One-Pot Species Release and Nanopore Detection in a Voltage-Stable Lipid Bilayer Platform

Biological nanopores have been used as powerful platforms for label-free detection and identification of a range of biomolecules for biosensing applications and single molecule biophysics studies. Nonetheless, high limit of detection (LOD) of analytes due to inefficient biomolecular capture into biological nanopores at low voltage poses practical limits on their biosensing efficacy. Several approaches have been proposed to improve the voltage stability of the membrane, including polymerization and hydrogel coating, however, these compromise the lipid fluidity. Here, we developed a chip-based platform that can be massively produced on a wafer scale that is capable of sustaining high voltages of 350 mV with comparable membrane areas to traditional systems. Using this platform, we demonstrate sensing of DNA hairpins in alpha-hemolysin nanopores at the nanomolar regime under high voltage. Further, we have developed a workflow for one-pot enzymatic release of DNA hairpins with different stem lengths from magnetic microbeads, followed by multiplexed nanopore-based quantification of the hairpins within minutes, paving the way for novel nanopore-based multiplexed biosensing applications.