Versatile Biosynthetic Approach to Regioselective Enzyme Patterning for Direct Bioelectrocatalysis Application

Abstract Due to the outstanding attributes of oxidoreductases, they have been utilized as biomaterials for bioelectrocatalytic systems. Herein, a simple and versatile biosynthetic approach that can designate binding position of enzymes on electrode with their surface‐orientation is suggested. In this regard, material‐selective properties of gold‐binding peptide (GBP) are exploited and genetically fused GBP to enzyme. To optimize the design of synthetic enzyme, a variable repeat number of GBP are fused to flavin adenine dinucleotide‐dependent glucose dehydrogenase gamma‐alpha complex (GDHγα) and their catalytic and gold‐binding activities are determined. The substrate catalysis and direct electrocatalytic capability of selected construct, GDHγα with three GBP repeats (GDHγα‐3GBP), are investigated on electrode. In an inorganic‐binding characterization, GDHγα‐3GBP exhibits fourfold higher affinity on gold (Au) surface and 215‐fold lower binding affinity for silicon dioxide (SiO₂) than wild‐type GDHγα. Utilizing those regioselective features, fusion GDHγα is incorporated into nanotemplates comprising Au nanopatterns and SiO₂ background. Thereby, nanoscale patterned GDHγα‐3GBP molecules are successfully obtained with their binding locations controlled specifically by Au nanopatterns, not SiO₂. The results reveal that genetic SBP fusions enable highly selective template‐based surface assembly of biomolecules with electrically intimate cofactor‐surface interfaces. The proposed technology has remarkable potential to fabricate small‐scale biochips applied for enzyme‐based bioelectronics.