the emergence of micro- and nano-
metal/polypyridine complex. Damaged segments of process technology, indirect electrochemical methods in
DNA can also be detected by measuring changes in the simple biosensors to monitor enzyme activity have
redox signals of base residues in DNA immobilised on gradually been replaced by more direct, but more com-
carbon electrodes. Covalently closed circular DNA can plex processes.
be attached to an electrode surface to obtain a sensor Methods for the preparation of electrochemical elec-
that detects a single break in the DNA sugar-phos- trodes are well established. Some of these techniques
phate backbone, or for the detection of agents leaving are used to prepare the conductive supporting sub-
the DNA backbone such as hydroxyl radicals, ionising strate, while others are employed to achieve an efficient
radiation or nucleases (Palecek et al., 1998). electrical communication between the chemical reaction
DNA sensing protocols, based on different modes of site and the electrode surface, high levels of integra-
nucleic acid interaction have been reviewed by Wang et tion, sensor miniaturisation, measurement stability, se-
al. (1997). The review describes recent efforts to couple lectivity, accuracy and precision. In addition, the
nucleic acid recognition layers to electrochemical technique used to immobilise the biological recognition
transducers. components of the sensor can affect biosensor perfor-
Peptide nucleic acids (PNAs) have been found to mance significantly.
exhibit unique and efficient hybridisation properties
that may offer significant advantages for sequence-spe- 3.1. Electrode fabrication
cific recognition compared to their DNA counterparts.
The advantages include higher sensitivity and specific- The electrode supporting substrate can be a noble
ity, faster hybridisation at room temperature and mini- metal (gold or platinum), carbon rod or paste, or an
mal dependence upon ionic strength. The use of PNA organic conducting salt or polymer. Techniques used
incorporated with a Co(phen)(3)(3+) redox indicator for the production of conductive supporting substrates
on a carbon electrode for the detection of sequence can be roughly classified as: (1) printing, (2) deposition,
specific DNA has been discussed by Wang et al. (3) polymerisation, (4) plasma induced polymerisation,
(1996). (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-
3. Techniques for the preparation of biosensors duction. Paste material is printed onto a matrix di-
rectly through a mask-net with a designed pattern. The
Design and construction technology and materials technique of carbon, or graphite, screenprinting is now
science are intimately linked in biosensor development. frequently used to prepare electrodes for biosensors.
Therefore, discussions of biosensor design and fabrica- Turner’s group recently improved this technique by
278 S. Zhang et al. / Biosensors & Bioelectronics 15 (2000) 273–282

using solvent resistant materials, heat stabilised contact with the electrode surface. This process allows
polyester sheet, carbon base tracks and an epoxy-based for a reduced applied potential of only 200 mV, the
polymer (Kroger and Turner, 1997). These electrodes avoidance of inference from co-oxidisable species and
have no problems with solvent induced baseline shift the minimisation of electrode fouling (Silber et al.,
and are therefore suitable for work in water-miscible 1996b). By depositing a thin electropolymerised film of
organic solvents. poly(1,3-diaminobenzene), electrochemical interference
Sensor arrays for the detection of more than one from ascorbate, urate, acetaminophen and other oxi-
parameter by different sensing techniques, or to assem- disable species can be greatly diminished (Madaras and
ble a package of sensors to measure the same parame- Buck, 1996). Sirkar and Pishko (1998) reported that a
ter, have potential practical applications. For this photo-initiated free-radical polymerised redox hydrogel
purpose, the screen printing technique has been used to polymer entrapped enzymes efficiently and increased
prepare a seven-channel electrode for simultaneous am- the transfer of electrons from enzyme oxidation/reduc-
perometric and potentiometric measurements. The ar- tion reactions through the gel to the electrode surface.
ray contains 14 gold working and counter electrodes (4) Plasma-induced polymerisation is performed un-
and one Ag/AgCl reference electrode (Silber et al., der high vacuum. The principle is to introduce func-
1996a). This sensor can be used to analyse blood and tional groups onto the substrate surface and then
serum electrolytes and metabolites. ‘polymerisable’ gas plasma is coated onto this surface
Khan (1996a) introduced a technique to produce a to form a layer of film. Plasma-polymerised film, gen-
printable biosensor. A printable paste is prepared by erated in a glow discharge, or plasma in a vapour
mixing glucose oxidase adsorbed organic charge trans- phase, may offer a new alternative for biosensor inter-
fer complex crystals with a binder and a solvent. This face design. The advantage of this technique is that it
paste is printed onto a matrix cavity and dried under can produce an extremely thin (B 1 mm) film that
vacuum. A thin layer of gelatin is then cast on adheres firmly to substrates. Furthermore, the film is
the electrode. The developed sensor provides a huge pin-hole free and both mechanically and chemically
response current with minimum interference from oxy- stable, and it allows a large amount of biological
gen and an extended linear range up to 100 mM material to be loaded onto the surface (Muguruma and
glucose. Karube, 1999).
