Bilirubin oxidase oriented on novel type three-dimensional biocathodes with reduced graphene aggregation for biocathode

Jing Tang, Xiaomei Yan, Wei Huang, Christian Engelbrekt, Jens Øllgaard Duus, Jens Ulstrup, Xinxin Xiao*, Jingdong Zhang

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Aggregation of reduced graphene oxide (RGO) due to π-π stacking is a recurrent problem in graphene-based electrochemistry, decreasing the effective working area and therefore the performance of the RGO electrodes. Dispersing RGO on three-dimensional (3D) carbon paper electrodes is one strategy towards overcoming this challenge, with partial relief aggregation. In this report, we describe the grafting of negatively charged 4-aminobenzoic acid (4-ABA) onto a graphene functionalized carbon paper electrode surface. 4-ABA functionalization induces separation of the RGO layers, at the same time leading to favorable orientation of the blue multi-copper enzyme Myrothecium verrucaria bilirubin oxidase (MvBOD) for direct electron transfer (DET) in the dioxygen reduction reaction (ORR) at neutral pH. Simultaneous electroreduction of graphene oxide to RGO and covalent attachment of 4-ABA are achieved by applying alternating cathodic and anodic electrochemical potential pulses, leading to a high catalytic current density (Δjcat:193 ± 4 μA cm−2) under static conditions. Electrochemically grafted 4-ABA not only leads to a favorable orientation of BOD as validated by fitting a kinetic model to the electrocatalytic data, but also acts to alleviate RGO aggregation as disclosed by scanning electron microscopy, most likely due to the electrostatic repulsion between 4-ABA-grafted graphene layers. With a half-lifetime of 55 h, the bioelectrode also shows the highest operational stability for DET-type MvBOD-based bioelectrodes reported to date. The bioelectrode was finally shown to work well as a biocathode of a membrane-less glucose/O2 enzymatic biofuel cell with a maximum power density of 22 μW cm−2 and an open circuit voltage of 0.51 V.

Original languageEnglish
Article number112500
JournalBiosensors and Bioelectronics
Number of pages10
Publication statusPublished - 2020


  • 4-Aminobenzoic acid monolayer
  • Bilirubin oxidase
  • Carbon paper
  • Direct electron transfer
  • Gas diffusion bioelectrode
  • Reduced graphene oxide


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