Electrostatically Enhanced Single-Molecule Sensing of Aliphatic and Aromatic Polyfluorinated Carboxylic Acids Isomers Using A260K Aerolysin Nanopore

Abstract

Per- and polyfluoroalkyl carboxylic acids (PFCA) are persistent organic pollutants with complex structures and strong biological toxicity. Single-molecule sensing using Aerolysin (Ael) nanopore and cationic probe enables standard-free quantification of PFCA homologues, but its identification performance is limited by the relatively short analyte dwell time in wild type (WT) Ael. Herein, we engineered the nanopore sensing interface by introducing lysine (K) and arginine (R) at three sites near the trans exit barrier of Ael (T240, E258, and A260) to prolong analyte dwell time and enhance resolution. All-atom molecular dynamics simulation revealed that the electrostatic potentials at trans exit shifted from negative (WT) to strong positive for all mutants. The K-substitutions significantly enhanced the signal difference among analytes, as experimentally validated by two aliphatic isomers. In particular, A260K exhibited the most superior sensing capability: dwell time was increased by 50-fold and the sigma of blockade was reduced by 80%, leading to nearly 30-fold improvement in resolutions compared to WT Ael. Further evaluation with seven aromatic PFCA isomers showed that the identification accuracy increased from 57.71% for WT Ael to 91.58% for A260K, with the aid of multi-feature classification. This study provides a new strategy towards comprehensive standard-free detection of PFCA.

Keywords

Per- and polyfluorinated carboxylic acid / Engineered nanopore / Machine learning / Isomer

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