Length-Dependent, Single-Molecule Analysis of Short Double-Stranded DNA Fragments through Hydrogel-Filled Nanopores: A Potential Tool for Size Profiling Cell-Free DNA.

Abstract

Fast sampling followed by sequence-independent sensing and length-dependent detection of short double-stranded DNA fragments, the size of those found in blood and other bodily fluids, is achieved using engineered molecular sensors, dubbed hydrogel-filled nanopores (HFNs). Fragments as short as 100 base pairs were blindly sampled and concentrated at the tip of an HFN before reversing the applied potential to detect and distinguish individual molecules based on fragment length as they translocate out of the nanopore. A remarkable 16-fold increase in the signal-to-noise ratio was observed in the eject configuration compared to the load configuration, enabling the resolution of fragments with a size difference of 50 nucleotides in length. This fast and versatile technology offers great tunability for both sampling and detection. While increasing sampling time leads to an increase in the local DNA concentration at the tip prior to detection, a linear correlation between the peak current and DNA fragment size enables good resolution of fragments up to 250 bp long.