Single Molecule Surface-Enhanced Raman Spectroscopy: A Critical Analysis of the Bianalyte versus Isotopologue Proof

Verification of single-molecule (SM) detection for surface-enhanced Raman spectroscopy (SERS) requires the use of two analytes via either the bianalyte or isotopologue approach. For both approaches, the preferential observation of the individual analytes over a combination of both analytes is used to conclude that SM detection has been achieved. Isotopologues are preferred because they have identical surface binding affinities and Raman cross sections, whereas bianalyte pairs typically do not. We conducted multianalyte SERS studies to investigate the limitations of the bianalyte approach. The bianalyte partners, Rhodamine 6G (R6G-d0) and crystal violet (CV-d0), were directly compared, while SM detection was verified (or disproved) using their corresponding isotopologues (R6G-d4, CV-d12). We found that the significant difference in counts between R6G and CV can provide misleading evidence for SMSERS. We then rationalized these results using a joint Poisson-binomial model with unequal detection probabilitie...