Salt Gradient Modulation of MicroRNA Translocation through a Biological Nanopore

In resistive pulse sensing of microRNA biomarkers, selectivity is achieved with polynucleotide-extended DNA probes, with the unzipping of a miRNA–DNA duplex in the nanopore recorded as a resistive current pulse. As the assay sensitivity is determined by the pulse frequency, we investigated the effect of cis/trans electrolyte concentration gradients applied over α-hemolysin nanopores. KCl gradients were found to exponentially increase the pulse frequency, while reducing the preference for 3′-first pore entry of the duplex and accelerating duplex unzipping, all manifestations of an enhanced electrophoretic force. Unlike silicon nitride pores, a counteracting contribution from electro-osmotic flow along the pore wall was not apparent. Significantly, a gradient of 0.5/4 M KCl increased the pulse frequency ∼60-fold with respect to symmetrical 1 M KCl, while the duplex dwell time in the nanopore remained acceptable for pulse detection and could be extended by LiCl addition. Steeper gradients caused lipid bilayer destabilization and pore instability, limiting the total number of recorded pulses. The 8-fold KCl gradient enabled a linear relationship between pulse frequency and miRNA concentration for the range of 0.1–100 nM. This work highlights differences between biological and solid-state nanopore sensing and provides strategies for subnanomolar miRNA quantification with bilayer-embedded porins.