Single-monosaccharide resolution glycan profiling enabled by the nanopipette.

Glycans encode critical biological information through variations in monosaccharide composition, branching architecture, and chemical modifications. However, decoding these structural features with single-monosaccharide precision at physiological (nM-µM) concentrations remains a major challenge. Herein, we develop a rectification-guided nanopipette strategy that identifies individual monosaccharide differences in heparosan oligosaccharides at picomolar concentrations. This capability arises from an optimized ionic current rectification ratio, which results in a suitable balance between electroosmotic flow (EOF) and electrophoretic force (EPF), thereby enabling enhanced capture efficiency and signal-to-noise ratio. The success rate of single-molecule glycan detection increases from below 20% to over 80%, enabling reliable discrimination of linear and branched oligosaccharides with single-monosaccharide resolution and over 98% accuracy. Combining high sensitivity with single-monosaccharide resolution, this method provides a promising platform for nanopore-based glycomics and sequencing.