Single-Molecule Monitoring of Nucleic Acid Dynamics Using Raman Correlation Spectroscopy in Plasmonic Nanowells

Label-free monitoring of single molecules by single-molecule surface-enhanced Raman spectroscopy (SM-SERS) in plasmonic nanopores can track the molecular dynamics and gain insight into its internal mechanism for applications including catalysis and sequencing. However, challenges including unstable plasmonic hot spot, fast molecule movement, and citrate interference hinder the SM-SERS data analysis and biomedical applications. In this study, we report a new SM-SERS method by sticking a single gold nanoparticle in a gold nanowell in air to generate a fixed plasmonic gap-mode hot spot on the particle surface for continuous single-molecule readout and long-term monitoring of DNA diffusion. The unlimited resident time of the DNA in the hot spot revealed unidirectional and back-and-forth diffusion patterns of different DNAs at single-base resolution depending on their sequences as well as cooccupation of the hot spot by citrate and DNA. Significantly, the spatial resolution of the hot spot was found to be able to cover 2 neighboring nucleobases, 1 sugar–phosphate backbone in the DNA, and 1 citrate. By using Raman correlation spectroscopy, the diffusion times of nucleobases in the DNAs were calculated as 5–22 s depending on molecular structures, while those of citrate were 0.1–7 s. Our results were so promising for monitoring biomolecular dynamics that they could be used to investigate oligonucleotide hybridization kinetics and may set the basis for developing SM-SERS sequencing technologies.