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.
Surface-enhanced Raman scattering (SERS) is a powerful technique to study the biological molecules and structures.
SERS of proteins is always difficult due to their complex, flexible and diverse structures. This difficulty is one of the
major obstacles hindering the applicability of SERS for the label-free detection and identification. In this study, we have
employed several sample preparation approaches involving the packing AgNPs with protein molecules in a proper
manner to allow the polarization of the electron system of proteins in coherence with the nanostructured noble metal
system. The applicability of heat denaturation kinetics is perused for the detection and identification of proteins in
model protein mixtures. Human serum albumin, transferrin, hemoglobin and the binary mixtures of these proteins are
used as models. We have found that the SERS spectrum of each protein in the protein mixture is rather different at an
increased temperature, which could be used to distinguish a protein in the protein mixture.
Detection and identification of biomacromolecules is of critical importance in many fields ranging from biotechnology
to medicine. Surface-enhanced Raman scattering (SERS) is an emerging technique for the label-free detection and
identification of biological molecules and structures with its fingerprinting properties and high sensitivity. However,
there are a number of obstacles for its applications for biological macromolecules due to their complexity. In this report,
manipulation of microscopic processes in play during the drying of a sessile droplet as a tool to influence the
nanoparticle-macromolecule packing, which has dramatic effect on SERS performance, before the SERS acquisition is
demonstrated. A process known as the coffee ring phenomenon jams all particles and molecular species to the edges of
the droplet during drying. This uncontrolled process has dramatic effects on a SERS experiment, using colloidal metal
nanoparticles as substrates, by sweeping everything to the edges and influencing the packing of nanoparticles in the
droplet area. A plastic tip was dipped into a drying sample droplet to influence the uncontrolled piling up. A negatively-charged
protein, BSA, a positively-charged protein, cytochrom c, and a 20-base long oligonucleotide, were used as
model biomacromolecules in this study. While a minimum of one order of magnitude lower concentration improvement
in detection limit was observed with negatively-charged biomacromolecules, no significant improvement was observed
with positively-charged ones compared to a sample droplet left on the surface without any interference. With the
demonstrated approach, picomolar-level biomolecular detection using SERS is possible.
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