AmpemmetricEnzymeElectmds:Mediac#.

Design

Tony Cass

The basic principle that underlies all amperometric enzyme electrodes is the flow
of electrons between the analyte and the electrode under the influence of a
controlled potential. In most cases there are large kinetic barriers to electron
transfer between organic compounds and solid electrodes resulting in slow
electron exchange a n d hence small c u r r e n t s and/or high
overpotentials.Furthermore in a complex analytical matrix there is the possibility
of more than one compound being oxidised or reduced a t the applied potential.
Under these circumstances the enzyme serves the dual fbnction of acting as a
specific catalyst and does so by reacting with the analyte:

Enzyme(ox.or red.) + Substrate 4 Enzyme(red or ox.} + Product

Thus the reducing or oxidising equivalents in the analyte are transferred to the
enzyme.
At this stage the problem of specificity has been largely overcome but the electrode
kinetics is now between the enzyme and the electrode and this process may be
even less favourable than with the initial substrate. We can classify redox
enzymes into 3 broad groups:
0 Those that employ a small difisible redox cofactor.
0 Those that have a bound redox centre that reacts with
small
molecules.
0 Those that have a bound redox centre that reacts with other redox
proteins.
The first group of enzymes is exemplified by dehydrogenases and are by far the
most numerous, the latter two groups are a varied collection of molecules and
have been the main focus of application in biosensors based upon amperometric
techniques. Redox proteins that interact with other proteins often have their
centres at the surface of the molecule and are therefore available t o interact with
suitably prepared electrodes. This are of promoter electrochemistry of redox
proteins is of great fundamental interest and has been extensively reviewed. In
most applications of amperometric enzyme electrodes we are usually employing
the middle group of enzymes, those that interact with small donor or acceptor
redox partners. In vivo these partner substrates are species such as glutathione
or oxygen, particularly the latter. It is here that the use of mediator technology is
most fruitful although often mediators can react with proteins that have
macromolecular redox partners.
The classic example of redox proteins that use oxygen as an in vivo electron
acceptor are the flavoenzyme oxidases. This group of some 15-20 enzymes catalyse
the oxidation reaction:
SH2 + 0 2 4 S + H202
Centre for Biotechnology, Biochemistry Department, Imperial College, London
sw7 2Az
c ,.
In the context of enzyme electrodes the hydrogen peroxide can then be
determined electrochemically by its oxidation:
H202+ 0 2 + 2e- + 2H+
Problems with this approach include a dependence of the current on oxygen
tension, pH and the possible oxidation of other compounds in the analyte a t the
potentials needed to oxidise peroxide. Replacement of the oxygen by a non-
physiological oxidant can bypass some or all of these problems.
The use of artificial electron donors o r acceptors has a long and distinguished
history in biochemistry although the choice of mediator was oRen determined by
the need for a large change in optical properties, rather than criteria based on
heterogeneous kinetics, and hence organic dyes tended t o dominate.
Comprehensive lists of mediators for biochemical applications have been
published.
In selecting mediators for enzyme electrodes factors such as stability and rapid
kinetics with the enzyme are still important but additional influences including
rapid heterogeneous (electrode) kinetics, lack of reactivity with oxygen and
suitability for incorporation into the enzyme/electrode environment assume
importance. Ideally the electrochemistry of the mediator should be that of a one
electron, zero proton couple with a low redox potential. The former feature
eliminates a pH dependence in the mediator electrochemistry whilst the latter
avoids potential interference from other reducing agents in the sample. There
have been a number of reviews on mediator technology that describe the extent to
which these criteria have been met.

Although careful choice of mediator can improve the performance of enzyme

electrodes the simple addition of the soluble mediator suffers several drawbacks:
0 Loss of mediator to the bulk solution.
0 Side reactions of the mediator with other constituents of the sample
matrix.
0 Currents that are limited by the diffusion of mediator between enzyme
and electrode.
To circumvent these difficulties various chemistries have been employed to hold
the mediator in the vicinity of the enzymdeledrode interface. These are:
0 Modification of the electrode with covalently attached mediator.
0 Modification of the enzyme with the mediator.
0 Entrapment of the enzyme within a polymer that cames redox active
(mediator) groups.
The first and last of these draw upon the extensive research of the past 20 years in
chemically modified electrodes (CME's) whilst the second approach is an
extension of protein chemical modification.
Perhaps the most useful of the mediators employed in enzyme electrodes are those
based on ferrocene:
The advantages of this organometallic compound include rapid heterogeneous
kinetics, a versatile "peripheral" chemistry of the cyclopentadiene rings and a low
potential one elctron zero proton redox couple. The versatility of ferrocenes in
making enzyme electrodes is shown by their use in the forms of soluble mediator,
covalently bound mediator, and polymeric mediator.
A variety of examples of these configurations will be discussed and compared
with other types of mediation scheme.