Identification of chiral amino acids using an electrostatically asymmetric nanopore

Chiral molecules play a key role in chemical and biological processes. In view of the great significance of chiral amino acids, many strategies focusing on peptide chiral recognition have been developed. However, due to the same molecular weight and similar physicochemical properties, existing technologies including circular dichroism, capillary electrophoresis, and even liquid chromatography-mass spectrometry encounter great challenges in peptide epimer discrimination. Recently, the single-molecule approach based on nanopore has revealed the capacity of sensing chiral molecules by establishing a specific chiral environment, but it is hard to generalize towards peptide epimers. Herein, we designed an asymmetrically stereo-confined space utilizing the natural peptide-folded structure inside OmpF, an outer membrane porin from E. coli. The negatively charged pocket with the opposite arginine ladder in the constriction zone of OmpF forms an asymmetric potential distribution. The resulting lateral electrostatic field forces the amino-acid sidechains in a single peptide to specific orientations within OmpF, causing distinct ionic current fluctuations. Using statistical analysis of the distinct ionic current variations allows discriminating the presence and the position of one single chiral amino acid. Furthermore, the disease-related peptide β-Amyloid and its D-Asp1 mutant and a mixture of the icatibant peptide drug and its D-Ser7 impurity have been distinguished, demonstrating the potential application of OmpF as a chiral sensor. Our studies highlight a novel sensing mechanism for identifying chiral amino acids in peptide epimers and even for achieving single-molecule protein sequencing.