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.
KEYWORDS: Raman spectroscopy, Raman scattering, Molecules, Biopolymers, System on a chip, Signal generators, Photonics, Image resolution, Gold, Americium
Tip-enhanced Raman scattering (TERS) is a promising optical and analytical technique. TERS signals generated by a gap-mode configuration where a tip is coupled with a gold substrate can resolve DNA and RNA molecules at a single-molecule resolution. Through our deposition procedure, the molecules can be stretched, uncoiled, and attached to the substrate by its phosphate groups while exposing its nucleobases to the tip. The proof-of-principle RNA sequencing approach significantly advances a direct RNA sequencing technique without RNA labeling or amplification via reverse transcriptase RT-PCR. The demonstrated technique holds promise for next-generation DNA and RNA sequencing.
Interferometric scattering microscopy is a newly emerging alternative to fluorescence microscopy in biomedical research and diagnostic testing due to its ability to detect nano-objects such as individual proteins, extracellular vesicles, and virions individually through their intrinsic elastic light scattering. To improve the signal-to-noise ratio, we developed photonic resonator interferometric scattering microscopy (PRISM) in which a photonic crystal (PC) resonator is used as the sample substrate. The scattered light is amplified by the PC through resonant near-field enhancement, which then interferes with the <1% transmitted light to create intensity contrast. Importantly, the scattered photons assume the wavevectors defined by PC’s photonic band structure, resulting in the ability to utilize a non-immersion objective without significant loss at illumination density as low as 25 W/cm2. We demonstrate virus and protein detection, including highly selective capture and counting of intact pseudotype SARS-CoV-2 from saliva with sensitivity equivalent to conventional nucleic acid tests. The results showcase the promise of nanophotonic surfaces in the development of resonance-enhanced interferometric microscopies, and as a single step, room temperature, and rapid viral detection technology.
Newly demonstrated advanced biosensor imaging technologies utilize the unique electromagnetic capabilities of photonic metamaterials to enhance the interaction between light and biological matter. The resulting capabilities address gaps in existing technologies for biomolecular analysis that rely upon enzymatic and chemical amplification, costly instrumentation, and complex assay protocols. Through amplification of the excitation/extraction efficiency of light emitting tags, absorption efficiency of nanoparticle tags, and scattering efficiency of biological analytes, technology platforms have been demonstrated that are capable of ultrasensitive, digital-resolution, room temperature, isothermal, rapid, and highly quantitative biomolecular analysis.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.