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The impact of methanol on oxygen reduction activity is studied using a mediated biocathode catalyzed by laccase from Trametes versicolor. The sensitivity of oxygen reduction current density to the presence of methanol at such electrodes depends strongly on mediator redox potential. This study demonstrates that the selectivity of laccase cathodes toward oxygen reduction can allow methanol feed concentration up to 5 M in direct methanol fuel cells. Within the 0-5 M concentration range, methanol primarily affects enzyme kinetics and not the electron transport via the mediator. For methanol concentrations of 0-2.5 M, laccase activity toward oxygen was largely maintained; approximately 30% loss of activity occurred in the 2.5-5 M range, and irreversible loss of enzyme activity was observed beyond 7.5 M. The presence of methanol primarily decreases the turnover number of the enzyme rather than altering substrate binding, suggesting a noncompetitive inhibition mechanism. It is proposed that this reduction occurs due to changes in the electron transfer environment near the T1 binding pocket due to the presence of methanol.

Homogeneous, sub-micron thick bioactive films containing redox hydrogels and the enzyme laccase were prepared on gold-coated glass slides via convective self-assembly. Sub-micron film thickness (∼10-50 nm) allowed higher mediator and enzyme utilization by lowering transport limitations. This study demonstrates control of redox hydrogel film morphology by manipulating hydrophilicity, spreading and wetting properties of the hydrogel/electrode interface. Use of a non-ionic surfactant (Triton X-100) enabled spreading of enzyme containing precursor solutions with no negative impact on enzyme activity. Ellipsometry was used to measure filmswelling properties of these systems, square wave voltammetry was employed to estimate the amount of electroactive redox polymer, and cyclic voltammetry enabled estimation of apparent electron diffusivity. This study impacts the design of thin, catalytic films for bioelectronic applications.

The kinetics of glucose oxidation by glucose oxidase (GOx) were evaluated with mediation by two different osmium redox polymers and in the presence and absence of oxygen (O2). A one-dimensional model was designed to account for the presence of dissolved oxygen, which competes with the mediator for adsorption sites on GOx and electrons from glucose oxidation. Kinetic parameters were determined by fitting the experimental current density measurements for varying potential, glucose and O2 concentrations, accounting for competitive inhibition and competitive reaction. It was demonstrated that the presence of O2 does not change the turnover number, kcat, of the enzyme, indicative of a competitive inhibition mechanism. However, improved fitting of the model was observed when considered the competitive reaction of O2. Kinetic parameters specific to the mediator reaction were an order of magnitude higher than other homogeneous mediated systems, leading to high mediation efficiency. Bimolecular rate constants for the GOx-oxygen reaction were found to be an order of magnitude lower than that of the free solution, which can be attributed to the reduced activity of immobilized GOx.

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