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

Abstract

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-d 0) and crystal violet (CV-d 0), were directly compared, while SM detection was verified (or disproved) using their corresponding isotopologues (R6G-d 4, CV-d 12). 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 probabilities and adjusted the relative concentrations of R6G and CV to achieve a comparable distribution of SMSERS counts. Using this information, we outlined the necessary considerations, such as accounting for the differences in molecular properties, for reliable SMSERS proofs. Moreover, we showed that multianalyte experiments at the SM level are achievable, opening the opportunity for new types of SM studies.

SERS speciation of the electrochemical oxidation–reduction of riboflavin

“…The signal for this species is much lower in intensity than the oxidized and semi-quinone form, suggesting either a lack of affinity for the surface 42, 44 or significantly lower cross-section. 45 …”

“…It is worth noting that in SERS experiments, the signal intensity arises from a number of factors, including the Raman cross-section 45 and the affinity of the molecule for the SERS surface. 42, 44 SERS discriminates between the large number of molecules in the bulk solution in favor of the small number located in “hotspots” in the focal volume on the electrode surface.…”

Review of surface enhanced Raman spectroscopic (SERS) detection of synthetic chemical pesticides

“…From the substrate point of view, the variation in sensitivity of SERS detection can 170 largely be attributed to the variations in "hot spots" on substrates [20]. These hot spots are found at the interstitial gaps between metallic nanoparticles,and are able to produce intense local field enhancements due to localized surface plasmon resonance (LSPR).The LOD of SERS methods can sometimes reach a single molecule detection level through a rational design of the SERS substrate and the experiment [21][22][23][24].…”

Plasmonic nanoparticles in chemical analysis

“…by about 15 orders of magnitude in comparison to the cross-section of normal Raman scattering) making possible observation of SERS spectra even of a single molecule. [66][67][68] Although, as far as we known, single molecule SERS spectroscopy has not been used for practical analysis due to the poor reproducibility of the measured signal (for example see Fig. 7, which shows single molecule SERS spectra of adenine measured from the solution with the concentration of 10 À12 M), 69 its strong uctuation and possibility of the complete disappearance of the signal due to the photodecomposition of the investigated molecule, SERS is actually one of the most sensitive analytical tools with the limit of detection for some analytes of the order of 10 À18 mol dm À3 .…”