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4. fuel cell

Biosensors & Bioelectronics 17 (2002) 441– 456
www.elsevier.com/locate/bios
Review
Mediated biosensors
Asha Chaubey *, B.D. Malhotra
Biomolecular Electronics and Conducting Polymer Research Group, National Physical Laboratory, Dr. K.S. Krishnan Marg, New Delhi 110 012,
India
Received 14 August 2001; received in revised form 26 October 2001; accepted 31 October 2001
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1. Biosensors . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1.1. Electrochemical biosensors . . . . . . . . . . .
1.1.1.1. Conductometric biosensors . . . .
1.1.1.2. Potentiometric biosensors. . . . . .
1.1.1.3. Amperometric biosensors . . . . . .
1.1.2. Functioning of amperometric biosensors .
2. Significance of mediated systems . . . . . . . . . . . . . . .
3. Mediators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1. Characteristics of an ideal mediator . . . . . . . . .
3.2. Advantages of using mediators . . . . . . . . . . . .
3.3. Some commonly used mediators . . . . . . . . . . .
3.3.1. Ferrocene and derivatives . . . . . . . . . . .
3.3.2. Tetracyanoquinodimethane(TCNQ) . . . .
3.3.3. Tetrathiafulvalene(TTF) . . . . . . . . . . . . .
3.3.4. Conducting salts . . . . . . . . . . . . . . . . . .
3.3.5. Quinones . . . . . . . . . . . . . . . . . . . . . . .
3.3.6. Ferri/Ferrocyanide . . . . . . . . . . . . . . . . .
4. Mechanism for electron transfer . . . . . . . . . . . . . . .
4.1. Mediated electron transfer . . . . . . . . . . . . . . . .
4.1.1. Homogeneous mediation . . . . . . . . . . . .
4.1.2. Heterogeneous mediation . . . . . . . . . . . .
5. Enzyme electrodes . . . . . . . . . . . . . . . . . . . . . . . . .
6. Modified electrodes . . . . . . . . . . . . . . . . . . . . . . . .
7. Commercialization of mediated biosensors . . . . . . .
8. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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* Corresponding author. Fax: + 91-11-585-2678.
E-mail address: [email protected] (A. Chaubey).
0956-5663/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 9 5 6 - 5 6 6 3 ( 0 1 ) 0 0 3 1 3 - X
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442
A. Chaubey, B.D. Malhotra / Biosensors & Bioelectronics 17 (2002) 441–456
Abstract
Direct electrode transfer between enzyme and the electrode in biosensors requires high efficiency therefore, synthetic
replacement for oxygen led to the development of enzyme mediators and modified electrodes in biosensor fabrication. In this
context, a number of electron acceptors and complexes have been used. Present paper gives an overview of various methodologies
involved in the mediated systems, their merits and wide applications. © 2002 Elsevier Science B.V. All rights reserved.
1. Introduction
Traditional chemical and biological analytical techniques involve reactions that take place in solutions on
addition of reagents and samples. In recent years, much
work has been performed in the field of ‘reagentless
systems’ (Schuhmann et al., 1993; Schmidt and Schuhmann, 1995; Senillou et al., 1999; Leech and Feerick,
2000; Wilson and Hu, 2000). In such systems, the
reagents are already immobilized in the system and
therefore need not be added by the user. In such systems
the reaction takes place at the surface of the electrode and
are commonly called as biosensors. By definition, biosensors are devices comprising of an analyte and a selective
interface in close proximity or integrated with a transducer, which relays an interaction between the surface
and analyte either directly or through a mediator (Hall,
1992). The analyte selective interface is a bioactive
substance e.g. enzyme, antibody or micro-organism etc.
These are capable of recognizing their specific analytes
and also regulate the specificity and sensitivity of the
device. The transducer converts the biochemical signal
into an electronic signal, which can be suitably processed
and output. Biosensors promise low cost, rapid and
simple to operate analytical tools. They therefore, represent a broad area of emerging technology ideally suited
for health care analysis.
The biosensors are highly selective due to the high
substrate specificity of the enzyme and the interference
free indication of the reaction product. They offer the
possibility of real-time analysis which is important for the
rapid measurement of body analytes. The potential
application of biosensors usually lies in the clinical
analysis for health care. The modern concept of biosen-
sors evolved in 1962 when Clark and coworkers proposed
that enzymes could be immobilized at the electrochemical
detectors to form enzyme electrodes and these may be
utilized to sense their specific analytes.
1.1. Biosensors
Biosensors are functional analogs that are based on the
direct coupling of an immobilized biologically active
compound with a signal transducer and an electronic
amplifier. The main function of a transducer is to convert
the physico-chemical change in the biologically active
material resulting from the interaction with the analyte
into an output signal. Fig. 1 shows a general configuration of a biosensor. Based on the type of transducer used,
biosensors have been divided into optical, calorimetric,
peizoelectric and electrochemical biosensors. Optical
biosensors are based on the measurement of light absorbed or emitted as a consequence of a biochemical
reaction. In such a biosensor, the light waves are guided
by means of optical fibers to suitable detectors (Peterson
and Vurek, 1984; Seitz, 1987). They can be used for
measurement of pH, O2 or CO2 etc. A commercial optical
biosensor, which is the hybrid electrochemical/optical
LAPS (light addressable potentiometric sensor) was
developed by the company Molecular Devices in Palo
Alto, USA (Tiefenthaler, 1993). Calorimetric biosensors
detect an analyte on the basis of the heat evolved due to
the biochemical reaction of the analyte with a suitable
enzyme. Recently, integrated circuit temperature sensitive structures have been modified with enzymes. Different substrates, enzymes, vitamins and antigens have been
determined using thermometric biosensors. The most
commonly used approach in the thermal enzyme probes
Fig. 1. Schematic of a biosensor.
