Nanopore-Enhanced Positioning of Molecules in Zero-Mode Waveguides

Abstract

Zero-mode waveguides are metal nanostructures that define zeptoliter illumination volumes for low-background imaging of diffusing fluorescent molecules. Despite their numerous advantages for DNA sequencing and other applications, diffusion-based loading is slow and limited by Poisson statistics. This inherently limits the fractional efficiency of zero-mode waveguide devices, and further presents a limit on the minimum input requirements for practical observations. Solid-state nanopores, on the other hand, are sensitive biomolecular detectors which focus and position individual charged biomolecules with nanometer precision. Here we demonstrate zero-mode waveguides fabricated atop ultrathin freestanding solid-state membranes, such that each waveguide has a sub-5 nm solid-state nanopore at its base. Applying voltage across the device allows us to actively load and position a molecule in the excitation volume of each zero-mode waveguide. Using synchronous optical and electrical recordings, we demonstrate the reversible, voltage-driven positioning and ejection of individual DNA-protein complexes. Finally, we show that voltage-enhanced loading of DNA into the waveguides is at least three-orders of magnitude more efficient than diffusion loading. When combined with SMRT-sequencing platforms, this enhancement can pave the way for probing epigenetics in non-amplified mammalian DNA fragments.