Fluorescence origami paper–based analytical device based on strand displacement assay for SARS-CoV-2 cDNA detection

Abstract

A fluorescence origami paper–based analytical device (Flu-oPAD) based on a strand displacement assay as a point-of-care testing (POCT) sensing platform for DNA detection is presented. This device facilitates multiple steps in a single device, including sample loading, incubation, and washing. The detection zone was immobilized with a 6-FAM-modified probe (F-probe), which formed a complex with a BHQ1-modified probe (Q-probe) to minimize background signals. In the presence of target DNA, hybridization with the F-probe releases the Q-probe, resulting in a fluorescence response proportional to the target DNA concentration. The sensor exhibits good selectivity for target DNA, with a linearity range from 0.1 nM to 10 μM and an experimental detection limit of 0.1 nM, completing all procedures within 15 min. The real-world applicability is demonstrated by successfully detecting complementary DNA (cDNA) from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a cause of the COVID-19 pandemic. This study further introduces a multi-array Flu-oPAD for simultaneous detection of SARS-CoV-2 ORF1ab, N, and E gene cDNAs. This multi-array Flu-oPAD is applied to nasopharyngeal swab samples, showing good agreement with the standard RT-PCR method. Overall, the developed Flu-oPAD has shown great potential as an effective POCT tool for DNA screening. It offers simplicity, portability, accessibility, and cost-effectiveness, offering its potential impact on addressing pressing healthcare needs.