Electrocatalysis for Fuel Cells at Enzyme-Modified Electrodes - Fuel Cell Catalysis: A Surface Science Approach - page 598

methionine in laccase from Trametes villosa, for instance, results in a drop of the
T1 midpoint potential of only 100 mV [Xu et al., 1999]. Clearly, other factors must
come into play, such as solvent accessibility at the active site and charge distribution
within the protein. This is an example of the role that subtle changes in the protein
environment can play in exerting a strong influence over the catalytic properties of
an enzyme active site. The reduction potentials of the other Cu sites in laccase are
less well characterized.
Laccases usually have optimum activity under weakly acidic conditions ( pH 4 - 5),
whereas the optimum for bilirubin oxidase activity is close to neutral pH.
Electrocatalytic O 2 reduction by laccases, as for Pt, is generally inhibited by chloride,
and the enzymes typically retain only 40% activity at 100 mM NaCl [Barton et al.,
2002]. In contrast, bilirubin oxidase retains substantial activity in the presence of
chloride [Mano et al., 2003]. Bilirubin oxidase is therefore the more suitable
enzyme for operation in most biological fluids. This enzyme is also tolerant to certain
levels of alcohols, and has been used at the cathode of a membraneless ethanol/O 2 fuel
cell operating at 1 mM ethanol in buffered aqueous solution [Topcagic and Minteer,
2006]. Laccase also has significant tolerance to alcohols: an RDE modified with lac-
case in an Os-containing redox hydrogel operating in 5 M methanol in aqueous buffer
(pH 4) retained almost 80% of the activity recorded in methanol-free solution [Hudak
and Barton, 2005]. A carbon paper cathode modified with laccase in the same redox
hydrogel has been tested in a fuel cell in which either methanol or H 2 is oxidized at the
Pt anode (separated from the cathode by a Nafion 1135 membrane). The cell polariz-
ation curves are almost identical for operation on 1 and 10 M methanol at the anode,
indicating that the behavior of laccase is not significantly impaired by the increase in
fuel crossover to the cathode compartment expected at the higher fuel concentration
[Hudak and Barton, 2005].
17.2.1.1 Attachment Strategies It has been shown that a film of adsorbed
laccase will exchange electrons directly with a PGE electrode, leading to electrocata-
lytic O 2 reduction, but the adsorbed film is very unstable [Blanford et al., 2007].
Several approaches have been employed to generate films of laccase that are stable
for many days and show higher electrocatalytic current density.
A redox hydrogel approach for immobilizing and mediating electron transfer to
glucose oxidase was suggested by Heller and co-workers in 1989, and this team has
subsequently developed and elaborated upon the method for application to a
number of redox enzymes, including laccase and bilirubin oxidase, and also ascorbate
oxidase, horseradish peroxidase, and fructose dehydrogenase (see Section 17.3.1)
[Pishko et al., 1990; Barton et al., 2004; Heller, 2006]. The technique involves attach-
ment of an Os III/II complex to a hydrophilic polymer that can be crosslinked to form a
redox-active hydrogel (Fig. 17.6a). An enzyme dissolved in the precursor solution with
the redox polymer and crosslinker becomes incorporated into the hydrogel, and, upon
crosslinking, is immobilized in the hydrogel film. Redox hydrogels are readily formed
from soluble polymers such as polyvinylimidazole and polyvinyl alcohol, or from
insoluble polymers such as polyvinylpyridine that can be made soluble by quaterni-
zation [Kenausis et al., 1996; Mano et al., 2003; de Lumley-Woodyear et al., 1995].
Copolymers of polyvinylimidazole and polyacrylamide have also been reported,