Optimising the adsorption of bilirubin oxidase using dual polarisation interferometry and an electrochemical quartz crystal microbalance with dissipation analysis

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Abstract

This thesis investigates the adsorption mechanisms and changes in activity of bilirubin oxidase from Myrothecium verrucaria immobilised to gold- and silica-modified surfaces. This enzyme is used as an efficient bioelectrocatalyst for the four-electron oxygen reduction in fuel cells. An electrochemical quartz crystal microbalance with dissipation monitoring (E–QCM–D) was used to show how applying a constant potential to bilirubin oxidase adsorbed to a carboxylate-terminated gold electrode resulted in activity loss attributed to structural rearrangement of the adsorbed enzyme layer. When a varying potential was applied, rapid enzyme deactivation occurred, with no mitigation of activity loss through covalent attachment to the electrode.The E-QCM-D was further used to observe how changing enzyme concentration affects the adsorption mechanics and catalytic activity of the adsorbed layer. An optimum concentration produced greatest activity and stability, with lower concentrations denaturing more readily, and higher concentrations adopting an unfavourable geometry for electron transfer. Surface functionality showed adsorption to hydrophobic methyl- terminated electrodes revealed a rigid layer with reduced catalytic activity. Ammonium terminated surfaces resisted denaturation, but misorientated the enzyme for efficient electrocatalysis. Increasing the chain length of the surface modifiers increased the enzyme–electrode distance; this decreased activity for the carboxylate surface and removed the activity for methyl- or ammonium-terminated surfaces. Dual polarisation interferometry further showed no enzyme denaturation when it was adsorbed to amine and sulfonic acid surfaces.Enzyme adsorption under an applied constant potential caused a decrease in both mass loading and activity when compared to open circuit potential adsorption. The presence of an applied potential did not cause increased layer denaturation, but changed the orientation of the enzyme in a position unfavourable for electron transfer. Lower applied potential give lower mass loadings, yet similar surface mechanics and activity per adsorbed enzyme.Chemical modification to pristine graphene showed targeted interactions with biomolecules such as proteins and fluorophores. This surface modification has the potential to be adapted towards adsorption of bilirubin oxidase for fuel cell catalysis and other electrochemical sensing applications.The observations in this thesis show how the E–QCM–D and DPI can provide a more expansive picture of the applicability of redox enzymes in fuel cell systems. Preventative steps need to be taken in order to maintain an enzyme’s structural integrity and in turn its catalytic competency. Without such provisions the observations above suggest that redox enzymes have a finite lifetime when under conditions approximating fuel cell systems.

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