Multiplexed Molecular Counters using a High-Voltage Transmembrane Pore Platform

Abstract

Synthetic andBiological nanopores have been used as powerful platforms for label-free detection and identification of a spectrum of biomolecules, which are used in applications that range from biosensing, single-molecule biophysics, and DNA/RNA sequencing. Nonetheless, their relatively high limit of detection (LOD) has inhibited further development of nanopore biosensors. Traditionally, biological nanopores are incorporated into planar lipid membranes painted on PTFE apertures, and thus their LOD is limited by the maximum voltage bias that can be applied across the lipid bilayer and protein nanopore. Here, by using grayscale lithography, we developed a polymer support for lipid bilayers that consists of an aperture with a unique 3D profile. We find that lipid bilayer membranes formed on the support exhibit longer lifetimes and a consistent ability to sustain voltages as high as 350 mV, without compromising the diameter of the aperture. By increasing the applied voltage, in addition to improving the signal-to-noise ratio, the LOD of DNA hairpins in α-hemolysin nanopores was pushed to the nM regime. To harness this platform, we demonstrate the use of different length DNA hairpins as multiplexed reporter molecules for biosensing. As proof-of-concept, we demonstrate DNA hairpin release from magnetic microbeads through a site-specific enzymatic digestion reaction, followed by direct quantification of the hairpins using a nanopore on our platform at high voltage. This convenient approach, which is general for any ELISA-type biomarker assay and does not require further cleanup steps following digestion, paves the way for development of a novel nanopore-based multiplexed biosensing strategies in which small concentrations/volumes are required. Finally, since our polymer support chips are housed on a silicon-based chip, our devices are compatible with optical sensing, as well as integration with silicon-based microfluidic elements.