We report an integrated system for rapid sample-to-answer detection of a viral pathogen in a droplet of whole blood comprised of a two-stage microfluidic cartridge for sample processing and nucleic acid amplification, and a clip-on detection instrument that interfaces with the image sensor of a smartphone. The cartridge is designed to release RNA from the Zika virus in whole blood using chemical lysis, followed by mixing with the assay buffer for performing reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) reactions in six parallel microfluidic compartments. The battery-powered instrument heats the compartments from below, while LEDs illuminate from above. Fluorescence generation in the compartments is dynamically monitored by a smartphone camera. We characterize the assay time and detection limits for detecting Zika RNA and gamma-irradiated Zika virus spiked into buffer and whole blood and compare the performance of the same assay when conducted in conventional PCR tubes. Our approach for kinetic monitoring of the fluorescence-generating process in the microfluidic compartments enables spatial analysis of early fluorescent “bloom” events for positive samples. We show that dynamic image analysis reduces the time required to designate an assay as a positive test to 22 minutes, compared to ~30-45 minutes for conventional analysis of the average fluorescent intensity of the entire compartment. We achieve a total sample-to-answer time in the range of 17-32 minutes, while demonstrating a viral RNA detection as low as 2.70×102 copies/ul, and a gamma-irradiated virus of 103 virus particles in a single 12.5 microliter droplet blood sample.
We report a single-step, room-temperature, five to ten 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.
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 palm-sized 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 AirPods™ 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.
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