Biomedical Engineering Reference
In-Depth Information
9.4.1.3 Amperometric Enzyme Electrode
Common strategies that an amperometric enzyme electrode is constructed for mon-
itoring a substrate are as follows:
1. The fi rst-generation enzyme biosensors [Figure 9.6(b)] were the extensions
of the oxygen electrode where an oxygen consuming enzyme is immobilized
to a platinum electrode and the reduction of oxygen at the electrode pro-
duces a current that is inversely proportional to the analyte concentration.
Typically, oxidase enzyme is immobilized on to membrane at the surface
of a platinum electrode. The consumption of O
2
or the formation of H
2
O
2
is subsequently measured at a platinum electrode. The detection limit for
H
2
O
2
-based sensors is generally better than for oxygen sensing systems. A
problem with this arrangement is the loss in selectivity between the biorec-
ognition event and the amperometric H
2
O
2
detection. Furthermore, it suf-
fers from slow response characteristics, diffi culties in miniaturization, and
low accuracy and reproducibility. However, a major limitation of the H
2
O
2
detection approach is the high oxidizing potential (700 mV versus Ag/AgCl
reference electrode) necessary for H
2
O
2
oxidation results in substantial in-
terference from the oxidation of other compounds such as ascorbic acid
(also called vitamin C), uric acid, and acetaminophen in complex matrices.
2. The second generation is focused on lowering the working potential by
means of an artificial electron mediator [Figure 9.6(b)]. The electron ac-
ceptor is replaced by the mediator, which shuttles the electrons involved
in the redox process from the enzyme toward the electrode or vice versa.
Most oxidases are not selective with respect to oxidizing agent, allowing
the substitution of a variety of artificial oxidizing agents. An example of
glucose oxidase is given in the following reaction:
GOX-FADH + Mediator
GOX-FAD+Mediator
2
ox
red
These mediators catalyze the oxidation of H
2
O
2
at the electrode of biosen-
sors. Reagents such as ferrocyanide, ferrocene derivatives, quinones quinoid-like
dyes, organic conducting salts, and viologens have been coimmobilized as media-
tors between H
2
O
2
and electrodes. A kind of ferric enzyme, horseradish peroxi-
dase (HRP), is also used. In these electrodes a reduction current, resulting from
either the direct or mediated electron transfer, is measured at low applied potential,
thereby circumventing the interference problems encountered during the electro-
chemical oxidation of H
2
O
2
.
The selection of mediators with appropriate redox potentials allows a better
performance of the working electrode in a potential range where other components
in the sample matrix are not oxidized or reduced. Low O
2
solubility in aqueous
solutions and the difficulty associated with controlling the O
2
partial pressure were
disadvantages of biosensors based on the O
2
/H
2
O
2
reaction. When a highly solu-
ble artificial mediator is used, the enzyme turnover rate is not limited by the co-
substrate (O
2
) concentration. Hence, eliminating the O
2
dependence facilitates the
control of the enzymatic reaction and sensor performance. Furthermore, the use of
mediators other than O
2
allows an exploitation of other oxidoreductase enzymes
