Biomedical Engineering Reference
In-Depth Information
inability of the monomers to penetrate the growing film matrix beyond a certain thickness
to be oxidized at the electrode surface. Other significant advantages previously mentioned
are that electropolymerizations can be controlled via the applied potential as well as spa-
tially, occurring solely at the electrode surface. Finally, these monomers have the advantage
of being soluble in purely aqueous solution, allowing electropolymerization without the
need for additional harsh polymerization initiators or oxidizing agents. This latter feature
makes it a Green Chemistry method preferred over chemical synthesis for making poly-
meric films. It also has the advantage that many mainly hydrophilic biological molecules
can be added into the solution in the electrochemical cell during the electropolymerization
step, leading to their stable incorporation by physical entrapment within the growing film
matrix. As an immobilization method, this approach is simple, convenient, and somewhat
unique. It has real value since many biological molecules, especially proteins, often have
their fragile native structures and biological activities stabilized upon immobilization. Once
entrapped in thin films or immobilized by other methods upon an electrode, a biological
macromolecule can then be accessed by electron transfer methods at a specific potential.
Biosensor output, typically the current, then can reveal the particular electroactive analyte
concentration being detected.
A requirement for this type of biosensor is that the biological molecule involved in
electron transfer be efficiently coupled electronically to the electrode surface by a suit-
able transfer mechanism. It is known that electron transfer rates between electron dona-
tor and receptor species decrease exponentially as their separation distance increases, as
described by Marcus theory. This requirement for close approach of the electroactive
enzyme active site to the electrode surface has spurred the development of a number of
innovative methods to enhance electronic coupling via transfer agents, thereby elimi-
nating the need for physical proximity of the active site to the electrode surface. One
specific example involves the use of hydrophilic polymers containing repeating spaced
osmium redox centers to shuttle electrons between the enzyme active site and the elec-
trode surface via transfers between successive osmium redox groups along the polymer
chain (67).
We have created and studied an enzyme-based biosensor of the small molecule ana-
lyte H
2
O
2
. The enzyme horseradish peroxidase [HRP: (68) review] was physically
entrapped during electropolymerization of various thin films formed from the phenolic-
based monomers shown in Figure 1.22. All of the monomers shown—phenol, substi-
tuted phenols, and tyrosine-based amino acid derivatives—possessed the phenol ring
moiety, with the tyrosine monomers having this moiety as a pendant portion of their
amino acid side chains (69). The films were all formed via cyclic potential sweeping
across the potential range for oxidation of the monomers, which had broad peak max-
ima between
0.8 V at neutral pH. In this electrochemical process, films were
formed by a free radical-based coupling of monomers. The insoluble polymerization
products that formed were either direct phenol ring-ring C-C linkages, ether ring C to
ring -OH linkages or a mixture of both linkages and the film formation rates were found
to be monomer concentration dependent (70,71). The films electropolymerized on the
electrode surface were to varying extents self-limiting in thickness and pinhole free. We
studied the enzyme electrode sensitivities to H
2
O
2
at two different potentials. The first
corresponds to enzymatic catalysis sensing at
0.6 and
0.05 V. Following the direct two-electron
electroreduction of H
2
O
2
by entrapped HRP, the HRP active oxidation state is regener-
ated at -0.05 V and this current was shown to be proportional to H
2
O
2
concentration. In
Figure 1.23, we show that the current detection sensitivities for HRP-entrapped tyrosine
derivative films are in the low millimolar range for H
2
O
2
and could result in micromo-
lar determinations at nanoampere current detection levels. Clearly, there is background
detection of H
2
O
2
by direct electroreduction at the bare Pt electrode or at the electrode
