Bionic nanopore recognition receptors for single-molecule enantioselectivity studies of chiral drugs

Background: Enantiodiscrimination of chiral drugs is critical for understanding physiological phenomena and ensuring medical safety. Although enantiomers of these drugs share identical physicochemical properties, they exhibit significant differences in pharmacodynamic, pharmacokinetic, and toxicological properties due to the differences in their three-dimensional shapes. Therefore, the development of effective methods for chiral recognition is of great significance and has been a hot topic in chemo/biological studies. Results: In this study, we designed a recognition receptor comprising a α-hemolysin (α-HL) nanopore and sulfobutyl ether-β-cyclodextrin (SBEβCD) for identifying the enantiomers of the antidepressant duloxetine at the single-molecule level. Chiral molecules were discriminated based on the different current blockages within the recognition receptor. The results indicated a strong interaction between R-duloxetine and the recognition receptor. By combining the experimental data and molecular docking results, we explored the recognition mechanism of the designed nanopore recognition receptor for chiral drug molecules. It was found that hydrophobic and electrostatic interactions play key roles in chiral recognition. Additionally, by comparing the binding kinetics of enantiomers to cyclodextrins in confined nanospace and bulk solution, we found that enantiomeric identification was better facilitated in the confined nanospace. Finally, the enantiomeric excess (ee) of the enantiomeric duloxetine mixture was measured using this recognized receptor. Significance: This strategy has the advantages of low cost, high sensitivity, and no need for additional derivative modifications, providing a new perspective on the development of chiral recognition sensors with excellent enantioselectivity in drug design, pharmaceuticals, and biological applications.