Salt gradient modulation of microRNA translocation through a biological nanopore
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 0.1−100 nM. This work highlights differences between biological and solid-state nanopore sensing and provides strategies for sub-nanomolar miRNA quantification with bilayer-embedded porins.
nanopores, alpha-hemolysin, resistive pulse sensing, microRNA, molecular diagnostics, biosensor
8822-8829
Ivica, Josip
aacf91f9-3876-4a95-8914-98e94cc524d7
Williamson, Philip T.F.
0b7715c6-b60e-4e95-a1b1-6afc8b9f372a
De Planque, Maurits R.R.
a1d33d13-f516-44fb-8d2c-c51d18bc21ba
5 September 2017
Ivica, Josip
aacf91f9-3876-4a95-8914-98e94cc524d7
Williamson, Philip T.F.
0b7715c6-b60e-4e95-a1b1-6afc8b9f372a
De Planque, Maurits R.R.
a1d33d13-f516-44fb-8d2c-c51d18bc21ba
Ivica, Josip, Williamson, Philip T.F. and De Planque, Maurits R.R.
(2017)
Salt gradient modulation of microRNA translocation through a biological nanopore.
Analytical Chemistry, 89 (17), .
(doi:10.1021/acs.analchem.7b01246).
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 0.1−100 nM. This work highlights differences between biological and solid-state nanopore sensing and provides strategies for sub-nanomolar miRNA quantification with bilayer-embedded porins.
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Ivica_accepted_manuscript
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More information
Accepted/In Press date: 27 July 2017
e-pub ahead of print date: 27 July 2017
Published date: 5 September 2017
Keywords:
nanopores, alpha-hemolysin, resistive pulse sensing, microRNA, molecular diagnostics, biosensor
Identifiers
Local EPrints ID: 414055
URI: http://eprints.soton.ac.uk/id/eprint/414055
ISSN: 0003-2700
PURE UUID: a22a9ff7-a22b-4ccc-84c4-5220957897ba
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Date deposited: 13 Sep 2017 16:31
Last modified: 16 Mar 2024 05:35
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Author:
Josip Ivica
Author:
Maurits R.R. De Planque
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