Immobilization of bilirubin oxidase on graphene oxide flakes with different negative charge density for oxygen reduction. The effect of GO charge density on enzyme coverage, electron transfer rate and current density

Previously we showed that an effective bilirubin oxidase (BOD)-based biocathode using graphene oxide (GO) could be prepared in 2 steps: 1. electrostatic adsorption of BOD on GO; 2. electrochemical reduction of the BOD-GO composite to form a BOD-ErGO (electrochemically reduced GO) film on the electrode. In order to identify an optimal charge density of GO for BOD-ErGO composite preparation, several GO fractions differing in an average flake size and zeta-potential were prepared using centrifugation and consequently employed for BOD-ErGO biocathode preparation. A simple way to express surface charge density of these particular GO nanosheets was developed. The values obtained were then correlated with biocatalytic and electrochemical parameters of the prepared biocathodes, i.e. electrocatalytically active BOD surface coverage (Gamma), heterogeneous electron transfer rate (k(s)) and a maximum biocatalytic current density. The highest bioelectrocatalytic current density of (597 +/- 25) mu A cm(-2) and the highest Gamma of (23.6 +/- 0.9) pmol cm(-2) were obtained on BOD-GO composite having the same moderate negative charge density, but the highest ks of (79.4 +/- 4.6) s(-1) was observed on BOD-GO composite having different negative charge density. This study is a solid foundation for others to consider the influence of a charge density of GO on direct bioelectrochemistry/bioelectrocatalysis of other redox enzymes applicable for construction of biosensors, bioanodes, biocathodes or biofuel cells. (C) 2016 Elsevier B.V. All rights reserved.