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-3carbonitrile (o-MPN) and 2-mercaptopyridine-5-carbonitrile (pMPN) are highly effective as SERS reporter molecules that can be captured by silver or gold nanoparticles to give intense SERS spectra, each with a distinctive nitrile peak at 2230 cm-1. p-MPN is a more sensitive reporter and can be detected at low nanomolar concentrations. An assay validation study synthesized two novel substrate molecules, Glc-o-MPN and Glc-p-MPN, and showed that they can be cleaved efficiently by beta-glucosidase (Km = 228 and 162 mu M, respectively), an enzyme with broad industrial and biomedical utility. Moreover, SERS detection of the released reporters (o-MPN or p-MPN) enabled sensing of beta-glucosidase activity and beta-glucosidase inhibition. Comparative experiments using a crude almond flour extract showed that the presence of beta-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.

Automated summary

This study finds that 2-mercaptopyridine-carbonitriles can serve as highly effective SERS reporter molecules that can be captured by silver or gold nanoparticles, providing intense SERS spectra with high sensitivity and functional group selectivity. It also demonstrates the potential use of these molecules in enzyme sensing, specifically in detecting beta-glucosidase activity and inhibition.