A. Chaubey, B.D. Malhotra / Biosensors & Bioelectronics 17 (2002) 441–456
(Danielsson and Mosbach, 1987; Weaver et al., 1976)
was related to the enzyme directly attached to the
thermistor. It was observed that the majority of the
heat evolved in the enzymatic reaction was lost to the
surrounding solution without being detected by thermistor resulting in the decrease in sensitivity of the
biosensor. Piezoelectric biosensors operate on the principle of generation of electric dipoles on subjecting an
anisotropic natural crystal to mechanical stress. Adsorption of the analyte increases the mass of the crystal
and alters its basic frequency of oscillation. They are
used for the measurement of ammonia, nitrous oxide,
carbon monoxide, hydrogen, methane and certain
organophosphorus compounds (Abad et al., 1998; Minunni et al., 1994). All these biosensors suffer from
certain drawbacks. For example, optical biosensors,
though very sensitive, however, cannot be used in turbid media. Thermal biosensors cannot be utilized with
systems with very little heat change. Moreover, they are
not easy to handle. Electrochemical biosensors have
emerged as the most commonly used biosensors. They
have been found to overcome most of the disadvantages, which inhibit the use of other types of biosensors.
These biosensors are rapid, easy to handle and are of
low cost.
1.1.1. Electrochemical biosensors
Electrochemical biosensors are the most commonly
used class of biosensors. These are based on the fact
that during a bio-interaction process, electrochemical
species such as electrons are consumed or generated
producing an electrochemical signal which can in turn
be measured by an electrochemical detector. Electrochemical biosensors have been widely accepted in
biosensing devices. These biosensors can be operated in
turbid media, have comparable instrumental sensitivity
and are more amenable to miniaturization. Electrochemical biosensors are usually based on potentiometry
and amperometry. Ion selective electrodes (ISE), ion
selective field effect transistors (ISFET) and pH electrodes are usually based on the oxidation of the substrate/product e.g. oxygen electrode, detection of H2O2.
Depending upon the electrochemical property to be
measured by a detector system, electrochemical biosensors may further be divided into conductometric, potentiometric and amperometric biosensors.
1.1.1.1. Conductometric biosensors. Conductometric
biosensors measure the changes in the conductance
between a pair of metal electrodes as a consequence of
the biological component (Sukeerthi and Contractor,
1994). The electrochemical biosensors that have recently attracted much attention can be broadly
classified into several types depending on the mode of
detection.
443
Fig. 2. The scheme of mediated and unmediated electron transfer.
1.1.1.2. Potentiometric biosensors. Potentiometric
biosensors (Papastathopoulos and Rechnitz, 1975;
Mascini, 1995; Senillou et al., 1999; Koncki et al., 2000)
consist of measurement of potentials at the working
electrode with respect to the reference electrode. They
function under equilibrium conditions and monitor the
accumulation of charge, at zero current, created by
selective binding at the electrode surface. For example,
ISE detect ions such as Na+, K+, Ca2 + , H+ or NH+
4
in complex biological matrices by sensing changes in
electrode potential when the ions bind to an appropriate ion exchange membrane.
1.1.1.3. Amperometric biosensors. Amperometric biosensors measure the changes in the current on the working
electrode due to direct oxidation of the products of a
biochemical reaction. Amperometric techniques are linearly dependent on analyte concentration and give a
normal dynamic range and a response to errors in the
measurement of current. Oxygen and H2O2 being the
co-substrate and the product of several enzyme reactions, are detected for amperometric estimation (Lindgren et al., 2000; Davis et al., 1995). Electrochemical
biosensors are based on mediated or unmediated electrochemistry for electron transfer (Fig. 2). Ferrocene
and its derivatives, ferricyanide, methylene blue, benzoquinone and N-methyl phenazine etc. are most commonly used mediators in mediated biosensors
(Karyakin et al., 1994; Turner, 1988; Kulys and Cenas,
1983; Kajiya et al., 1991; Cass et al., 1984; Cenas et al.,
1984; Jaffari and Turner, 1997; Gregg and Heller, 1991;
Garjonyte et al., 2001). Various conducting polymers
such as polyaniline, polypyrrole etc. have also been
used for fabricating electrochemical biosensors (Foulds
and Lowe, 1986; De Taxis du Poet et al., 1990; Contractor et al., 1994; Schuhmann, 1995; Chaubey et al.,
2000a,b).
Amperometric biosensors may be based on direct/indirect systems. Indirect sensors exploit conventional
detectors to measure the metabolic substrate or product
A. Chaubey, B.D. Malhotra / Biosensors & Bioelectronics 17 (2002) 441–456
444
of biological material. Direct amperometry involves a
biological redox reaction, having an intimate relationship between biology and electrochemistry. These involve the utilization of modified electrodes, usually an
electron acceptor in place of the natural electron
donors. Fig. 3 relates to designing of the modified
electrodes and the reagentless electrodes used in the
fabrication of biosensors. The amperometric biosensors
are known to be reliable, cheaper and highly sensitive
for the clinical, environmental and industrial purposes.
It is postulated that direct e-transfer occurs between the
redox center of GOD immobilized in poly(Nmethylpyrrole) and a gold electrode surface (De Taxis
du Poet et al., 1990). Usually this electron transfer is
achieved by small and mobile molecules of redox species (mediators) present (apart from an enzyme in polymer layer) not by the polymer chain itself. This has
been observed in the case of polymers modified with
chemical mediators or by the use of redox polymers
(Degani and Heller, 1989; Gregg and Heller, 1990;
Persson et al., 1993; Okawa et al., 1999).
Such biosensors were dependent on the ambient concentration of O2. The simplest amperometric biosensors
are based on the Clark oxygen electrode. This consists
of a platinum cathode (where oxygen is reduced) and a
Ag/AgCl reference electrode when a potential of −0.6
V versus Ag/AgCl electrode is applied to the Pt electrode, a current proportional to the oxygen concentration is produced.
Ag anode:
4Ag+ 4Cl− “ 4AgCl + 4e−
(2)
Pt cathode:
O2 + 4H+ + 4e− “ 2H2O
(3)
In this case the rate of electrochemical reduction of O2
depends on the rate of diffusion of the oxygen from the
bulk solution, which in turn is dependent on the concentration gradient and hence the bulk oxygen
concentration.
