Preparation of surface-enhanced resonance Raman scattering-active substrates: applications to heme-containing proteins

摘要

Surface-enhanced resonance Raman scattering (SERRS) spectroscopy combines the advantages unique to resonance Raman scattering (RRS) with those of surface-enhanced Raman scattering (SERS). Although much of the initial studies concentrated on elucidating the mechanism (s), considerable attention has recently been directed at exploiting the enormous potential of the technique for analytical and biological purposes. However, the practical applicability of SERRS to many investigations remains limited because experimental conditions corresponding to the effect have not been fully characterized;This problem is especially apparent when SERRS is used for the study of biological systems. In many cases, the biomolecule species is perturbed upon adsorption onto a SERRS-active metal substrate. Hence, SERRS is not a viable method if the resulting spectra correlate to perturbed biological structures. It is the aim of our research to develop procedures for preserving the native structure and activity of proteins at SERRS-active substrates including, silver island-films, electrochemically-roughened silver electrodes, and citrate-reduced sols. In particular, our work has focused on the heme-containing proteins, cytochrome c, cytochrome P-450[subscript] b, myoglobin, and cytochrome c[subscript]3. Depending upon the biomolecule, the native state of the protein can be retained by choosing the appropriate adsorption conditions;Another aspect of our research has been concerned with optimizing and characterizing the experimental parameters in the preparation of SERRS/SERS-active silver-island films. Our results show that for both SERS and SERRS, the intensity of the observed signals is dependent upon the evaporation rate used in the preparation of the silver-island film. The optical densities of the films as well as the resulting surface morphologies were also studied as a function of deposition rate. Specific applications of silver-island films as SERRS-active substrates included Raman microprobe SERRS investigations and the characterization of covalently modified electrodes by using the SERRS technique.