Techniques (2), (3), (4) and (5) are thick- /thin-film (5) Photolithography techniques have long been used
techniques that are used in biosensors to form mono- in the semiconductor industry to produce integrated
or multi-layers of conducting film onto a supporting chips. Light passes through a photo-mask and is cast
substrate in order to obtain a direct electrical commu- upon a photodegraded material surface to form a pat-
nication between the chemical/biochemical reaction site tern. Nussbaum et al. (1997) used this technique to
and the supporting surface. Factors affecting electron fabricate micro-lens arrays for sensors. The manufac-
transfer from biological molecules to electrode surfaces ture of integrate transducer arrays for measurements of
have been reviewed by Cardosi and Turner (1991). a single parameter, or for several different parameters,
(2) Traditional chemical, or electrochemical, deposi- is now possible by means of photolithography and
tion methods can be used to deposit an electroconduc- plasma technology. These techniques have been used to
tive film on a supporting substrate. The deposited film increase the dynamic range and sensitivity of urea
can be metal such as platinum, catalytic material such sensors (Steinschaden et al., 1997).
as TiO, or a metal complex. Biological elements can (6) Nano-techniques have recently appeared with the
also be simultaneously coupled during the film deposi- maturation of modern technologies such as surface
tion process. Lorenzo et al. (1998) discussed the analyt- probe microscopy and lithography, atomic force mi-
ical strategies for various electrodeposited films. croscopy (AFM), AFM lithography and lateral force
(3) Polymerisation takes place due to the condensa- microscopy (LFM). A brief historical overview was
tion of small molecules in monomers, or by free radical given by Skladal and Macholan (1997), in which recent
creation and reaction by rearranging the bonds within developments in miniaturisation, microfabrication,
each monomer. Free radicals are produced when the nanotechnology, immuno-sensors and gene-sensors are
double bond is broken by initiation activated by heat, discussed. Sugimura and Nakagiri (1997) produced
light, or electro-chemicals. Electrical conductivity can patterns with resolutions in the nanometer range based
be achieved by the introduction of metal powder into upon photo- and AFM- lithography with an orgaosi-
the monomer before polymerisation, or through elec- lane monolayer resist.
trons that are not conjugated in the monomer. A new The combination of techniques mentioned above
technique has been developed to generate poly- leads to multilayer structures that may well prove to be
methylene blue-modified thick-film on gold electrodes useful in the development of new types of biosensor.
by electropolymerisation to form an eletrocatalytically Bilayer polymer coatings consisting of polypyrrole acid
active conducting layer that is in intimate and stable poly(o-phenylenediamine) on a supporting substrate
S. Zhang et al. / Biosensors & Bioelectronics 15 (2000) 273–282 279

may improve selectivity and reduced inference from 3.2. Immobilisation methods
electroactive species like uric and ascorbic acids that are
often present in biological samples (Vidal et al., 1999). Considerable progress has been made in the develop-
Kranz et al., (1998) proposed a multilayer architecture ment of new methods of immobilising biological recog-
to predefine electron-transfer pathways, integrate redox nition elements onto sensor surfaces. The use of
mediators, immobilise enzymes and restrict diffusional self-assembled mono- and multi-layers (SAMs) is in-
access by interfering compounds. A multilayer wafer creasing rapidly in various fields of research, and this
can also be formed by depositing a thin functionalized applies especially to the construction of biosensors.