Alternatively, the rate of production of H2O2 directly
by applying a potential of 0.68 V versus Ag/AgCl to the
Pt electrode.
Pt anode:
Ag cathode:
1.1.2. Functioning of amperometric biosensors
Amperometric enzyme electrodes have been used
widely as they are capable of directly transducing the
rate of reaction into a current. Amperometric biosensors function by the production of a current when a
potential is applied between two electrodes. The electrodes are used to measure the concentration of O2 or
the product H2O2 (Clark and Lyons, 1962; Guilbault
and Lubrano, 1973)
GOD
Glucose +O2 “ Gluconolactone +H2O2
(1)
Fig. 3. Modified electrodes and reagentless electrodes used in the
fabrication of biosensors.
H2O2 “ O2 + 2H+ + 2e−
2AgCl+ 2e “ 2Ag + 2Cl
−
(4)
−
(5)
These ‘first generation biosensors’ are dependent on the
concentration of dissolved oxygen in the bulk solution
(Updike and Hicks, 1967; Guilbault and Lubrano,
1973; Harrison et al., 1988; Shimizu and Morita, 1990).
In order to overcome these problems, the concept of
using artificial electron acceptors, evolved in the ‘second generation biosensors’ which can avoid the reduction of oxygen (Ianniello et al., 1982; Kulys and Cenas,
1983; Cass et al., 1984; Umana and Waller, 1986;
Foulds and Lowe, 1988; Yokoyama et al., 1989; Hill
and Sanghera, 1990; Kajiya et al., 1991; Pandey et al.,
1993a; Garjonyte et al., 2001). In these biosensors, all
the substances having conversion potential lower than
the electrode potentials contribute to the overall electrochemical signal. The most common example is that
at the oxidation potential of +600 mV alongwith H2O2
other metabolites such as uric acid, ascorbic acid, glutathion etc. also get oxidized and interfere with the
electrochemical signal. It is therefore, essential to apply
an electrode potential as low as possible. It was hence
thought of using electrochemically active electron acceptors to which the enzyme can donate electrons. In
this context, some artificial electron acceptors having
low oxidation potentials were discovered. These artificial electron acceptors are commonly called mediators.
This approach leads to a considerable reduction of
electrochemical interferences and the development of
mediated biosensors. Thus, mediated biosensors can be
constructed with the enzymes that can donate electrons
to electrochemically active artificial electron acceptors
(Pandey et al., 1997). Mediated electron transfer from
an enzyme to an electrode may be studied in rapid
systems using direct current (dc) cyclic voltammery and
A. Chaubey, B.D. Malhotra / Biosensors & Bioelectronics 17 (2002) 441–456
445
Table 1
Some mediators with their oxidation potentials used with different
enzymes
Enzyme
Mediator
Redox potential (versus SCE)
Glucose oxidase
1,1-dimethyl ferrocene
Ferrocene
Vinyl ferrocene
Ferrocene carboxylic acid
Hydroxy methyl ferrocene
[Ru(CN)6]4−
Benzoquinone
NMP
TTF
TCNQ
[Fe(CN)6]4−
Hydroxy methyl ferrocene
Ferrocene carboxylic acid
[Fe(CN)6]4−
[Fe(CN)6]4−
Ferrocene carboxylic acid
Hydroquinone
Ferrocene carboxylic acid
Ferrocene carboxylic acid
[Fe(CN)6]4−
Ferrocene carboxylic acid
[Fe(CN)6]3−
TMPD
Ferrocene carboxylic acid
[Fe(CN)6]3TMPD
Ferrocene carboxylic acid
N-methyl phenazene
Ferrocene carboxylic acid
1,1-dimethyl ferrocene
N-ethyl phenazene
TMPD
Ferrocene carboxylic acid
1,1-dimethyl ferrocene
100
165
250
275
185
685
39
−161
300
127
180
190
275
Cholesterol oxidase
Lactate oxidase
Pyruvate oxidase
Xanthine oxidase
Lactate dehydrogenase
NADH dehydrogenase
Alcohol dehydrogenase
Glucose dehydrogenase
Galactose oxidase
established reaction kinetics (Cass et al., 1984; Davis,
1989).
The development of second generation biosensor
(Cass et al., 1984; Cardosi and Turner, 1987) involves a
two step procedure in which the enzyme takes part in
first redox reaction with the substrate and is in turn
reoxidized by a mediator and finally the mediator is
oxidized by the electrode.
Glucose + GOD/FAD
“ Gluconolactone + GOD/FADH2
(6)
GOD/FADH2 +2M “GOD/FAD + 2 M* + 2H+
(7)
2M* “ 2M+ 2e−
(8)
where FAD represents a flavin redox center in GOD
and the mediator, M/M* are the oxidized and reduced
forms of mediator. e.g. Ferrocene, ferricyanide, NMP
etc. are the commonly used mediators (Table 1).
180
280
60
275
275
180
275
180
−10
275
180
−10
275
−156
275
100
−172
−10
275
100
The immobilization of the redox species can be carried out by adsorption, polymer coating and covalent
attachment (Murray, 1980; Bard, 1983; White and
Turner, 1997). The amperometric procedure leads to
the utilization of a lower redox potential that can be
used for H2O2 detection (Cass et al., 1984). If the fixed
concentration of an electron acceptor is retained within
the enzyme layer, the operational stability of the sensor
can be increased. The amperometric biosensors incorporating immobilized mediators therefore provide an
interesting alternative to peroxide detecting systems
(Romette and Boitieux, 1984; Shichiri et al., 1982,
1984).
Some electrodes have been developed which can directly oxidize the reduced enzyme and do not require
any exogenous mediator (Kulys et al., 1980; Lotzbeyer
et al., 1986; Contractor et al., 1994; Schuhmann, 1995;
Khan, 1996; Chaubey et al., 2000a,b). These have been
called ‘third generation biosensors’. Such enzyme electrodes can be prepared by the coating of the electronic
446
A. Chaubey, B.D. Malhotra / Biosensors & Bioelectronics 17 (2002) 441–456
conductors (conducting salts) and are stable for several
months. Conducting salts such as NMP+TCNQ−
could also be used as the electrode materials (Kulys et
al., 1980; Cenas and Kulys, 1981). These electrodes are
often used for recations involving NAD(P)+ dependent
dehydrogenases as they allow the electrochemical oxidation of the reduced forms of these coenzymes. For
example, the reduced enzyme GOD/FADH2 can be
directly oxidized (Albery and Bartlett, 1985; Albery et
al., 1985).
