Label-free SERS detection of small proteins modified to act as bifunctional linkers

Two double-cysteine mutants of a small protein judiciously modified so that the cysteines appear at axially opposite sides of the native fold were prepared such that different axes were defined in the two mutants. Upon reduction, the disulfide bonds are broken, and the proteins act as bifunctional ligands toward Ag nanoparticles, encouraging their assembly into nanoparticle dimers and small aggregates such that, when excited with laser light, the proteins are automatically located at electromagnetic hot spots within the aggregates. Because the protein molecules are small (similar to 2.3 nm) and because the electromagnetic energy at a hot spot tends to increase as the size of the interparticle gap decreases, this nanoparticle-protein- nanoparticle geometry significantly enhances the Raman emission at the metallic surface. Exploiting this effect, we have recorded surface-enhanced Raman spectra (SERS) of the proteins at near-single-molecule level. The observed SERS spectra were dominated,by the vibrations of molecular groups near the anchor points of the proteins.