Rapid, sensitive detection of intact SARS-CoV-2 using DNA nets and a smartphone-linked fluorimeter

We report a single-step, room-temperature, 5-10 minute SARS-CoV-2 saliva self-monitoring method that overcomes the limitations of existing approaches through the use of fluorophore-releasing Designer DNA Nanostructures (DDNs) that bind with the multivalent pattern of spike proteins on the exterior intact virions and an inexpensive smartphone-linked, pocket-size fluorimeter, called a "V-Pod” for its resemblance to an Apple AirPod™ headphone case. We characterize the V-Pod fluorimeter performance and the DDN-based assay to demonstrate a clinically relevant detection limit of 104 virus particles/mL for pseudo-typed WT SARS-CoV-2 and 105 virus particles/mL for real pathogenic variants, including Delta, Omicron, and D614g. © 2023 SPIE.

V-Pods: Rapid, Sensitive Detection of Intact SARS-CoV-2 using DNA Nets and a Smartphone-Linked Fluorimeter

Rapid, simple, inexpensive, and sensitive self-testing for SARS-CoV-2 is expected to be an important element of controlling the ongoing COVID pandemic. We report a novel approach in which saliva is mixed at room temperature with a Designer DNA Nanostructure (DDN) engineered to create a net-like structure that positions an array of highly specific nucleic acid aptamer-quencher locks at the locations of the trimeric spike proteins. When the spike proteins selectively unlock aptamers on the DDN, fluorescent reporter molecules are unquenched, generating an intense and easily measured optical signal. The fluorescence intensity, proportional to the virus concentration, is detected by a battery-powered palmsized fluorimeter, whose functions are managed wirelessly with a Bluetooth-linked smartphone. Because the single-step, room temperature, test is performed in a conventional 0.2 mL PCR tube that is inserted into the fluorimeter, which resembles an Apple AirPodsT headphone case, we call the technology (DDN+fluorimeter+App) a "V-Pod." We show that DDNs are highly specific only for detection of SARS-CoV-2 in both its initial form as well as common variants. The approach achieves a detection limit of 10,000 genome copies/mL, consistent with laboratory-based PCR, while requiring only one reagent and a 5-10 minute incubation time with saliva. Because DDNs are inexpensively synthesized, structurally stable nucleic acid constructs, and the V-Pod instrument is comprised of inexpensive electronic and photonic components, the approach offers potential for rapid self-monitoring of viral infection with integrated capability for contact tracing and interaction with health services.