Glucose + GOD/FAD
“ Gluconolactone+ GOD/FADH2
(9)
Electrode:
GOD/FADH2 “ GOD/FAD + 2H+ +2e−
(10)
The current produced in the amperometric biosensors
can be related to the rate of reaction (VA) by the
equation
i= nFAVA
(11)
where n is the number of electrons transferred, A is the
electrode area and F is Faraday constant.
Usually the rate of reaction is a diffusion controlled
phenomenon where external membranes are used,
therefore the current produced is proportional to the
analyte concentration and independent of both the
enzyme and electrochemical kinetics.
2. Significance of mediated systems
Mediated enzyme electrodes are known to be less
susceptible to interfering substances due to lower electrode potentials. Gorton et al. (1991) have investigated
a system in which hydrogen peroxide generated during
the oxidation of glucose by glucose oxidase can be
detected at a peroxidase modified electrode. During the
process, a polymer bound mediator is oxidized by the
peroxidase with the subsequent reduction of the mediator at the working electrode. Okamoto’s group has
designed highly flexible ferrocene containing siloxane
polymers. These facilitate electron transfer from the
reduced flavin co-factor of several oxidases. Highly
stable amperometric biosensors based on these polymeric systems have been reported (Hale et al., 1989,
1991, 1993; Inagaki et al., 1989; Gortan et al., 1990).
During the past few years, water insoluble cation exchange polymers have been used to elaborate amperometric biosensors. This approach has following
advantages:
(i) The enzymes can be immobilized during casting
procedure of the polymer enzyme solution at the
electrode;
(ii) Negative charges of the polymer enzyme film can
act as a barrier for the negatively charged biological interferents present in biological fluids;
(iii) The polymer enzyme film offers the possibility of
incorporation by ion exchange, positively charged
electron mediators which can be used for shuttling for electron transport from the redox site of
the enzyme to the electrode surface (Chen et al.,
1992; Fortier et al., 1992; Mizutani et al., 1996).
In both cases when hydrophobic and cationic mediators are incorporated in the polymer GOD films, an
increase in the permeability to blood interferents is
observed and can be related to the neutralization of the
polymer charge by the mediator. During the process,
reorganization of the polymer structure takes place
leading to an increase in the porosity of polymer films.
In order to avoid the leaching of the mediator from the
enzymatic film and to block the entry of anionic biological interferents, the enzymatic film was covered by
Nafion polymer, which forms a repulsive barrier. During the electrochemical reaction, the electrons pass from
an enzyme based biological component to the amplifier
or a microprocessor component. The biological component immobilized onto the surface of the transducer
provides adaptability, reliability and thus immobilization is expected to have effects such as specificity, good
stability to temperature, pH and ionic strength etc.
Ferrocene has been known to be used for this purpose.
Higgins et al. (1984) have reported a true mating of the
sensor and the biological component by the use of
mediator sandwich molecules between the sensor and
the biological component i.e. with ferrocene. It has
been suggested that overloading of the surface support
with the biological component should be avoided. This
is because the activity increases with loading initially
but may decrease with high loading due to restricted
access. This may be overcome by a porous surface on
the support (Kennedy et al., 1973). A large number of
dehydrogenases utilize NADH as a cofactor. Therefore,
its electrochemical oxidation at low potentials is of
particular interest. It has been reported that the direct
oxidation of NADH at the above electrode requires
large overpotentials (Moiroux and Elving, 1978;
Jaegfeldt, 1980). However, these overpotentials can be
reduced using redox mediators. These mediators are
capable of bringing the oxidation process closer to the
thermodynamic value estimated to be − 0.32 V versus
NHE (Clark, 1960).
3. Mediators
Mediators are artificial electron transferring agents
that can readily participate in the redox reaction with
the biological component and thus help in the rapid
electron transfer. It is a low molecular weight redox
couple, which shuttles electrons from the redox center
A. Chaubey, B.D. Malhotra / Biosensors & Bioelectronics 17 (2002) 441–456
of the enzyme to the surface of the indicator electrode.
During the catalytic reaction, the mediator first reacts
with the reduced enzyme and then diffuses to the
electrode surface to undergo rapid electron transfer.
For example:
Glucose + FAD +H2O “Gluconic acid+FADH2
(12)
FADH2 + Mox “FAD +Mred +2H+
At electrode:
Mred “Mox
(13)
(14)
The rate of production of the reduced mediator (Mred)
is measured amperometrically by oxidation at the
electrode.
A mediator is expected to be stable under required
working conditions and should not participate in the
side reactions during electron transfer. The mediator
should be chosen in such a way that it has a lower
redox potential than the other electrochemically active
interferents in the sample. The redox potential of a
suitable mediator should provide an appropriate potential gradient for electron transfer between enzyme’s
active site and electrode. The redox potential of the
mediator (compared to the redox potential of enzyme
active site) should be more positive for oxidative biocatalysis or more negative for reductive biocatalysis.
Direct current voltammetry is a useful technique to
study the properties of mediators and it helps in selecting a suitable mediator for an amperometric biosensor
(Nakaminami et al., 1997; Gilmartin and Hart, 1995).
