Enzyme Technology
Amperometric biosensors
Amperometric biosensors function by the production of a
current when a potential is applied between two electrodes. They generally have
response times, dynamic ranges and sensitivities similar to the potentiometric
biosensors. The simplest amperometric biosensors in common usage involve the
Clark oxygen electrode (Figure 6.5). This consists of a platinum cathode at
which oxygen is reduced and a silver/silver chloride reference electrode. When a
potential of -0.6 V, relative to the Ag/AgCl electrode is applied to the
platinum cathode, a current proportional to the oxygen concentration is
produced. Normally both electrodes are bathed in a solution of saturated
potassium chloride and separated from the bulk solution by an oxygen-permeable
plastic membrane (e.g., Teflon, polytetrafluoroethylene). The following reactions
occur:
Ag anode 4Ag0 + 4Cl−
4AgCl + 4e− [6.13]
Pt cathode O2 + 4H+ +
4e− 2H2O [6.14]
The
efficient reduction of oxygen at the surface of the cathode causes the oxygen
concentration there to be effectively zero. The rate of this electrochemical
reduction therefore depends on the rate of diffusion of the oxygen from the bulk
solution, which is dependent on the concentration gradient and hence the bulk
oxygen concentration (see, for example, equation
3.13). It is clear that a
small, but significant, proportion of the oxygen present in the bulk is consumed
by this process; the oxygen electrode measuring the rate of a process which is
far from equilibrium, whereas ion-selective electrodes are used close to
equilibrium conditions. This causes the oxygen electrode to be much more
sensitive to changes in the temperature than potentiometric sensors. A typical
application for this simple type of biosensor is the determination of glucose
concentrations by the use of an immobilised glucose oxidase membrane. The
reaction (see reaction scheme [1.1]) results in a reduction of
the oxygen concentration as it diffuses through the biocatalytic membrane to the
cathode, this being detected by a reduction in the current between the
electrodes (Figure 6.6). Other oxidases may be used in a similar manner for the
analysis of their substrates (e.g., alcohol oxidase, D- and L-amino acid oxidases,
cholesterol oxidase, galactose oxidase, and urate oxidase)
Figure
6.5. Schematic diagram of a simple amperometric biosensor. A potential
is applied between the central platinum cathode and the annular silver anode.
This generates a current (I) which is carried between the electrodes by means of
a saturated solution of KCl. This electrode compartment is separated from
the biocatalyst (here shown glucose oxidase, GOD) by a thin plastic membrane, permeable only to oxygen.
The analyte solution is separated from the biocatalyst by another membrane,
permeable to the substrate(s) and product(s). This biosensor is normally
about 1 cm in diameter but has been scaled down to 0.25 mm diameter using a Pt
wire cathode within a silver plated steel needle anode and utilising dip-coated
membranes.
Figure 6.6. The response of an amperometric biosensor
utilising glucose oxidase to the presence of glucose
solutions. Between analyses the biosensor is placed in oxygenated buffer devoid of glucose. The steady rates of oxygen depletion may be
used to generate standard response curves and determine unknown samples. The
time required for an assay can be considerably reduced if only the initial
transient (curved) part of the response need be used, via a suitable model and
software. The wash-out time, which roughly equals the time the electrode spends
in the sample solution, is also reduced significantly by this process.
An alternative method for determining the rate of this reaction is
to measure the production of hydrogen peroxide directly by applying a potential
of +0.68 V to the platinum electrode, relative to the Ag/AgCl electrode, and
causing the reactions:
Pt anode H2O2
O2 + 2H+ +
2e− [6.15]
Ag cathode 2AgCl + 2e−
2Ag0 +
2Cl−[6.16]
The major problem with these
biosensors is their dependence on the dissolved oxygen concentration. This may
be overcome by the use of 'mediators' which transfer the electrons directly to
the electrode bypassing the reduction of the oxygen co-substrate. In order to be
generally applicable these mediators must possess a number of useful properties.
