Quantifying electro-osmotic flow in mutant MspA nanopores

Nanopores have emerged as a powerful tool for single-molecule sensing and sequencing. In a typical nanopore experiment, a charged analyte such as DNA is driven through the pore by an electrophoretic force generated by the applied electric field. This can be problematic, however, when analytes such as proteins carry mixed charges. An alternative driving force can be from electro-osmotic flow (EOF), the water flow that arises from an asymmetric migration of cations and anions through the nanopore. However, despite much recent interest, there is no straightforward experimental method to quantify EOF in nanopores. Here, we trap a dipolar peptide in an MspA nanopore, where studying its escape rate versus voltage allows us to extract the work done by the EOF in various MspA mutants and wildtype MspA compared to a M2-MspA reference. For the 30 amino acid model peptide, this yields values of ~5.6-16.7 kBT for the work done by the EOF at 80 mV. Finite-element simulations confirm the trends observed in the experiments. Charged mutations close to the nanopore's constriction site are found to produce the highest EOF. Our novel method provides a quantitative metric of the EOF in nanopores, opening up its study and broad use in future nanopore sequencing and sensing applications.