Fig. 4 shows a typical cyclic voltammogram obtained in
a solution of glucose in the absence and presence of
glucose oxidase. A steep rise in the current is observed
in the presence of the enzyme. It is desirable to use a
low potential mediator with a high electrochemical rate
447
constant, the latter is important to ensure that the
response of the biosensor is not limited by electrode
kinetics. Ferrocene and its derivatives having oxidation
potential of 165 and 100 mV (dimethyl ferrocene) versus SCE have been commonly used in fabrication of
enzyme electrodes. Ferrocene carboxylic acid, TTF,
TCNQ etc. are also used (Mc Cann, 1987; Turner et al.,
1987; Kulys et al., 1992). Among soluble mediators,
ferricyanide, N,N,N%,N%tetramethyl-4-phenylene diamine (TMPD) and benzoquinone remain useful mediators in certain assays (Cardosi and Turner, 1987). Smit
and Rechnitz (1993) have reported that the tyrosine
substrate of tyrosinase can be replaced with an electrochemically regenerable mediator, ferrocyanide which
can act as an electron donor in the enzymatic reduction
of oxygen. Thus, the catalytic current for oxygen reduction is a measure of the tyrosinase electrode enzyme
activity.
3.1. Characteristics of an ideal mediator
(i) It should be able to react rapidly with the reduced
enzyme
(ii) It should exhibit reversible heterogeneous kinetics
(iii) The overpotential for the regeneration of the oxidized mediator should be low and pH
independent
(iv) It should have stable oxidized and reduced forms
(v) The reduced form should not react with oxygen.
3.2. Ad6antages of using mediators
(i) measurements are less dependent on oxygen
concentration
(ii) the working potential of the enzyme electrode is
determined by the oxidation potential of the
mediator
(iii) with the use of mediators at low oxidation potentials, the interference of unwanted species can be
avoided.
(iv) if the oxidation of reduced mediator does not
involve protons, it can make the enzyme electrode
relatively pH insensitive.
3.3. Some commonly used mediators
Fig. 4. Typical cyclic voltammogram of ferrocene monocarboxylic
acid in glucose: (a) without glucose oxidase; (b) with glucose oxidase.
Organic dyes such as methylene blue, phenazines,
methyl violet, Alizarin yellow, prussian blue, thionin,
azure A and C, toluidine blue and inorganic redox ions
such as ferricyanide have been widely used in a number
of biosensors (Brunetti et al., 2000; Aoyagi et al., 1997;
Dubinin et al., 1991; Karyakin et al., 1994, 1995;
Molina et al., 1999). They, however, suffer from a
number of problems such as poor stability and pH
448
A. Chaubey, B.D. Malhotra / Biosensors & Bioelectronics 17 (2002) 441–456
3.3.2. Tetracyanoquinodimethane (TCNQ)
It has been reported that TCNQ is an effective
electron transfer mediator (Cenas and Kulys, 1981;
Kulys, 1991; Kulys and D’Costa, 1991; Pandey et al.,
1993b; Murthy and Anita, 1994; Lima Filho et al.,
1996). Edge plane pyrolytic graphite electrode modified
with TCNQ by dip coating and voltage assisted immobilization (Murthy et al., 1994) has been found to be
sensitive to NADH.
Fig. 5. Amperometric response of the PAB/GOX electrode for glucose in the unmediated system (0.7 V) and in the presence of FCA
(0.4 V) and TTF (0.3 V) mediated systems.
dependence of their redox potentials (organic dyes).
The inorganic mediators have problems in that it is not
easy to tune their redox potentials and solubility by the
use of substituents. Recently, the use of ferrocene
derivatives as redox mediators for flavo and quino
enzymes has been worked out. Nakaminami et al.
(1997) studied the electrochemical sensitivity to cholesterol in the detection system using cholesterol oxidase
and these redox compounds, and the employment of
MPMS or thionin allows electrochemical determination
of cholesterol at a low electrode potential (0 V versus
SCE) in the concentration range 0.25– 0.5 mM. Ramanathan and coworkers (Ramanathan et al., 2000)
conducted amperometric response measurements on
glucose oxidase covalently coupled to poly(o-amino
benzoic acid) using ferrocene carboxylic acid and tetrathiafulvalene mediators for glucose. Fig. 5 shows the
response of the poly(o-amino benzoic acid)/GOX electrodes for the unmediated (0.7 V) and ferrocene carboxylic acid (0.4 V) and tetrathiafulvalene (0.3 V)
mediated systems as a function of glucose concentration. The results reveal a favorable reaction kinetics
and mediation in the presence of FCA system as compared to that of unmediated system.
3.3.1. Ferrocene and deri6ati6es
Ferrocene and derivatives are the most widely used
class of mediators in the fabrication of stable and
sensitive biosensors (Kajiya et al., 1991; Cass et al.,
1984; Turner, 1988; Luong et al., 1994; Hendry et al.,
1995). Galactose oxidase was co-immobilised with peroxidase by drop-coating on the surface of a graphite
electrode with adsorbed ferrocene (Tk’a c et al., 1999).
This system offers low detection limit—0.51 mg galactose/l and fast response: 44 s in phosphate buffer or 25
s in borate buffer. The response was linear in phosphate
buffer in the range 1– 110 mg/l, while in borate buffer
linear range was extended to 3–210 mg/l because of
chelating effect of borate.
3.3.3. Tetrathiaful6alene (TTF)
TTF has been recognized as an alternative to ferrocene derivatives as a mediator for amperometric
biosensors (Murthy and Anita, 1996; Palleschi and
Turner, 1990; Mulchandani et al., 1995; Turner et al.,
1987; Qing et al., 1997). It has been demonstrated that
TTF acts as a mediator between glucose oxidase and a
graphite electrode (Turner et al., 1987). Gunasingham
and Tan (1990) have developed a carbon paste TTF
amperometric enzyme electrode for glucose determination in flowing systems.
3.3.4. Conducting salts
Conducting salts such as tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ) and N-methyl
phenazinium-tetracyanoquinodimethane (NMP-TCNQ)
have widely been used as electron shuttling agents in
biosensors (Kulys, 1986; Kulys et al., 1980; Albery et
al., 1985). Kulys et al. (1980), Kulys (1981) first reported the use of conducting salts with redox enzymes.
