A GFET nitrile sensor using a graphene-binding fusion protein

A new route to single-step, non-covalent immobilization of proteins on graphene is exemplified with the first biosensor for nitriles based on a graphene field-effect transistor (GFET). The biological recognition element is a fusion protein consisting of nitrile reductase QueF from E. coli with an N-terminal self-assembling and graphene-binding dodecapeptide. Atomic force microscopy and analysis using a quartz crystal microbalance show that both the oligopeptide and the fusion protein incorporating it form a single adlayer of monomeric enzyme on graphene. The fusion protein has a 6.3-fold increase in binding affinity for benzyl cyanide (BnCN) versus wild-type QueF and a 1.4-fold increase for affinity for the enzyme’s natural substrate preQ0. Density functional theory analysis of QueF’s catalytic cycle with BnCN shows similar transition-state barriers to preQ0, but differences in the formation of the initial thioimidate covalent bond (ΔG‡ = 19.0 kcal mol–1 for preQ0 vs 27.7 kcal mol–1 for BnCN) and final disassociation step (ΔG = –24.3 kcal mol–1 for preQ0 vs ΔG = +4.6 kcal mol–1 for BnCN). Not only do these results offer a single-step route to GFET modification, but they also present new opportunities in the biocatalytic synthesis of primary amines from nitriles.