Label-free detection of proteins in ternary mixtures using surface-enhanced Raman scattering and protein melting profiles

Abstract

ABSTRACT. The multiplex detection of biologically important molecules such as proteins in complex mixtures has critical importance not only in disease diagnosis but also in other fields such as proteomics and biotechnology. Surface-enhanced Raman scattering (SERS) is a powerful technique for multiplex identification of molecular components in a mixture. We combined the multiplexing power of SERS and heat denaturation of proteins to identify proteins in ternary protein mixtures. The heat denaturation profiles of four model blood proteins, transferrin, human serum albumin, fibrinogen, and hemoglobin, were studied with SERS. Then, two ternary mixtures of these four proteins were used to test the feasibility of the approach. It was demonstrated that unique denaturation profiles of each protein could be used for their identification in the mixture.

Influence of Protein Size on Surface-Enhanced Raman Scattering (SERS) Spectra in Binary Protein Mixtures

“…We demonstrated this necessity in our previous reports. 17,18,29,30 Sizes of proteins are in nanometer scale; therefore, proteins can be thought of as nanostructures. Upon mixing with AgNPs, they coat the NPs and fill the voids among the AgNPs in the aggregates, which might have a quenching effect on surface plasmon formation.…”

“…29 In the other study, we used protein-melting temperatures to be able to detect each protein in ternary mixtures using SERS. 30 In that work, concentrations of the proteins were the same (50 μg/ml), and citrate reduced silver nanoparticles were used for SERS analysis. In order to show dissimilarity between spectra acquired at different temperatures, two-dimensional Euclidian distance plots were constructed.…”

“…29 In the other study, they detected proteins in ternary mixtures with the help of their melting profiles, and constructed two-dimensional Euclidian distance plots to show dissimilarity of the spectra acquired at different temperatures. 30 Zhou et al studied a binary mixture of proteins in terms of whether the mixture contains proteins with chromophores or not using a heat-induced SERS method. 31 Results were significant; on the other hand, the proteins they studied were mostly small proteins.…”

Bioanalytical applications of surface-enhanced Raman spectroscopy: de novo molecular identification

“…105 The conformational changes of the proteins were investigated for the temperature gradient of 30 to 70 °C. The two different mixtures were HSA-transferrin-Hb and HSA-fibrinogen-Hb.…”

“…However, at high temperatures, 60 and 70°C, an intense Tyr (640 cm −1 ) and C-C stretching (671 cm −1 ) vibrational modes were observed, suggesting a change in structure at 50°C. 105 …”