They used NMP-TCNQ, NMP(TCNQ)2, and NMATCNQ
(N-methyl
acridinium
tetracyanoquino
dimethane) as electrode materials either in the form of
pressed pellets or as a paste. These mediators were used
along with glucose oxidase, lactate dehydrogenase, peroxidase and xanthine oxidase etc., respectively. They
concluded from these studies that electron exchange
with LDH and with peroxidase proceeded directly at
the electrode. For GOD, their observation suggested
that the reaction proceeds in the solution with the
dissolution products from the electrode acting as mediators. Khan (1996) have described a printable glucose
sensor based on a paste containing glucose oxidase
adsorbed onto the crystals of charge transfer conducting mediator prepared from TCNQ and TTF solution
in acetonitrile on being treated with a binder. Such a
biosensor showed a response upto 100 mM glucose. A
micro-biosensor was constructed by incorporating the
organic conducting salt tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ) into a platinum
(Pt) wire and further covering with the electrochemical
polymerical heteropolypyrrolfilm, in which glucose oxidase (GOX) was entrapped (Li et al., 1999). The
enzyme electrode can sensitively determine glucose at a
low working potential, mainly based on the oxidation
of H2O2. The incorporated TTF-TCNQ can significantly improve the oxidation of H2O2 on the electrode,
A. Chaubey, B.D. Malhotra / Biosensors & Bioelectronics 17 (2002) 441–456
although a part of the TTF-TCNQ functions as a
mediator.
A novel electrode based on highly conducting organic metals has proved to be a very useful system for
the regeneration of NAD+ from NADH (Kulys, 1981).
These electrodes are based on stable charge transfer
complex formed by the partial transfer of electrons
from the donor such as 7,7,8,8-tetracyano-pquinodimethane (TCNQ) to an acceptor such as tetrathiafulvalene (TTF) or N-methyl phenazinium. An
electrochemical biosensor based on the incorporation of
enzymes into modified graphite paste has been described by Pandey and his coworkers (1993) to sensitively probe enzyme catalyzed reactions. In this system,
TCNQ, which is an electroactive molecule facilitated
the electron transfer from the active center of the redox
enzyme to the electrode surface. It has been suggested
that the oxidation of NADH at the NMP+TCNQ−
electrode takes place via a mediatory route (Kulys,
1981). Improvement of the performance of H2O2 oxidation at low working potential by incorporating TTFTCNQ into a platinum wire electrode for glucose determination has been reported by Li et al. (1999).
3.3.5. Quinones
Some reports on the use of quinone derivatives as
mediators are reported (Ikeda et al., 1989; Kulys and
Cenas, 1983; Zhao and Lennox, 1991). Generally orthoquinones have actively been utilized in electrocatalytic oxidation of NADH. Various derivatives
incorporating such a group have also been employed
(Huck and Schmidt, 1981; Ueda et al., 1982). Persson et
al. (1993) have described amperometric biosensor based
on redox polymer mediated electron transfer from
NADH to carbon paste electrodes, regenerating the
NAD+ needed for the dehydrogenase catalysed reaction. These sensors were shown to operate around 0
mV versus SCE, were capable to drive an unfavorable
equilibrium of a dehydrogenase catalysed reaction to
the product side. These are reagentless, therefore,
NAD+ need not be added to the analyte solution.
3.3.6. Ferri/ferrocyanide
Ferri/ferrocyanide is one of the most commonly used
inorganic mediator used in biosensors (Dubinin et al.,
1991; Shulga et al., 1994; Jaffari and Turner, 1997). It
is well known that lactate ions are oxidized to pyruvate
ions by hexacyanoferrate(III) ions in the presence of
LDH (Williams et al., 1970; Katrlik et al., 1997). Cassidy et al. (1993) developed a device consisting of a thin
layer twin electrode electrochemical cell with working
and auxillary electrodes facing each other. The electrolyte consisted of a solution of aqueous electrolyte
containing either glucose oxidase, cholesterol oxidase
and the mediator [Fe(CN)6]4 − . On addition of electrolyte, the following reaction takes place:
449
ChOx
Cholesterol+ O2 “ Cholestenone+H2O2
(15)
POD
2[Fe(CN)6]4 − + H2O2 + 2H+ “ 2[Fe(CN)6]3 − +2H2O
(16)
When the cell is on, the working electrode where
[Fe(CN)6]3 − is reduced to [Fe(CN)6]4 − is held at a
potential of − 0.1 V versus Ag/AgCl.
A glucose sensitive enzyme field effect transistor has
been fabricated by Shulga et al. (1994) using potassium
ferricyanide as an oxidizing substrate. Arun Kumar et
al. (Kumar et al., 2001) immobilized cholesterol oxidase
on dodecylbenzene sulphonate doped polypyrrole obtained by electrochemical method. These DBS-PPY
films with physically adsorbed cholesterol oxidase and
ferricyanide (0.1 M) have been characterized by UV–
visible and FTIR spectroscopy. The enzyme electrode
responds linearly to cholesterol concentration from 2 to
8 mM, have response time of about 60 s and are stable
for about 3 months at 4 °C.
4. Mechanism of electron transfer
It has been demonstrated that electrogenerated small
molecular reactants should be used to couple biological
redox couples to an electrode (Swartz and Wilson,
1971; Ito and Kuwana, 1971). The mediators serve to
facilitate a biological electron transfer, which is favorable thermodynamically but not kinetically.
MO + e− l MR
BO + e− “ BR
electrochemical
(17)
electrochemical (very slow reaction)
(18)
MR + BO “ MO + BR
chemical
(19)
where MO and BO are the oxidized forms and MR and
BR are the reduced forms of the mediator and the
biological molecule, respectively. The electrochemical
reaction occurs at the characteristic potential of the
mediator. As MO is regenerated close to the electrode
surface as a consequence of reaction (3), it does not
have to diffuse very far to again undergo electron
transfer. Therefore, a significant enhancement of the
current can be observed for only a small amount of BO
present if the chemical reaction is rapid. The observed
current may be related to the concentration of BO
present, and therefore, this approach has been widely
applied for the fabrication of biosensor. Since reaction
of the electrogenerated MR is not very specific, care
must therefore be taken to exclude other potential
oxidants that can compete with BO.
A. Chaubey, B.D. Malhotra / Biosensors & Bioelectronics 17 (2002) 441–456
450
and organic conducting salts come in this category of
mediators (Kulys, 1986; Khan, 1996; Li et al., 1999).
