Real-Time SERS Tracking of Single-Protein Orientational Change via Plasmonic Nanopore Confinement

The orientational and conformational changes of individual protein molecules are particularly attractive. However, current methodologies struggle to directly observe these transient states of single-molecule (SM) proteins. In this study, we developed a real-time dynamic SM surface-enhanced Raman scattering (SM-SERS) tracking system based on gold plasmonic nanopores with small orifices. This system enables continuous monitoring of protein orientation changes with subsecond temporal resolution. SM lysozymes (Lyz) were driven by ionic currents and trapped in the gold plasmonic nanopores, exhibiting typical residence times of a few seconds. The SM-SERS spectra were obtained with a 300 ms temporal resolution; Raman vibrational bands representing different chemical groups appear at different times, which represent the dynamic orientational changes of SM Lyz. Different plasmonic nanopores provided similar time-averaged SM-SERS spectra for SM Lyz, suggesting that the present system can reveal regular orientation states of SM proteins. Additionally, we observed a significant difference between the time-averaged SM-SERS spectrum and the multimolecule SERS spectrum, emphasizing the importance of attributing characteristic peaks to discover protein orientation. This study demonstrates great potential for elucidating the orientation of SM proteins and represents a promising advancement toward SM protein sequencing.