-
They must react rapidly with the reduced form of the
enzyme.
-
They must be sufficiently soluble, in both the oxidised and
reduced forms, to be able to rapidly diffuse between the active site of the
enzyme and the electrode surface. This solubility should, however, not be so
great as to cause significant loss of the mediator from the biosensor's
microenvironment to the bulk of the solution. However soluble, the mediator
should generally be non-toxic.
-
The overpotential for the regeneration of
the oxidised mediator, at the electrode, should be low and independent of pH.
-
The reduced form of the mediator should not readily react with
oxygen.
The ferrocenes represent a commonly used family of mediators
(Figure 6.7a). Their reactions may be represented as
follows,
[6.17]
Electrodes have now been developed which can remove the electrons
directly from the reduced enzymes, without the necessity for such mediators.
They utilise a coating of electrically conducting organic salts, such as N-methylphenazinium
cation (NMP+, Figure 6.7b) with tetracyanoquinodimethane radical anion
(TCNQ.- Figure 6.7c). Many flavo-enzymes are strongly
adsorbed by such organic conductors due to the formation of salt links,
utilising the alternate positive and negative charges, within their hydrophobic
environment. Such enzyme electrodes can be prepared by simply dipping the
electrode into a solution of the enzyme and they may remain stable for several
months. These electrodes can also be used for reactions involving
NAD(P)+-dependent dehydrogenases as they also allow the
electrochemical oxidation of the reduced forms of these coenzymes. The three
types of amperometric biosensor utilising product, mediator or organic
conductors represent the three generations in biosensor development (Figure
6.8). The reduction in oxidation potential, found when mediators are used,
greatly reduces the problem of interference by extraneous material.
Figure 6.7. (a) Ferrocene (e5-bis-cyclopentadienyl iron), the parent compound of a
number of mediators. (b) TMP+, the cationic part of conducting organic
crystals. (c) TCNQ.-, the anionic part of conducting
organic crystals. It is a resonance-stabilised radical formed by the
one-electron oxidation of TCNQH2.
Figure 6.8.
Amperometric biosensors for flavo-oxidase enzymes illustrating the three
generations in the development of a biosensor. The biocatalyst is shown
schematically by the cross-hatching. (a) First generation electrode
utilising the H2O2 produced by the reaction. (E0 =
+0.68 V). (b) Second generation electrode utilising a mediator (ferrocene)
to transfer the electrons, produced by the reaction, to the electrode.
(E0 = +0.19 V). (c) Third generation electrode directly
utilising the electrons produced by the reaction. (E0 = +0.10 V). All
electrode potentials (E0) are relative to the Cl−/AgCl,Ag0
electrode. The following reaction occurs at the enzyme in all three biosensors:
Substrate(2H) + FAD-oxidase
Product + FADH2-oxidasefi [6.18]
This is followed by the processes:
(a)
biocatalyst
FADH2-oxidase + O2
FAD-oxidase + H2O2
[6.19]
electrode
H2O2
O2 + 2H+ + 2e− [6.20]
(b)
biocatalyst
FADH2-oxidase + 2 Ferricinium+
FAD-oxidase + 2
Ferrocene +
2H+ [6.21]
electrode
2 Ferrocene
2 Ferricinium+ + 2e− [6.22]
(c)
biocatalyst/electrode
FADH2-oxidase
FAD-oxidase + 2H+ + 2e− [6.23]
The current (i) produced by such amperometric biosensors is
related to the rate of reaction (vA) by the expression:
i = nFAvA
(6.6)
where n represents the number of electrons
transferred, A is the electrode area, and F is the Faraday. Usually the rate of
reaction is made diffusionally controlled (see equation 3.27) by use of external
membranes. Under these circumstances the electric current produced is
proportional to the analyte concentration and independent both of the enzyme and
electrochemical kinetics.
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This page was established in 2004 and last updated by Martin
Chaplin on
6 August, 2014
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