4.1. Mediated electron transfer
Mediators have a wide range of structures, properties
and a range of redox potentials. During the process of
electron shuttling between the active site of the enzyme
and the electrode, the mediator is cycled between its
oxidized and reduced forms. The mediator competes
with the enzyme’s natural substrate (often molecular
oxygen), effectively and efficiently diverting the flow of
electrons to the electrode. However, some redox
mediators can only work in a deoxygenated
environment. The criteria, which must be met for an
ideal redox mediator for sensor applications has been
discussed (Cenas and Kulys, 1981; Albery and Craston,
1987). The mediated bioelectrochemistry includes both
homogeneous mediation and heterogeneous mediation.
In the homogeneous system, the mediator and the
enzyme freely diffuses in the solution. This is frequently
used to determine the rate constants for the enzyme
mediator reaction (Szentrimay et al., 1977; Turner et
al., 1987; Davis, 1985) and to measure the reaction
rates between mediators and redox proteins (Davis,
1989; Dicks et al., 1986).
4.1.1. Homogeneous mediation
At the electrode, the mediator M* is converted into
M. M diffuses away from the electrode and reacts with
the enzyme, E2 to give E1 and regenerate M*. Finally
the enzyme is reconverted into E2 form by reaction
with substrate S.
At the electrode:
Km
M + E2 “ M* + E1
KE
E1 + S “ E2 + P
M* “M
(20)
(21)
Km
Kcat
S+ El [E1S] l P+ E2
k
E2 + M“ E1 +M*
At the electrode:
(23)
(24)
M* “ M
(25)
where S is substrate, P is the product, E1 and E2
represent two redox states of the enzyme and M/M*
couple is a mediator or natural redox partner. If we
consider the case when glucose oxidase (GOX) converts
glucose to gluconolactone, M/M* couple may be the
natural redox partner (O2/H2O2) or some added mediator couple such as ferrocene/ferricinium. The immobilized enzyme converts the substrate into product, P and
is generated by reaction with either its natural redox
partner or an added mediator, M generating M* within
the film. Ferrocene derivatives of varying charge and
solubility with redox potentials between 100 and 400
mV versus SCE have also been shown to accept electrons from the GOX (Cass et al., 1984). A number of
cases have been reported where the enzymes and soluble mediators both are immobilized at the electrode
surface leading to heterogeneous electron transfer. An
early report (Williams et al., 1970) describes mediated
electron transfer between the enzyme entrapped in a
layer on electrode using the benzoquinone/hydroquinone redox couple. Reports are available where
the mediators have been incorporated within the growing conducting polymer films. Foulds and Lowe (1988)
have investigated immobilization of GOX in polymeric
films containing ferrocenyl substituted pyrrole.
(22)
For flavo and quino enzymes the redox reaction of
the prosthetic group is a two electron transfer process.
Hence, for one electron transferring mediators such as
the ferrocenes, two molecules of a mediator are required to complete the catalytic cycle. Cyclic voltammetry is a useful technique to assess the effectiveness of a
particular mediator– enzyme combination for the reaction between the enzyme and mediator (Nicholson and
Shain, 1965; Davis, 1985, 1989). The use of enzyme
mediation schemes in the development of amperometric
immunoassays has also been reported (Leech, 1994;
Gleria et al., 1986, 1989).
4.1.2. Heterogeneous mediation
There are two possibilities of heterogeneous systems:
(i) when the mediator is added to the bulk solution; (ii)
when the mediator is present in the electrode not in the
bulk solution. In this case, the mediator incorporated
into the electrode diffuses into the solution during the
measurements. Ferrocenes (Feinberg and Ryan, 1981)
5. Enzyme electrodes
A simplest enzyme electrode consists of a thin layer
of enzyme held in close proximity to the active surface
of a transducer, a suitable reference electrode and a
circuit for measuring either by potentiometry or amperometry. While carrying out the measurements, the enzyme electrode is immersed into the analyte to be
detected and the steady state potential or current is
read. In general, a logarithmic relationship is observed
for the potentiometric, and a linear behavior for the
amperometric, electrodes. Generally, the electrocatalysts are immobilized onto an electrode surface by
adsorption (Huck and Schmidt, 1981; Jaegfeldt et al.,
1981), polymerization (Jaegfeldt et al., 1983; Degrand
and Miller, 1980) or electrodeposition (Persson et al.,
1993). The peak potentials for NADH electrocatalysis
for different electrocatalysts was reported by Lorenzo
et al. (1998). They found dramatic potential shifts as
compared to the NADH oxidation at the bare glassy
carbon electrode (0.7 V).
A. Chaubey, B.D. Malhotra / Biosensors & Bioelectronics 17 (2002) 441–456
Fig. 6. Electron transfer pathway in a Os containing redox polymer.
For a kinetically controlled biochemical reaction
catalyzed by the immobilized enzyme, the steady state
current is proportional to the initial rate of enzymatic
process (Mell and Maloy, 1975). In this case a plot of
I versus substrate concentration S yields a typical
Michaelis –Menten
type
response.
A
linear
Lineweaver– Burke plot, 1/I versus 1/S is the diagnostic technique for kinetic control of the electrochemical
response. The response of a biosensor is typically dependent on the amount of active enzyme immobilized.
A low molecular weight soluble mediator is disadvantageous as it can leach out of the electrode and be
lost to the bulk solution. This may lead to a significant signal loss (Schuhmann et al., 1990) and is considered as a serious problem for in vitro applications.
To overcome this, several groups have investigated
the use of immobilized mediators either with enzyme
in solution or with co-immobilized enzyme.
6. Modified electrodes
A number of modified electrodes for the regeneration of oxidized enzymes have been used. These are
based on the redox polymers containing p- and o-quinone groups adsorbed onto the surface of electrodes
(Cenas et al., 1983; Cenas and Kulys, 1984). Fig. 6
shows the mechanism of electron transfer in the Os
containing redox polymer. It has been found that the
enzymes such as GOX, LOD and xanthine oxidase
etc. could be reoxidized in the range 0.05– 0.5 V versus Ag/AgCl at pH 7. The use of these redox polymers is not advantageous as they lose their
electrocatalytic activity after a short period of time
(Cenas et al., 1984). A review on the chemically
Fig. 7. Ferrocene containing cross-linked polyallylamine.
