Multi-in situ Electrochemical Impedance Analysis of Biofuel Cell Performance (Supporting Information)

Electrochemical impedance spectroscopy (EIS) provides valuable insights into the interfacial kinetics and mass-transport behavior of an electrochemical system. However, conventional steady-state EIS cannot sufficiently capture transient processes in enzymatic biofuel cells (EBFCs), wherein electrode/electrolyte interactions evolve dynamically. In this study, we demonstrate a multi-in situ impedance method applicable to glucose/O2 EBFCs that employ MgO-templated carbon electrodes. Chronopotentiometry reveals that lower current densities induce higher glucose utilization efficiencies, reflecting the balance between the rates of substrate diffusion and surface reactions. In situ impedance analysis further differentiates the electrode-specific degradation: the cathode exhibits progressively increasing charge-transfer resistance attributable to enzyme and mediator leaching, whereas the anode displays non-monotonic resistance changes linked to overpotential-driven kinetics. Equivalent circuit modeling confirms that the cathodic overpotential is responsible for accelerating charge-transfer processes, leading to smaller semicircular features in the Nyquist plot over time. These results highlight the utility of multi-impedance measurements in identifying the performance-limiting factors in an EBFC under operational conditions. This approach provides mechanistic insights into enzyme stability, mediator retention, and substrate transport, and it serves as a diagnostic tool for the rational design of next-generation bioelectrochemical energy devices.