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Stabilization of Bilirubin Oxidase in a Biogel Matrix for High-performance Gas Diffusion Electrodes.pdf (1.51 MB)

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

submitted on 30.03.2020 and posted on 31.03.2020 by Graziela Sedenho, Ayaz Hassan, Lucyano Macedo, Frank Crespilho
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-2 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.


FAPESP process 2015/22973-6

FAPESP process 2017/20493-2

FAPESP process 2019/12053-8

FAPESP process 2019/15333-1



Email Address of Submitting Author


University of São Paulo, São Carlos Institute of Chemistry



ORCID For Submitting Author


Declaration of Conflict of Interest

No conflict of interest


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