Enzyme Sensing Using 2‑Mercaptopyridine-Carbonitrile Reporters and Surface-Enhanced Raman Scattering

The high sensitivity and functional group selectivity of surface-enhanced Raman scattering (SERS) make it an attractive method for enzyme sensing, but there is currently a severe lack of enzyme substrates that release SERS reporter molecules with favorable detection properties. We find that 2-mercaptopyridine-3- carbonitrile (o-MPN) and 2-mercaptopyridine-5-carbonitrile (p-MPN) are highly effective as SERS reporter molecules that can becaptured by silver or gold nanoparticles to give intense SERS spectra, each with a distinctive nitrile peak at 2230 cm−1. p-MPN is amore sensitive reporter and can be detected at low nanomolar concentrations. An assay validation study synthesized two novelsubstrate molecules, Glc-o-MPN and Glc-p-MPN, and showed that they can be cleaved efficiently by β-glucosidase (Km = 228 and162 μM, respectively), an enzyme with broad industrial and biomedical utility. Moreover, SERS detection of the released reporters (o-MPN or p-MPN) enabled sensing of β-glucosidase activity and β-glucosidase inhibition. Comparative experiments using a crudealmond flour extract showed that the presence of β-glucosidase activity could be confirmed by SERS detection in a much shorter time period (>10 time shorter) than by UV−vis absorption detection. It is likely that a wide range of enzyme assays and diagnostic tests can be developed using 2-mercaptopyridine-carbonitriles as SERS reporter molecules.