Lateral flow assays (LFAs) providing rapid on-site detection of medically significant analytes couldbe extremely valuable for high-volume, high-speed population screening necessary to minimize the economicand societal impact of epidemic outbreaks. However, LFA performance has been mostly limited to colorimetricbinary diagnostics, primarily providing yes/no testing for clinically relevant analytes present at highconcentrations (µM-nM) and cannot satisfy sensitivity requirements for reliable virus detection (fM-aM) which ispresently achieved only using laboratory techniques (ELISA, PCR). Though signal amplification throughfluorescence has steadily gained traction to improve sensitivity by 1-2 orders of magnitude in comparison withstandard colorimetric tests, the inherent autofluorescence of materials used to construct LFA test strips interfereswith test performance and introduces variability. By enabling rapid, attomolar sensitivity, fluorescence-basedLFAs could facilitate a revolutionary shift in the analytical capability of point-of-care tests to provide quantitativeactionable data to inform health authorities to launch timely responses. The pathway to this development involvesadvancement of the fluorescence detection system and material properties of the reporters. This proposal aimsto strategically address both of these directions by advancing a recently emerged LFA platform based onfluorescent nanodiamond (FND) reporters. FNDs possess ideal features for an LFA reporter such as bright non-bleaching fluorescence and unsurpassed ruggedness. A prototype using FND as a novel LFA reporter wasrecently demonstrated by Debina Diagnostics in detecting Ebola virus glycoprotein at the picograms level, usinga custom-made optoelectronic reader (Axxin, Inc.) and off-the-shelf 200 nm FND produced by AdámasNanotechnologies. The most striking attribute of FND envisioned to improve FND-based LFA sensitivity by 2-3orders of magnitude arises from the uniquely coupled magneto-optical properties of the particles, where nitrogen-vacancy (NV) color centers with an electron spin allow the intensity FND fluorescence to be modulated by anexternal magnetic field. Based on this unique property, the FND-related signal can be separated frombackground autofluorescence in the frequency domain through lock-in analysis which is widely used in signalprocessing to extract small periodic signals present below noise levels. Lock-in signal processing of FND canallow up to 100x increase in sensitivity, as was demonstrated in bioimaging including recent results in LFA.Adámas recently developed a method further increasing magnetic modulation contrast by 3-5 folds. In thisproposal, Adámas and Debina join their efforts to tailor FND material properties in order to eliminate non-specificretention of particles on the LFA strip by modifying their physical (shape, size) and chemical (coating) properties,while optimizing brightness and magneto-optical properties (aim 1). After implementation and optimization of thelock-in analysis including interfacing the set-up with the opto-electronic reader (aim 2), we aim to demonstrate>300x improvement in LOD of Ebola virus antigen and <1pg LOD for detection of SARS-CoV-2 antigen (aim 3).
Public Health Relevance Statement: Narrative. Lateral flow assays (LFAs) providing rapid on-site detection of medically significant analytes would be extremely valuable for the high-volume, high-speed population screening necessary to minimize the economic and societal impact of epidemic outbreaks. However, current LFA performance lacks the sensitivity required for reliable virus detection (fM-aM) which is currently accessible only to laboratory techniques (ELISA, PCR). This proposal develops advanced fluorescent LFA platform capable to detect infectious diseases (including SARS- CoV-2) with aM sensitivity and detection time <15min enabling a powerful point of care tool for control of epidemics.
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