Single-Feature Identification of α2–8 Linked Sialoglycans Using Engineered Aerolysin Nanopores: A Paradigm for Glycan Linkage Analysis

Nanopore single-molecule sensing has emerged as a transformative platform in glycoscience and nanotechnology because of its inherent potential to decode complex "glycocodes" with high sensitivity, single-molecule resolution, and real-time throughput. However, realizing true sequencing across the vast diversity of glycans demands continuous enhancement of the nanopore resolution and applicability. In particular, conceptual advancements are needed to overcome the limitations of current profiling analysis-based approaches. Here, we propose a single-feature paradigm for nanopore glycan linkage analysis and demonstrate the specific recognition of α2-8 glycosidic linkage using an engineered aerolysin nanopore, K238Q. The N262-Q238-E258 region establishes multiple synergistic hydrogen-bonding interactions and an electrostatic barrier with the α2-8 motif, selectively decelerating α2-8 sialoglycans and enabling the characterization and fingerprinting of this subclass at the submonosaccharide level. We also discover the complementary behavior of a K238N variant, which responds to longer or highly branched sialoglycans in a dual-pore logic-gate assay. This assay combines the readouts from K238Q and K238N to extract key structural information from unknown glycans. Converged with nanopore-compatible preprocessing and machine learning, this approach enables proof-of-concept identification and quantification of sialoglycans in serum. This work not only establishes engineered aerolysin nanopores as a powerful platform for the selective discrimination of sialoglycans in complex biological matrices but also opens a new avenue for nanopore-based glycan sequencing.