An optimized procedure is described for the acquisition of 785 nm excited SERS spectra of dried bloodstains and shown to offer great potential for rapid, portable, highly sensitive and specific,...

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Surface enhanced Raman spectra (SERS) excited at 785 nm are reported for hemoglobin, myoglobin, two heme B proteins, and cytochrome c, a heme C protein, solubilized in a variety of solvent systems and then placed on Au and Ag substrates. These solvent environments include H2O pH 7.4, HCl/water pH 2, HCl/50% ethanol pH 2, 50% acetic acid and the organic layer of a pH 2 HCl/butanone mixture. To obtain the most intense SERS spectra of heme B proteins (Mb and Hb) it is not sufficient to be in a low pH (pH ~ 2) denaturing environment. A hydrophobic solvent component is additionally required in order to efficiently solubilize the cleaved heme moiety and consequently observe intense SERS spectra indicative of these heme B proteins. Although both heme B proteins exhibit virtually identical, metal (Au, Ag) specific SERS spectra, Cyt c is much weaker in the low pH environments and only displays a Ag‐like SERS spectrum on both metals indicating that this heme C protein is not cleaved in any of these solvation environments. The strong SERS signals of hemoglobin and myoglobin are due to hemin and ferric acetate heme formed in pH 2 HCl/ethanol and pH 2/50% acetic acid solutions respectively. SERS vibrational features confirm oxidation state, coordination number and ligands. SERS signals due to globin protein aggregation effects are reduced in low pH solvent systems with polarities lower than pure water. These results confirm the mechanism of a successful acetic acid protocol previously developed for the highly sensitive SERS detection of dried blood for forensic applications and indicate how it may be further improved to achieve even higher SERS sensitivity for heme protein detection.

Surface‐enhanced Raman spectra (SERS) of protoporphyrin IX (PPIX) and analogous metal‐containing PPIX compounds excited at 785 nm exhibit robust, strongly enhanced (~5 × 109) spectra that are highly dependent on the nanostructured metal (Au or Ag). Although the relative intensities of the 785‐nm SERS vibrational spectral features are very similar for all observed PPIX‐based compounds, the relative intensities are dramatically different on Au and Ag SERS substrates. Time‐dependent density functional theory (TDDFT) calculations are carried out for Au2/Ag2–PPIX complexes to understand the origins of this chemical enhancement contribution to the observed SERS spectra. The observed metal dependence of the SERS spectra in the heme ring stretching region (1,500–1,620 cm−1) are reproduced by calculated resonance Raman intensities due to low‐lying metal (σ) to molecule (π*) charge transfer excitations of the metal dimer–PPIX complexes when the metal dimer is near a porphyrin ring Cβ position. The nuclear coordinate dependence of electronic transition moments is essential for capturing the effects of these CT excitations. A simple method for determining the importance of the transition moment normal mode dependence relative to Franck–Condon factors (A term) is described and implemented. The consequences of this vibronic analysis for the observation of overtone/combination bands in SERS spectra are discussed.

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