NIR surface enhanced Raman spectra of biological hemes: Solvation and plasmonic metal effects

Abstract

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.

Use of Nanoparticle Decorated Surface-Enhanced Raman Scattering Active Sol–Gel Substrates for SALDI-MS Analysis

“…17,18 When extracted by the 50% acetic acid solution, the SERS spectra are primarily due to ferric heme acetate. 31 As described previously, the SERS spectra have very different patterns of relative intensities on Au and Ag substrates (Figure 1) owing to their different mechanisms of chemical enhancement and their different points of physisorption on the metal surface. 17 Consequently, the largest SERS feature of dried blood on gold substrates is the 1543 cm −1 , 1514 cm −1 doublet corresponding to ν 11 and ν 38 symmetric porphyrin ring stretching modes of the cleaved heme moiety.…”

“…The SERS performance of these substrates for a variety of applications has been well characterized. − A simple 50% acetic acid microextraction procedure was found to result in robust, intense 785 nm excited SERS spectra of neat dried blood on Au and Ag NP substrates as shown in Figure . , When extracted by the 50% acetic acid solution, the SERS spectra are primarily due to ferric heme acetate . As described previously, the SERS spectra have very different patterns of relative intensities on Au and Ag substrates (Figure ) owing to their different mechanisms of chemical enhancement and their different points of physisorption on the metal surface .…”

“…In the neat dried blood extracts, small amounts of hemin are also observed on the gold substrate and silver heme adducts on the silver substrate (Figure S3). Heme is a common biomarker for blood, and the acetic acid extraction leads to the formation of ferric acetate heme, which is responsible for most signals in SERS . In the SALDI-MS analysis, ferric acetate heme is observed as a sodiated adduct ( m / z 698.180) on the gold substrates and has ion signal that is 18.2% of the heme molecular ion.…”

“…Heme is a common biomarker for blood, and the acetic acid extraction leads to the formation of ferric acetate heme, which is responsible for most signals in SERS. 31 In the SALDI-MS analysis, ferric acetate heme is observed as a sodiated adduct (m/z 698.180) on the gold substrates and has ion signal that is 18.2% of the heme molecular ion. Hemin and the ferric acetate heme could be an origin of the observed heme molecular ion, but the anion might have been lost during SALDI-MS.…”

Tribute to Derek Long: An instant snapshot of the development of Raman spectroscopy and its application in the fields of instrumentation and methodology, solid‐state materials, cultural heritage, DFT modeling and applications in biology, microbiology, and medicine

“…Ingraham et al ( NIR surface enhanced Raman spectra of biological hemes: Solvation and plasmonic metal effects , Boston University, USA) [ 42 ] review the application of SERS excited at 785 nm for the analysis of hemoglobin, myoglobin, two heme B proteins, and cytochrome c , a heme C protein, solubilized in a variety of solvent systems (water, butanone at various pHs) with Au and Ag substrates. 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 the intense SERS spectra indicative of these heme B proteins.…”