Biomedical Engineering Reference
In-Depth Information
such as dehydrogenases and peroxidases. Unlike oxidases, these enzymes do not
use O
2
as an electron-accepting cosubstrate. An example is the lactic acid biosensor
base on lactate dehydrogenase, which catalyses the following reaction:
Lactate dehydrogenase
+
Lactate +
NAD
Pyruvate+
NADH
Another way to measure H
2
O
2
at a low oxidation potential is by using car-
bon with dispersed rhodium ruthenium or iridium particles. Although effective in
lowering the operating potential, most mediated electrodes still suffer from some
ascorbic acid and uric acid interference. Furthermore, mediators are small mol-
ecules, and excessive diffusion out of the film immobilized on the electrode surface
results in a mediator loss, which results in a loss of catalytic activity. In addition,
there can be competition between the oxidized mediator and oxygen for the oxida-
tion of the active site.
3. The third generation is based on the direct electron transfer between the
active site of a redox enzyme and the electrochemical transducer [Figure
9.6(c)]. Hence, the signal transduction eliminates the oxygen consumption
at the electrode. One approach involves binding redox-active centers (me-
diators) and enzymes in a polymeric matrix immobilized on an electrode
surface. A series of such enzyme-based systems are developed and are gen-
erally referred to as “wired” enzyme electrodes. The enzyme was, in effect,
wired by the mediator to an electrode. The wired enzymes were able to
transfer redox equivalents from the enzyme's active site through the media-
tor to an electrode. Wired enzyme electrodes were originally developed by
Adam Heller as a solution to prevent the diffusion of the mediators out
of the fi lm. The mediators for these systems are osmium bipyridine com-
plexes, which are cationic and hence bind electrostatically to the anionic
glucose oxidase.
The wired-enzyme principle resulted in subsequent development of enzyme-
immobilizing redox polymers. The coimmobilization of enzyme and mediator is
accomplished by the redox mediator labeling of the enzyme followed by enzyme
immobilization in a redox polymer or an enzyme and mediator immobilization in a
conducting polymer (such as polypyrols). Different configurations of a cholesterol
biosensor with a cholesterol oxidase entrapped in a polypyrrole film have also
been developed. These polymers effectively transfer electrons from glucose-reduced
GOX flavin sites to polymer-bound redox centers. A series of chain redox reactions
within and between polymers transfer the equivalents to an electrode surface. This
allows the easy exchange of electrons between the osmium centers of the complexes
and the active site of the enzyme.
For these possibilities, immobilization technique is important to ensure that
the redox center is sufficiently close to the electrode to allow rapid electron trans-
fer. For large redox enzymes, such as glucose oxidase, this is difficult to realize as
their active sites are hidden inside the protein structure. For these enzymes it be-
comes important that they are immobilized on a compatible electrode surface in a
way that makes electron transfer from the catalytic center to the electrode feasible
