Abstract
Surface-enhanced Raman scattering (SERS) is a method in which analytes are adsorbed onto nanoscopically structured metallic surfaces and excited by strong electric fields that are induced by surface plasmon resonance. It has great potential as an analytical tool. The technique has facilitated the study of molecular structures at dilute concentrations1 and also of the dynamic behaviour of single molecules.2 It thus can be employed to investigate ligand-receptor binding—of significance for drug-protein complexes—and heterogeneity in individual molecules, such as catalysis rates of a single enzyme. However, issues concerning the irreproducibility of SERS spectra, which is more pronounced at ultra-low concentrations, have not been fully addressed, and the many confounding factors have not been identified. This hinders the quantitative application of this otherwise-powerful technique. For this reason, we have undertaken studies that detail the nano-environment in which the Raman-active analytes reside. Much of the theoretical and experimental work with SERS has concentrated on elaborating general mechanisms of the phenomenon3, 4 and in optimizing effective substrates. Only recently has the SERS activity of molecules at low concentrations been studied meticulously. However, there is a dearth of detailed knowledge. For example, how would heating generated by localised surface plasmon resonance (SPR) contribute to measured SERS signals or to the large fluctuations observed in previous single-molecule SERS (SM-SERS) experiments? Is the thermal energy associated with SPR-heating comparable to the activation energy barrier between conformation minima in bio-molecules? How would heating influence the binding constant of antigenantibody complexes? These and similar issues have been largely neglected. Interpretations of SERS spectra in previous reports are based mainly on the assumption that the analyte molecule is rigid. Figure 1. (a) SERS decay curve under prolonged laser irradiation. (b) A degradation profile versus irradiation time. Acquisition time for each spectrum is 40s. Laser power = 2.5mW, 20× 0.4 NA objective. Sample = 1mM Crystal violet in phosphate-buffer.
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