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.