Stabilization of bilirubin oxidase in a biogel matrix for high-performance gas diffusion electrodes

Enzyme immobilization on solid conducting surfaces faces challenges for practical applications in technologies such as biosensors and biofuel cells. Short-term stability, poor electrochemical performance, and enzyme inhibition are some unsolved issues. Here, we show a simple methodology for bilirubin oxidase (BOD) immobilization on carbon-based gas diffusion electrode for four-electron electrochemical oxygen reduction reaction. BOD is incorporated into a Nafion® matrix and crosslinked with glutaraldehyde by a one-pot reaction in buffered solution, producing a stable BOD-based biogel. The biogel provides stable electrode performance and allows the direct electron-transfer mechanism of multicopper centers buried in the enzyme. A biocatalytic reduction current of −1.52 ± 0.24 mA cm −2 at 0.19 ± 0.06 V was observed under gas diffusion conditions. Additionally, the bioelectrode showed unprecedented long-term stability under continuous operation combined with satisfactory catalytic current without redox mediator. The BOD-based biogel layer thickness and the entrapment of BOD into Nafion network are crucial for the biocathode stability, and BOD crosslinking by glutaraldehyde contributes to enhance the catalytic currents. Further, the BOD-based biogel provides a suitable microenvironment for long-term enzymatic activity involving three-phase interfacial reaction. The present study provides new insights into enzyme immobilization to overcome the critical short-term stability of enzyme-based electrochemical devices for practical applications. • BOD is incorporated into a polymeric matrix producing a BOD-based biogel. • The biogel allows the DET-type bioelectrocatalysis toward four-electron ORR. • BOD-GDE showed -1.52 mA cm-2 at 0.19 V and high stability under continuous operation. • Biogel layer thickness and BOD entrapment into Nafion network are crucial for biocathode stability. • Biogel provides suitable microenvironment for long-term enzymatic activity involving three-phase interfacial reaction.