Khozeymeh Sarbishe Foroogh, Khabarov Kirill, Blanco Formoso Maria, Baldi Ilaria Micol, Storari Veronica, Haka Henri, Mastrangeli Massimo, Difato Francesco, Armirotti Andrea, Villa Federica, Tantussi Francesco, De Angelis Francesco
Italian Institute of Technology, Genoa, 16162, Italy.
Department of Physics, University of Genoa, Genoa, 16146, Italy.
Adv Mater. 2025 Oct;37(39):e2504436. doi: 10.1002/adma.202504436. Epub 2025 Jul 9.
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.
利用表面增强拉曼散射（SERS）光谱技术无需标记就能识别单个蛋白质分子的能力，是生物医学诊断领域的一项重大进展。然而，大多数（生物）分子固有的拉曼散射截面很小，需要显著的信号放大才能成功检测，尤其是在单分子水平。本文介绍了一种用于制造适用于连续拉曼读出的等离子体纳米孔的新方法，即记录逐渐流入等离子体热点的分子部分的拉曼光谱。该方法基于金纳米颗粒（Au NPs）的毛细管辅助颗粒组装（CAPA），从而确保了高稳定性和成本效益。通过电泳驱动多肽通过这些纳米孔，使用单光子雪崩二极管（SPAD）相机实现实时拉曼检测，在1纳米浓度下以100微秒分辨率实现单分子检测。对转运时间、光子散射率和光谱数据的统计分析证实了停留时间、分子长度和拉曼信号强度之间的线性相关性。平均而言，记录到每个氨基酸6个光子的光子散射和每个氨基酸7微秒的转运时间。这些结果证明了连续拉曼读出的可行性，克服了SERS的关键局限性。该方法代表了朝着无标记、高分辨率分子识别迈出的重要一步，在蛋白质识别方面具有潜在的应用前景。
