Multiplex discrimination of single amino acid residues in polypeptides by single SERS hot spot.

Surface-enhanced Raman spectroscopy (SERS) is a sensitive label-free optical method that shows potential for single-molecule sequencing. However, SERS detection of protein sequences with single-residue sensitivity suffers from signal dominance of aromatic amino acid residues and backbones, impeding detection of non-aromatic amino acid residues. Here, we trap a gold nanoparticle in a plasmonic nanohole to generate a single SERS hot spot for single-molecule detection of 2 alike polypeptides (vasopressin and oxytocin) and 10 distinct amino acids that constitute the 2 polypeptides. Significantly, both aromatic and non-aromatic amino acids are detected and discriminated at single-molecule level either at individual amino acid molecules or within the polypeptide chains. Correlated with Molecule Dynamics simulation, our results suggest that the signal dominance due to large spatial occupancy of aromatic rings of the polypeptide sidechains on gold surfaces can be overcome by the high localization of the single hot spot. The superior spectral and spatial discriminative power of our approach can be applied to single-protein analysis, fingerprinting and sequencing.