Single-protein Nano-FTIR Vibrational Spectroscopy : Near-field Interactions Confined below the Tip Radius

Tip-enhanced vibrational spectroscopy has matured to the point of reliably achieving nanoscale spatial resolution, with tip-enhanced Raman spectroscopy reaching even atomic-level and submolecular sensitivity. Likewise, tip-enhanced infrared methods, such as nano-FTIR and AFM-IR spectroscopy, have enabled chemical analysis at the nanoscale for diverse systems. However, the fundamental spatial resolution and sensitivity limits of infrared nano-spectroscopy remain elusive, highlighting the need for a quantitative understanding of near-field interactions within infrared nanocavities. In this study, we apply nano-FTIR spectroscopy to detect the amide-I vibrational mode of a single protein composed of approximately 500 amino acids. By employing higher-order demodulation harmonics up to the seventh order, we observe a significant increase in vibrational resonance amplitude, attributed to geometrical effects confined below the tip radius and beyond conventional dipole models. This work provides a quantitative description of near-field interactions at the single-nanometer scale, paving the way for sub-nanometer and single-molecule infrared vibrational spectroscopy.