polypyrrole film on the supporting surface and then SAMs can be used as interface layers upon which
covalently bonding a redox dye of polymerised almost all types of biological components, including
quinoidic species to prevent electrode fouling. The top proteins, enzymes, antibodies and their receptors, and
of this layer is coated with polypyrrole with entrapped even nucleotides for DNA recognition, can be loaded
tyroinase. Electrons transfer from the quinone to the (Wink et al., 1997). The use of biomembranes as recog-
electrode surface via the immobilised redox dye. nition elements was introduced by the pioneering work
Other new techniques have been suggested that could of Mueller et al., (1962). Lipid membranes provide a
be useful in the design and construction of new biosen- relatively blocompatible surface and mass diffusion
sors. The most promising of these may be the forma- based sensors constructed with lipid membranes have
tion of a direct electrochemical communication between fast response rates and high sensitivities. However,
the active enzyme site and the electrode surface using a there was no practical use of lipid films until the
biocatalyst with a very low molecular weight, such as development of physically stable bilayer lipid mem-
microperoxidase MP-11, immobilised on a thio-mono- branes (BLMs). BLMs can be formed on metal sur-
layer. In this case, the distance between enzyme and faces, conductive polymer supports, or agar plates. The
electrode surface is greatly reduced in comparison with technique usually proceeds by two steps. First, the
earlier constructions and this modification significantly support substrate is coated with the lipid layer by
increases the strength of the output signal (Lotzbeyer et immersing it in a lipid solution and then placed in an
al., 1996). electrolyte solution to create the self-assembled lipid
Khan (1996b) reported a stable organic charge-trans- bilayer.
fer-complex (CTC) electrode for the direct oxidation of The incorporation of biological recognition
flavoproteins. To construct the CTC electrode, tetrathi- molecules into lipid layers and their immobilisation on
afulvalene-tetracyanoquinodimethane is grown at the BLMs depend upon the degree of access to reactive
surface of an electroconductive polyanion-doped sites on the molecules. The various techniques used for
polypyrrole film in such a way that it makes a tree- this purpose have been described by Nikolelis et al.
shaped crystal structure, standing vertically on the sur- (1999). Hianik et al. (1998) recently described the tech-
face. By immobilising a glucose enzyme on the CTC nique used to construct an immunosensor based on a
electrode, direct electron transfer is achieved between self-assembled BLM supported on a metal surface.
the active enzyme and the crystal electrode and this Avidin modified monoclonal antibodies, originating
leads to remarkably improved sensor performance. from the E2/G2 clone (AMab), and matched antigens
An electrically conductive and mechanically flexible were incorporated in the membrane. Amperometric
composite polymer was prepared to construct a glucose biosensors for glucose and urea measurement have also
sensor by Yamato et al. (1997). Using this technique, been produced by the immobilisation of glucose oxidase
fine palladium particles are dispersed in polypyrrole/ and urease into BLMs through the avidin–biotin inter-
sulfated poly(beta-hydroxyethers) by thermally decom- action (Hianik et al., 1997).
posing the bis(dibenzylideneacetone) – palladium A layer-by-layer deposition technique may be used to
complex. With Ag/AgCl as a reference electrode, a optimise enzyme loading in bienzyme systems (Chen et
conventional platinum electrode responded to glucose al., 1998). With up to ten monomolecular layers con-
at a working potential of 650 mV, whereas the new taining avidin, biotin residues and choline oxidase
electrode responded at 400 mV. (ChOx), and two superficial layers containing choline
Elrhazi et al., (1997) used fibrinogen film to provide esterase, the sensor exhibits an increased response to
a porous, non-reactive layer over a carbon paste elec- acetylcholine.
trode to control the mass transfer rate of diffusing A technique to prepare gold electrodes with nanome-
species and Muller et al. (1997) introduced the tech- ter-sized openings for the immobilisation of biological
nique of pulsed LASER deposition (PLD) with opti- recognition elements, such as antibodies, has been de-
mised LASER parameters and reaction atmosphere to scribed by Padeste et al., (1996). Latex spheres are used
obtain more efficient enzyme activities than the conven- as a masking material to create 60 nm diameter holes in
tional platinum film electrode produced by argon gold film evaporated onto a supporting substrate. The
sputtering. nanometer-scale proximity of the recognition compo-
280 S. Zhang et al. / Biosensors & Bioelectronics 15 (2000) 273–282

nents to the conducting surface may facilitate the devel- Atanasov, P., Gamburzev, S., Wilkins, E., 1996. Needle-type glucose
opment of biosensors without mediators. biosensors based on a pyrolysed cobalt – tetramethoxy – phenylpor-
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4. Conclusions
Chen, Q., Kobayashi, Y., Takeshita, H., Hoshi, T., Anzai, J., 1998.
Avidin-biotin system-based enzyme multilayer membranes for
Biosensor development and production are currently biosensor applications: optimisation of loading of choline esterase
expanding due to the recent application of several new and choline oxidase in the bienzyme membrane for acetylcholine
techniques, including some derived from physical chem- biosensors. Electroanalysis 10, 94 – 97.
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