RESUMEN
Getting information about the fate of immobilized enzymes and the evolution of their environment during turnover is a mandatory step toward bioelectrode optimization for effective use in biodevices. We demonstrate here the proof-of-principle visual characterization of the reactivity at an enzymatic electrode thanks to fluorescence confocal laser scanning microscopy (FCLSM) implemented in situ during the electrochemical experiment. The enzymatic O2 reduction involves proton-coupled electron transfers. Therefore, fluorescence variation of a pH-dependent fluorescent dye in the electrode vicinity enables reaction visualization. Simultaneous collection of electrochemical and fluorescence signals gives valuable space- and time-resolved information. Once the technical challenges of such a coupling are overcome, in situ FCLSM affords a unique way to explore reactivity at the electrode surface and in the electrolyte volume. Unexpected features are observed, especially the pH evolution of the enzyme environment, which is also indicated by a characteristic concentration profile within the diffusion layer. This coupled approach also gives access to a cartography of the electrode surface response (i.e., heterogeneity), which cannot be obtained solely by an electrochemical means.