Solid-state nanopore quantification of discrete sequence motifs from DNA and RNA targets in human plasma

Abstract

Fast and sensitive detection of target nucleic acid biomarker sequences in complex biofluids is essential for translational diagnostics. In this work, we report on the use of a solid-state nanopore assay to quantitate sequence motifs in human plasma. Extracted DNA or RNA is annealed to a biotinylated DNA oligonucleotide probe and then subjected to single-strand-specific enzymatic digestion to decompose off-target regions. The remaining duplex product is then bound to a protein tag that enables selective detection via resistive pulse sensing. We first demonstrate our approach on single-strand DNA and single-strand RNA spiked into human plasma and then extend the methodology to double-strand DNA, expanding the range of motifs that can be targeted. These advancements position our assay as a tool for the analysis of viral, bacterial, and human genetic markers.