Solid-state nanopore counting of amplicons from recombinase polymerase isothermal amplification

Abstract

Single-molecule detection methods based on electrical readout can transform disease diagnostics by miniaturizing the downstream sensor to enable sensitive and rapid biomarker quantification at the point-of-care. In particular, solid-state nanopores can be used as single-molecule electrical counters for a variety of biomedical applications, including biomarker detection. Integrating nanopores with efficient DNA amplification methods can improve upon sensitivity and accessibility concerns often present in disease detection. Here, we present nanopores as biosensors downstream of a reverse-transcription recombinase polymerase amplification (RT-RPA)-based assay targeting synthetic SARS-CoV-2 RNA. We demonstrate the efficacy of nanopore-integrated RT-RPA for the direct electrical detection of target amplicons, and discuss challenges from RPA-based assays and adaptations that facilitate solid-state nanopore readout.