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

Highlights: • Hydrophilic bridged enzyme aggregate is introduced for bioelecotronics. • Sulfhydyl-maleimide conjugation is used for cross linking of enzymes. • The hindrance of mass transfer and inactivation of enzyme are mitigated. • The catalytic activity is enhanced 1.732 times through the use of GOx-BMPEG. • 94.5% of initial current density is maintained during 3 h. 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.