Plasmonic nanostructures have shown great potential for use in biosensing and single-molecule detection owing to their optical properties[cite: 136]. Their localized surface plasmon resonances (LSPR) provide many advantages in analytical spectroscopic techniques[cite: 137]. In surface enhanced Raman scattering (SERS), the Raman scattering signal of a molecule can be enhanced by several orders of magnitude due to high electromagnetic field enhancements as a consequence of the LSPR[cite: 137]. Very early work in SERS focused on electrochemical systems and simple analytes, while later studies identified the potential of plasmonic nanoparticles, particularly gold nanoparticles, as efficient SERS substrates for large molecules in biosensing applications[cite: 137]. SERS spectra of proteins, amino acids, nucleic acids and nucleic bases, and live cells have been reported using spherical gold nanoparticles[cite: 138]. Anisotropic nanostructures such as nanorods and nanostars have also been used to create different types hot-spots that provide very high field enhancements[cite: 139]. More complex nanostructures like shell-isolated nanoparticles, DNA-hybridized nanostructures, immobilized nanoparticle substrates and nanoshell arrays have been reported to show high stability of substrate-analyte interactions and thereby selectivity for probing different modes in the vibrational SERS spectra[cite: 139].

The detection of single DNA and protein molecules by SERS dates back for several decades, and includes the detection of single hemoglobin molecules, DNA bases, and amino acids[cite: 140]. The field of molecular plasmonics, specifically the improved control of molecule-nanostructure interactions by positioning biomacromolecules in high local fields of plasmonic nanostructures, e.g., by the use of DNA origami technology, helped to improve the high sensitivity for detection of specific functional groups by SERS[cite: 140].

Plasmonic nanopores emerged as plasmonic substrates that confine the biomolecule-metal nanostructure interaction when the biomolecules translocate through them[cite: 141]. Nanopore-based approaches were shown to be effective in distinguishing single molecule DNA bases and single amino acids in DNA and protein molecules, respectively[cite: 142]. However, the high fluctuation of SERS signals at high enhancement levels poses great challenges with respect to fully exploiting the vibrational information that is contained in the SERS data about specific molecular structure and interaction[cite: 143]. As example, the concentration-dependent interactions of a protein with a gold nanostructure were shown to greatly depend on the concentration and sequence of a protein, even at high sequence similarity[cite: 144].

As such, trapping of biomolecules in SERS hot spots while translocating them through plasmonic nanopores is an effective strategy to isolate a biomolecule of interest for SERS characterization[cite: 145]. This project focuses on the following main aspects to improve in this approach by implementing[cite: 145]:

An efficient temporal control of biomolecule translocation [cite: 146]
The design of optimized plasmonic and identification of excitation conditions that yield an actual vibrational structural characterization based on more than a few bands attained previously [cite: 147]
The interpretation of intrinsic variations that are present in any SERS spectra, to identify spectral fingerprints of different structures of biomolecule analytes, such as protein secondary structure[cite: 148].

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