Optimization of Parameters for Nanopore Resistive Pulse Sensing of MicroRNA

MicroRNAs are emerging as a new class of molecular biomarkers for a range of diseases. Nanopore resistive pulse sensing is a novel technique for the detection and quantification of these short oligonucleotides which, when electrophoretically driven through a nanopore, cause a transient decrease in electrical current. In the presence of a DNA probe complementary to the target miRNA sequence, pore capture and translocation events of miRNA-DNA hybrids are distinguished by the length and depth of the current-block pulse. Because quantification of the miRNA analyte requires a sufficiently high number of these pulses, optimization of the miRNA-DNA capture rate is important. We investigated the translocation rate and dwell time of miRNA-155 annealed to its complementary DNA probe for different electrolyte gradients across a lipid bilayer with an alpha-hemolysin nanopore. The translocation rate was found to increase with higher applied voltages and with steeper electrolyte gradients. The number of current block pulses was two orders of magnitude larger with 0.1 M / 4 M KCl than with 1 M / 1 M KCl electrolyte solutions, while the dwell time was significantly reduced at a bias of 120 mV, but not at higher voltages. By comparing DNA probes with 3′, 5′, or both 3′ and 5′ homonucleotide extensions we also established that the current block detail depends on the probe extension side, which is most likely related to different base pairing at the two miRNA-155 termini. The highest translocation rates were obtained with the dual extended DNA probe but the shortest dwell times with the 3′-only extension. These optimized parameters enable nanopore resistive pulse sensing of miRNA molecules over a larger concentration range.