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Salt Gradient Modulation of MicroRNA Translocation through a Biological Nanopore.

Josip Ivica1, Philip T F Williamson1, Maurits R R de Planque1

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Electrolyte gradients significantly boost microRNA detection sensitivity in nanopore sensing by increasing signal frequency. This method enhances microRNA quantification down to subnanomolar levels using biological nanopores.

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Area of Science:

  • Nanopore sensing
  • Biomarker detection
  • Molecular diagnostics

Background:

  • Resistive pulse sensing uses DNA probes for microRNA (miRNA) biomarker detection.
  • Assay sensitivity depends on the frequency of miRNA-DNA duplex unzipping events in the nanopore.

Purpose of the Study:

  • Investigate the impact of cis/trans electrolyte concentration gradients on nanopore sensing performance.
  • Optimize conditions for enhanced sensitivity and quantification of miRNA biomarkers.

Main Methods:

  • Utilized α-hemolysin nanopores embedded in lipid bilayers.
  • Applied potassium chloride (KCl) concentration gradients across the nanopore.
  • Measured resistive current pulses generated by miRNA-DNA duplex unzipping.
  • Analyzed pulse frequency and duplex dwell time under varying gradient conditions.

Main Results:

  • KCl gradients exponentially increased pulse frequency, enhancing signal.
  • Electrophoretic forces were identified as the primary driver of increased frequency.
  • A 0.5/4 M KCl gradient yielded a ~60-fold increase in pulse frequency.
  • Subnanomolar miRNA quantification (0.1-100 nM) was achieved with a linear relationship between pulse frequency and concentration.
  • Biological nanopores showed distinct behavior compared to solid-state pores regarding electro-osmotic flow.

Conclusions:

  • Electrolyte gradients are effective in significantly enhancing miRNA detection sensitivity in biological nanopores.
  • This approach offers a viable strategy for sensitive and specific subnanomolar miRNA quantification.
  • Understanding differences between biological and solid-state nanopores is crucial for sensor development.