Effects of Cross-linker\nChemistry on Bioelectrocatalytic\nReactions in a Redox Cross-linked Network of Glucose Dehydrogenase\nand Thionine

Enzyme-mediator bioconjugation is emerging as a building\nblock\nfor designing electrode platforms for the construction of biosensors\nand biofuel cells. Here, we report a one-pot bioconjugation technique\nfor flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH)\nand thionine (TH) using a series of cross-linkers, including epoxy, <i>N</i>-hydroxysuccinimide (NHS), and aldehydes. In this technique,\nFAD-GDH and thionine are conjugated through an amine cross-linking\nreaction to generate a redox network, which has been successfully\nemployed for the oxidation of glucose. The bioconjugation chemistry\nof cross-linkers with the amino groups on FAD-GDH and thionine plays\na vital role in generating distinct network structures. The epoxy-type\ncross-linker reacts with the primary and secondary amines of thionine\nat room temperature, thereby producing an FAD-GDH-TH-FAD-GDH hyperbranched\nbioconjugate network, the aldehyde undergoes a rapid cross-linking\nreaction to produce a network of FAD-GDH-FAD-GDH, while the NHS-based\ncross-linker can react with the primary amines of both FAD-GDH and\nthionine, forming an FAD-GDH-cross-linker-TH polymeric network. This\nreaction has the potential to enable the conjugation of a redox mediator\nwith a FAD-GDH network, which is particularly essential when designing\nan enzyme electrode platform. The data demonstrated that the polymeric\ncross-linked network based on the NHS cross-linker exhibited a considerable\nincrease in electron transport while producing a catalytic current\nof 830 μA cm<sup>–2</sup>. The cross-linker spacer arm\nlength also affects the overall electrochemical function of the network\nand its performance; an adequate spacer length containing a cross-linker\nis required, resulting in a faster electron transfer. Finally, a leaching\ntest confirmed that the stability of the enzyme electrode was improved\nwhen the electrode was tested using the redox probe. This study elucidates\nthe relationship between cross-linking chemistry and redox network\nstructure and enhances the high performance of enzyme electrode platforms\nfor the oxidation of glucose.