Biomedical Engineering Reference
In-Depth Information
covered with films containing no HRP. However, the HRP-containing films are overall
the most sensitive for H
2
O
2
detection. The second H
2
O
2
detection method we studied is
not formally a biosensor since it relies upon a process independent of the HRP, that is
direct electrode sensing of H
2
O
2
by a two-electron oxidation process at
0.85 V. Both
methods detect H
2
O
2
in the low millimolar range, although the enzymatic biosensor
detection method is more sensitive and is less prone to potential interferences from bio-
logical species such as uric acid and ascorbic acid.
It is worth noting that there is a rather large range of potential monomers that could be
electropolymerized to form biosensor films containing entrapped enzymes. As an exam-
ple, we studied a series of these including phenol, phenylenediamine,
o
-dianisidine, ben-
zene dialdehyde, and glutaraldehyde. These were electropolymerized to form films that
entrapped a range of protein and enzyme systems (72). These included: phycoerythrin,
which retained its native fluorescence spectra; alkaline phosphatase, which catalyzed the
organophosphorus pesticides paraoxon and methyl parathion to
p
-nitrophenol products
that were detected electrochemically at
0.8 V; and HRP and glucose oxidase enzymes
that were used to detect H
2
O
2
and glucose using a colorimetric approach.
It is interesting to consider that in the system just described, HRP is itself capable of
forming polymers by catalysis of phenolic monomers in the presence of H
2
O
2
. This is a
solution enzymatic process stoichiometrically dependent upon H
2
O
2
and no electron
transfer at the electrode surface is involved. However, it is of interest since it is an
aqueous-based Green Chemistry method for forming polymers and can be used as an
alternate method to electropolymerization of films. We have carried out a number of
studies of HRP-based polymerizations. In the case of LB monolayer films formed from
amphiphilic anilinic and phenolic monomers, such as 4-
o
-tetradecyloxyphenol, HRP
added to the subphase was shown to catalyze the conversion of the LB monomer film
to an LB polymer film (73-76). This process formed films with a high degree of order.
FT-IR experiments, using s and p polarized radiation as orientation probes, were per-
formed upon the monolayers picked up from the LB trough with a quartz substrate
and demonstrated a significant degree of order within the LB polymer film monolay-
ers. In fact, the ordered highly conjugated polymeric products produced by the LB
trough method possessed enhancements of their third-order nonlinear optical proper-
ties compared with bulk polymerized monomers (77,78). Following assembly, mono-
layers comprising these polymer films could be picked up from the LB trough surface
and used to create biosensors. In the case of the monomer discussed above, this pickup
process would rely upon the adhesion of the alkyl chains to a hydrophobic substrate
surface. As an example, we have shown using AFM and x-ray photoelectron spec-
troscopy that related amphiphilic phenol-based monomers quantitatively coat
hydrophobic gold surfaces and have significantly different morphologies before and
after HRP polymerization (79).
HRP is the most widely used of the peroxidase enzymes. Because of its broad substrate
specificity, it forms the basis for many commercial biotechnology applications (80). Using
genetic engineering techniques, its substrate specificity can be manipulated. For example,
the enantioselectivity of HRP for oxidation of alkyl aryl sulfides has been increased
through genetic engineering approaches (81). Many of the widespread HRP applications
occur in immunohistochemical assays for research and clinical use and involve colorimet-
ric- or fluorimetric-based product assays. In the colorimetric systems, an optical signal is
the output where the polymerization products have much increased extinction coefficients
at particular wavelengths in the visible. We have studied such an optical system, involv-
ing hydroxyquinoline-5-sulfonic acid as a substrate for HRP that could form the basis of a
biosensor for Fe(III) in the 10
5
M concentration range (82).
