Versatile Biosynthetic Approach to Regioselective Enzyme Patterning for Direct Bioelectrocatalysis Application

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 (GDH gamma alpha) and their catalytic and gold-binding activities are determined. The substrate catalysis and direct electrocatalytic capability of selected construct, GDH gamma alpha with three GBP repeats (GDH gamma alpha-3GBP), are investigated on electrode. In an inorganic-binding characterization, GDH gamma alpha-3GBP exhibits fourfold higher affinity on gold (Au) surface and 215-fold lower binding affinity for silicon dioxide (SiO2) than wild-type GDH gamma alpha. Utilizing those regioselective features, fusion GDH gamma alpha is incorporated into nanotemplates comprising Au nanopatterns and SiO2 background. Thereby, nanoscale patterned GDH gamma alpha-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.