Molecular Orientation Effects on Surface‐Enhanced Raman Scattering Spectra at the Single‐Molecule Level Revealed by DNA Origami Plasmonic Antennas

Characterizing single‐molecule signals in surface‐enhanced Raman scattering (SERS) remains challenging, particularly in identifying the sources of spectral fluctuations. While prior studies focused on nanoparticle composition and surface restructuring, they often overlook the molecule's behavior, partly due to limitations in controlling molecular positioning or the complexity introduced by bianalyte methods. Here, we employ theoretical methods to model the Raman spectra of a Cy5 dye, demonstrating that spectral variations occur when the molecule aligns parallel to the external electric field. By leveraging DNA's capability to immobilize and orient molecules, we experimentally acquired single‐molecule SERS spectra for dye molecules in parallel and perpendicular orientations to the DNA double helix. Notably, distinct spectral differences emerged between these orientations, suggesting that factors like functional group proximity to nanoparticle surfaces and the electric‐field intensity within nanocavities critically impact SERS outcomes. Density functional theory calculations further elucidate these effects, accounting for finite‐field effects, molecular orientation, and surface interactions. These findings enhance our understanding of single‐molecule behavior within plasmonic hot spots, offering new insights into the role of molecular orientation in SERS and informing future nanotechnology applications.