Stabilization of Bilirubin Oxidase in a Biogel Matrix for High-performance Gas Diffusion Electrodes

Abstract

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.

Challenges in Biomaterials Science for Electrochemical Biosensing and Bioenergy

“…18,19 This approach was used, for example, to improve the stabilization of bilirubin oxidase (BOD) in a Nafion salt/ glutaraldehyde gel, forming a biocatalytic gel layer added onto a porous and conducting carbon cloth substrate to generate a gas-diffusion electrode. 20 As an additional remark regarding enzyme kinetics in electrochemical sensing, the Michaelis−Menten constant (K M ) is often understated, being a direct measure of the catalytic efficiency of the sensor, independent of the enzyme amount, activity, and surface area. Therefore, one must take care to verify that obtained K M is not too small even when a high electron-transfer rate constant is calculated, which might suggest that the apparently good sensing performance is actually only due to enlarged surface area or redox-active impurities, hence of less real-world usefulness.…”

Effects of interactions between SPEEK or Nafion ionomers and bilirubin oxidase on O2 enzymatic reduction

“…Recently, the group of Crespilho et al formed a biogel matrix by mixing Nafion, glutaraldehyde and BOD, and showed an increased stability of the bioelectrode [17]. However, it must be noted that a huge amount of enzyme from solutions in the mM range was used at the electrode, which will limit any device development because of the induced cost.…”