β-Cyclodextrin-Functionalized Mycobacterium smegmatis Porin A for Steroid Discrimination and Gut Metabolite Monitoring.

Steroid metabolites like estradiol (E2) and bile acids (BAs) play pivotal roles in human metabolism but are challenging to distinguish due to their high structural homology. Herein, we integrated β-cyclodextrin (βCD) into the MspA nanopore via a hetero-octameric assembly following Michael addition. This engineered sensor, termed MspA-βCD, affords precise control over βCD conformation within the pore lumen, positioning βCD proximal to the pore constriction zone to enhance sensing resolution. The platform achieves high-resolution discrimination of 11 structurally analogous steroid metabolites. Automated machine learning classification achieved 99.7% validation accuracy. Single-molecule evidence revealed three key structural factors that exert a pivotal influence on the dissociation rate constants of BA-βCD complexes: the chirality of the 7-hydroxyl group, steric hindrance of the 12-hydroxyl group, and side-chain conjugation pattern of BAs. The platform was successfully validated for bile acid profiling in real fecal samples, highlighting its potential for point-of-care diagnosis of metabolic disorders.