Enzyme immobilization on solid conducting surfaces faces some challenges for practical applications in technologies such as biosensors and biofuel cells. Short-term stability, poor electrochemical performance, and enzyme inhibition are some issues that remain unsolved. Here, we propose a simple methodology for bilirubin oxidase (BOD) immobilization on carbon-based gas-diffusion electrodes for a four-electron electrochemical oxygen reduction reaction (ORR). The enzyme is incorporated into a Nafion® polymeric matrix and cross-linked with glutaraldehyde by a one-pot reaction in a buffered solution, producing a stable BOD-based biogel. The biogel prevents the formation of enzyme aggregates, producing a homogeneous bioelectrode surface, and allows access to the direct electron-transfer mechanism of multicopper centers buried in the enzyme. A biocatalytic reduction current of -1.52 ± 0.24 mA cm<sup>-2</sup> at 0.19 ± 0.06 V was observed under gas-diffusion conditions. Additionally, the bioelectrode showed an unprecedented long-term stability under continuous operation combined with satisfactory catalytic current without redox mediator, demonstrating that the BOD-based biogel provides a suitable microenvironment for long-term enzymatic activity involving a bio-three-phase interfacial reaction. Therefore, the present study contributes new insights into enzyme immobilization to overcome the critical short-term stability issue of enzyme-based electrochemical devices for practical applications.
Stabilization of Bilirubin Oxidase in a Biogel Matrix for High-performance Gas Diffusion Electrodes