Salt Gradient Modulation of MicroRNA Translocation through a Biological Nanopore

Abstract

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.

Recent advances in biological nanopores for nanopore sequencing, sensing and comparison of functional variations in MspA mutants

“…115 Importantly, it is used to analyze and point out the position of the mutations in DNA via non-functionalized PNA, 116 short fragments of DNA, 117 sequence-specic ssDNA detection with gold nanoparticles, 118 label-free detection of completely matched from mismatched DNA on the basis of enzymatic reaction 4 and to quantify multiple cancer biomarkers in blood samples at picomolar level. 119 Moreover, detection of micro-RNA 120,121 and identication of methylated cytosine by employing lithium salt-gradient have also been achieved. 113 Furthermore, identication of individual amino acids via their N-terminal derivatization, 122,123 polypeptide-pore interaction 124 and protein phosphorylation and dephosphorylation were well observed by utilizing the a-hemolysin nanopore.…”

Single‐Molecule Interaction of Peptides with a Biological Nanopore for Identification of Protease Activity

“…These results were consistent with the report of a salt gradient effect on DNA translocation which was more significant through nanopores. [ 35 ] Here, the NaCl gradients are thought to contribute to the regional electric funneling field in the cis side as a result of cation accumulation on the trans side, which satisfy the requirement for a constant current flow. [ 36,37 ] Therefore, the negatively charged peptides in the cis side of the pore experienced an electrophoretic force, allowing them to be easily captured and rapidly translocated through the pore.…”

Actinoporins: From the Structure and Function to the Generation of Biotechnological and Therapeutic Tools

“…The nanopores are embedded in membranes with two compartments (cis and trans) that contain a solution of electrolytes and electrodes. When a polarization voltage is applied across the membrane, the free passage of ions through the nanopores generates a constant current that is indicative of an open pore [103,104]. When a molecule diffuses through the pore, the ion flow is partially interrupted and results in a change in the ionic current that is detected as an electrical signal [105,106].…”