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A new route to single-step and 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 Escherichia 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 preQ 0 . Density functional theory analysis of QueF's catalytic cycle with BnCN shows similar transition-state barriers to preQ 0 , but differences in the formation of the initial thioimidate covalent bonding (∆G ‡ = 19.0 kcal mol −1 for preQ 0 vs 27.7 kcal mol −1 for BnCN) and final disassociation step (∆G = −24.3 kcal mol −1 for preQ 0 vs ∆G = +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.

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