Sulfhydryl-maleimide crosslinking for enhancing catalytic activity and duration of biocatalyst

Abstract Hydrophilic bridging of an enzyme using sulfhydryl conjugation to produce an aggregate is used to enhance the activity and operational lifetime of a glucose oxidase (GOx)-based biocatalyst. 1,8-Bismaleimido-diethyleneglycol (BMPEG) is used as an alternative to the conventional cross-linker glutaraldehyde (GA), which suffers from downsides in terms of catalytic activity and safety. To evaluate the effect of adopting BMPEG as the cross-linker, a reduced graphene oxide (RGO) and iron phthalocyanine (FePc) composite (RGO/FePc) support material was used as the under layer in the preparation of an [RGO/FePc]/[GOx-BMPEG] electrode for the hydrogen peroxide oxidation reaction. Its electrochemical behavior was verified by comparison with electrodes prepared with GOx alone ([RGO/FePc]/GOx) and a conventional GOx-GA aggregate ([RGO/FePc]/[GOx-GA]). [RGO/FePc]/[GOx-BMPEG] exhibited higher sensitivity (62.4 μW mM−1 cm−2) and longer operating duration (94.5% for 3h) than [RGO/FePc]/[GOx-GA] (52.3 μW mM−1 cm-2, 67.4% for 3 h), even though the amount of GOx on the [RGO/FePc]/[GOx-BMPEG] electrode was found to be around half that on [RGO/FePc]/[GOx-GA] in physiological condition (pH 7.4, 36.5 °C). The excellent performance of the [RGO/FePc]/[GOx-BMPEG] electrode was attributed to the hydrophilicity of the BMPEG cross-links, which mitigates the hindrance of mass transfer typically caused by hydrophobic cross-linkers such as GA, and the bonding of the thiol groups on GOx to BMPEG, which protects the GOx from inactivation. Thus, GOx-BMPEG is a superior alternative to GOx-GA for use in implantable bioelectronics.