Net-Shaped DNA Nanostructure-Based Lateral Flow Assays for Rapid and Sensitive SARS-CoV-2 Detection.

Lateral flow assay (LFA)-based rapid antigen tests are experiencing extensive global uptake as an expeditious and highly effective modality for the screening of viral infections during the COVID-19 pandemic. While these devices have played a significant role in alleviating the burden on the public healthcare system, their specificity and sensitivity fall short compared with molecular tests. In this study, we endeavor to address both limitations through the utilization of DNA nanotechnology in LFA format, wherein we substitute the target-specific antibody with designer DNA nanostructure-based molecular probes for recognizing the SARS-CoV-2 virus via multivalent, pattern-matching interactions. We meticulously designed a Net-shaped DNA nanostructure and strategically arranged trimeric clusters of aptamers that specifically recognize the spike proteins of SARS-CoV-2. This approach has proven instrumental in bolstering virus-binding affinity on the LFAs. Our findings indicate high LFA sensitivity, enabling the detection of viral loads ranging from 103 to 108 viral copies/mL. This notable sensitivity is maintained across various SARS-CoV-2 viral strains, obviating the need for intricate sample preparation protocols. The significance of this heightened sensitivity lies in the crucial role played by the designer DNA nanostructure, which facilitates the detection of extremely low levels of viral loads. This not only enhances the overall reliability of self-testing but also reduces the likelihood of false-negative results, especially in cases of low viral load within patient samples.

Net-Shaped DNA Nanostructures Designed for Rapid/Sensitive Detection and Potential Inhibition of the SARS-CoV-2 Virus.

We present a net-shaped DNA nanostructure (called "DNA Net" herein) design strategy for selective recognition and high-affinity capture of intact SARS-CoV-2 virions through spatial pattern-matching and multivalent interactions between the aptamers (targeting wild-type spike-RBD) positioned on the DNA Net and the trimeric spike glycoproteins displayed on the viral outer surface. Carrying a designer nanoswitch, the DNA Net-aptamers release fluorescence signals upon virus binding that are easily read with a handheld fluorimeter for a rapid (in 10 min), simple (mix-and-read), sensitive (PCR equivalent), room temperature compatible, and inexpensive (∼$1.26/test) COVID-19 test assay. The DNA Net-aptamers also impede authentic wild-type SARS-CoV-2 infection in cell culture with a near 1 × 103-fold enhancement of the monomeric aptamer. Furthermore, our DNA Net design principle and strategy can be customized to tackle other life-threatening and economically influential viruses like influenza and HIV, whose surfaces carry class-I viral envelope glycoproteins like the SARS-CoV-2 spikes in trimeric forms.