The ability to identify individual protein molecules using Surface-Enhanced Raman Scattering (SERS) spectroscopy, without the need for labelling, is a significant advancement in biomedical diagnostics. However, the inherently small Raman scattering cross-section of most (bio) mol
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The ability to identify individual protein molecules using Surface-Enhanced Raman Scattering (SERS) spectroscopy, without the need for labelling, is a significant advancement in biomedical diagnostics. However, the inherently small Raman scattering cross-section of most (bio) molecules necessitates significant signal amplification for successful detection, particularly at the single-molecule level. A novel approach is introduced for fabricating plasmonic nanopores suitable for sequential Raman readout, namely the recording of Raman spectra from portions of molecules, which are progressively flowing into plasmonic hot spots. The method is based on Capillary-Assisted Particle Assembly (CAPA)of gold nanoparticles (Au NPs), thus ensuring high stability and cost-effectiveness. By electrophoretically driving polypeptides through these nanopores, real-time Raman detection is achieved with a Single-Photon Avalanche Diode (SPAD) camera, attaining single-molecule detection at 1 nm concentration with 100 microsecond resolution. Statistical analysis of translocation times, photon scattering rates, and spectral data confirms a linear correlation between dwell time, molecular length, and Raman signal intensity. On average, a photon scattering of 6 photons/amino acid and a translocation time of 7 µs per amino acid is recorded. These results demonstrate the feasibility of sequential Raman readout, overcoming key limitations of SERS. This method represents a significant step toward label-free, high-resolution molecular identification, with future potential applications in protein identification.