Chiral Discrimination of all Proteinogenic Amino Acid Enantiomers by Nanopore Sensing.

D-amino acids, the stereoisomers of the more prevalent L-forms, are ubiquitously distributed across microorganisms, plants, and mammalian systems. Recent advances have uncovered their indispensable roles in biological processes, yet direct, single-molecule detection of D-amino acids has long been understudied. The simultaneous discrimination of all amino acid enantiomers remains a formidable analytical challenge. Here, we report a nickel-ion chelated hetero-octameric Mycobacterium smegmatis porin A (MspA) (MspA-NTA-Ni) nanopore sensor that achieves simultaneous identification of all 19 D-amino acids corresponding to proteinogenic L-isomers. By integrating supervised machine learning algorithms, this system demonstrated an overall recognition accuracy of 99.5%. The method was further validated in analyzing acid-hydrolyzed D-peptide products, yielding clear compositional profiles of amino acid enantiomers. Notably, the technology enables simultaneous sensing of all 39 amino acid enantiomers with an overall accuracy of 98.9%. This approach not only allows direct characterization of DL-amino acid mixtures but also provides a critical sensor component for nanopore-based exo-sequencing of D-peptides and D-proteins.