Nanoscale Chemical Imaging of Functionalized Monosaccharides Via Click-Chemistry by Tip-Enhanced Raman Spectroscopy

Direct molecular imaging of individual biomolecules, such as sugars, is critical for understanding their structural and functional diversity. Tip-enhanced Raman spectroscopy (TERS) offers nanoscale resolution and molecular fingerprints simultaneously, but imaging native monosaccharides is hindered by their weak Raman scattering and poor metal-surface adsorption. Here, azido sugars are conjugated with an alkyne-thiol reporter (4-EBMT) via click chemistry, enabling chemisorption on gold. This approach allows reproducible TERS mapping of six functionalized monosaccharides, namely, Gal, Neu5Ac, ManNAc, GlcNAc, Glc, and Xyl. Using density functional theory calculations, we assign sugar-backbone vibrations to bands at 1270-1450 cm-1. Unsupervised t-SNE analysis reveals distinct spectral clusters, and TERS imaging of mixed monolayers resolves nanoscale chemical heterogeneity, distinguishing Neu5Ac- from ManNAc-enriched regions with an apparent spatial resolution of approximately 5 nm (achieved under a 5 nm sampling interval). This work establishes a surface-anchored TERS strategy to probe local monosaccharide heterogeneity in model glycan-related systems.