Identification of Isomerically Diverse Ginsenosides Using Engineered Aerolysin Nanopore via Non‐Translocation Blockade Sensing

Abstract Typical nanopore sensing depends on slowed translocation through the pore to acquire effective blockade signals. However, this paradigm often suffers from low signal precision and poor resolution, making it challenging to resolve pools of analytes with diverse, similar structures. Here, we present a non‐translocation blockade sensing based on an engineered aerolysin S278K that enables the identification of isomerically diverse ginsenosides—a class of glycoconjugates whose structural characterization has been a persistent challenge in glycoscience. By introducing the S278K mutation, the aerolysin acquires a highly positively charged interior and generates intense electro‐osmotic flow and enhanced steric/enthalpic barriers, effectively trapping ginsenoside molecules in the K278‐R220 region and preventing their further translocation. This distinct blockade sensing mode significantly improves the detection capability of aerolysin by prolonging residence time (e.g., 43‐fold longer), enabling detailed molecular characterization. As a result, we demonstrate the unambiguous identification of 30 ginsenosides differing in glycosyl composition, isomerism, modification, and aglycone, as well as the quantitative analysis of complex ginsenosides in real samples by integrating deep learning. This work underscores the promise of non‐translocation nanopore sensing for deciphering structurally complex and diverse small molecule analytes.