Respiratory infections are the major cause of death from infectious disease worldwide. Multiplexed diagnostic approaches are essential as many respiratory viruses have indistinguishable symptoms. We created self-assembled DNA nanobait that can simultaneously identify multiple short RNA targets. The nanobait approach relies on specific target selection via toehold-mediated strand displacement and rapid readout via nanopore sensing. Here we show that this platform can concurrently identify several common respiratory viruses, detecting a panel of short targets of viral nucleic acids from multiple viruses. Our nanobait can be easily reprogrammed to discriminate viral variants with single-nucleotide resolution, as we demonstrated for several key SARS-CoV-2 variants. Last, we show that the nanobait discriminates between samples extracted from oropharyngeal swabs from negative- and positive-SARS-CoV-2 patients without preamplification. Our system allows for the multiplexed identification of native RNA molecules, providing a new scalable approach for the diagnostics of multiple respiratory viruses in a single assay.
High‐resolution analysis of biomolecules has brought unprecedented insights into fundamental biological processes and dramatically advanced biosensing. Notwithstanding the ongoing resolution revolution in electron microscopy and optical imaging, only a few methods are presently available for high‐resolution analysis of unlabeled single molecules in their native states. Here, label‐free electrical sensing of structured single molecules with a spatial resolution down to single‐digit nanometers is demonstrated. Using a narrow solid‐state nanopore, the passage of a series of nanostructures attached to a freely translocating DNA molecule is detected, resolving individual nanostructures placed as close as 6 nm apart and with a surface‐to‐surface gap distance of only 2 nm. Such super‐resolution ability is attributed to the nanostructure‐induced enhancement of the electric field at the tip of the nanopore. This work demonstrates a general approach to improving the resolution of single‐molecule nanopore sensing and presents a critical advance towards label‐free, high‐resolution DNA sequence mapping, and digital information storage independent of molecular motors.
Nanopores are one of the most successful label-free single-molecule techniques with several sensing applications such as biological screening, diagnostics, DNA and protein sequencing. In current nanopore technologies, stochastic processes influence both the selection of the translocating molecule, translocation rate and translocation velocity. As a result, single-molecule translocations are difficult to control spatially and temporally. Here we present a novel method where we engineer precise spatial and temporal control into the single-molecule experiment. We use a glass nanopore mounted on a 3D nanopositioner to spatially select molecules, deterministically tethered on a glass surface, for controlled translocations. By controlling the distance between the nanopore and the glass surface, we can actively select the region of interest on the molecule and scan it a controlled number of times and at controlled velocity. Decreasing the velocity and averaging thousands of consecutive readings of the same molecule increases the signal-to-noise ratio (SNR) by two orders of magnitude compared to free translocations. We applied our method to various DNA constructs, achieving down to single nucleotide gap resolution. The spatial multiplexing combined with the sub-nanometer resolution could be used in conjunction with micro-array technologies to enable screening of DNA, improving point of care devices, or enabling high-density, addressable DNA data storage.
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