Mapping and Modeling Interfacial pH Dynamics at Enzymatic Microelectrode

Redox enzymes are promising biocatalysts for energy conversion and sensing, particularly when immobilized on electrodes to facilitate charge transfer and enhance stability. While they offer the advantages of operating under mild conditions and near-neutral pH, high reaction rates can profoundly alter the immobilized enzyme microenvironment. This requires understanding the interplay between localized concentration gradients and enzyme activity at the electrocatalytic interface. Here, we elucidate this dynamic coupling between reaction kinetics and local pH at an enzyme-modified electrode through a combined modeling and experimental approach. We present a detailed 2D-axisymmetric finite element model of a graphite microelectrode modified with bilirubin oxidase from Myrothecium verrucaria catalyzing the oxygen reduction reaction via direct electron transfer. The model explicitly accounts for buffer equilibria through ion activities and incorporates experimentally determined pH-dependent kinetic parameters, including apparent turnover and Michaelis constants. The simulations were validated experimentally using in situ and operando fluorescence confocal laser scanning microscopy (FCLSM) with the pH-sensitive dye fluorescein in weakly buffered electrolytes. Both numerical and experimental results reveal substantial interfacial alkalinization, with the local pH increasing by more than two units under the weakest buffering conditions. The model successfully reproduces cyclic voltammetry and quasi-steady-state chronoamperometry profiles using a single enzyme-coverage-dependent parameter. Furthermore, spatial fluorescence profiling closely corroborates the simulated pH gradients, while revealing evidence of natural convection occurring over extended time scales. Our study provides a quantitative framework crucial for designing stable, high-performance bioelectrochemical interfaces, which can be readily extended to other key redox enzymes, particularly those involved in CO2 and H2 conversion.