451
modified electrodes was presented by Murray (1984)
and the kinetics of such electrodes has been discussed
in detail by Bartlett (1987).
In this context, Haemmerli et al. (1992) have suggested to link the mediators to a suitable polymer
backbone. The polymer may also serve to solidify the
carbon paste used to construct the enzyme electrode.
Koide and Yokoyama (1999) have electrochemically
characterized an enzyme (GOX) electrode based on a
ferrocene containing redox polymer. This redox polymer was based on cross-linked polyallylamine with
covalently attached ferrocene. Such a ferrocene containing cross-linked polyallylamine has been shown in
Fig. 7. Haemmerli et al. (1992) observed that the inclusion of polymeric mediator in preparation of the
carbon paste can enhance the sensor response significantly. Moreover, it is an improved method to enhance the performance of peroxidase based biosensor
for flow injection analysis applications. The problem
of electron transfer between the enzyme and the polymer has also been recently investigated (Degani and
Heller, 1989; Park et al., 1997).
Piro et al. (2000) studied the electro-oxidation of a
new aminonaphthalene compound modified with a
carboxylic acid function, 1-(5-aminonaphthyl)ethanoic
acid (ANEA), with a view to binding GOX covalently. Its electro-oxidation leads to thin films
(PANEA) on bare glassy carbon (GC), platinum (Pt)
electrodes and also on pre-polymerized aminonaphthoquinone (PANQ) films. Polymer-modified bilayer
electrodes Pt/PANQ/PANEA were then constructed.
GOX was covalently bound onto these electrodes,
leading to a Pt/PANQ/PANEA/GOX glucose sensor.
The amperometric response to glucose was monitored
in aerated and deaerated conditions. The results show
very efficient covalent binding of GOX and good enzyme activity on the electrode surface. Gorton et al.
(1991) have reported that H2O2 generated in a system
during the oxidation of glucose by GOX can be detected at peroxidase modified electrode. In this process, a polymer bound mediator is oxidized by the
peroxidase and the subsequent reduction of the mediator at the working electrode is measured.
Co-immobilization of enzymes and redox mediators
onto the electrode surfaces has attained considerable
attention (Gregg and Heller, 1990, 1991; Wang and
Heller, 1993; Ohara et al., 1994). In this context, the
molecular wiring of GOX in hydrogel of osmium redox mediator bound to polyvinylpyridine (Gregg and
Heller, 1990, 1991) or polyvinyl imidazole (Ohara et
al., 1993, 1994) backbone has already been achieved.
Apart from this wiring of LOD (Wang and Heller,
1993) and cellobiose oxidase (Elmgren et al., 1993)
has also been reported.
452
A. Chaubey, B.D. Malhotra / Biosensors & Bioelectronics 17 (2002) 441–456
7. Commercialization of mediated biosensors
Professor Clark (Clark, 1956) has been known as the
father of ‘Biosensor concept’ since he published his
definitive paper on the oxygen electrode in 1956. Later,
Clark and Lyons coined the term enzyme electrode
which was followed by Updike and Hicks (1967) when
they experimentally detailed the fabrication of a
functional enzyme electrode for glucose. The first
commercial biosensor for estimation of glucose was
launched by Yellow Springs Instrument Company
(Ohio) in 1975. This was based on the amperometric
detection of H2O2. In 1976, La Roche, Switzerland
introduced the lactate analyser (LA 640) in which the
soluble mediator, hexacyanoferrate was utilized for
electrons shuttling from LDH to the electrode. This
gave rise to the new generation of mediated biosensors.
Later, use of ferrocene and its derivatives as an
immobilized mediator with oxidoreductases was
explored for the fabrication of enzyme electrodes (Cass
et al., 1984). The glucose analysers commercialized by
Medi Sense (Abbott Laboratories, USA) in 1987 and
Boehringer Mannheim and Bayer are also mediated
biosensors. National Physical Laboratory, India has
patented a technology based on mediated electron
transfer for glucose estimation. This is the first
amperometric Indian glucose biosensor available in the
market.
8. Conclusions
The electrical communication between the redox enzymes and the electrodes can be established by using
biologically active or synthetic charge carriers as mediators. These artificial electron donor or acceptor
molecules are usually referred to as electron transfer
mediators. They are capable of accepting the redox
enzymes in place of their natural oxidants or reductants. The mediators are expected to have reversible
electrochemistry to provide rapid reactions with the
redox enzyme, oxidizing or reducing the active site of
the enzyme. The total efficiency of the electron transfer
depends on the mediator properties as well as on the
whole system. These mediated systems have resulted in
the development of numerous amperometric biosensors
and bioelectrocatalytic systems including bioreactors
and biofuel cells. Attempts have been made to reoxidize
the resulting reduced enzyme– cofactor complex directly
by electrochemical method. Later on, electron acceptors
were used to shuttle electrons from the catalytic site to
the electrode. Biosensors sometimes have some disadvantages such as small dynamic range, short lifetime,
sensitivity to interferents and inhibitors, pH dependency etc. These may be overcome by incorporating
them in a flow injection analysis (FIA) system. Using
this technique, the sample may be automatically collected from the bioreactor and conditioned according
to the requirements of the sensor. Mediated enzyme
electrodes are known to be less susceptible to interfering substances due to lower electrode potentials. For
the sensors in flow injection analysis, special attention is
given to the immobilization of the mediator.
Acknowledgements
We are thankful to Dr Krishan Lal, The Director,
NPL for his encouragement towards biosensor field.
AC thanks CSIR for the award of Senior Research
Fellowship. Financial support received from DST sponsored project (SP/S2/M-52/96), Indo-Polish (INT/POL/
P.015/2000) and Indo-Japan project (INT/IJJC/I-17/97)
is gratefully acknowledged.
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