SBIR, Phase I (R43): Ultrasensitive Lateral Flow Assays Enabled by Fluorescent Nanodiamond Labels

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Summary. Lateral flow assays (LFAs) providing rapid on-site detection of medically significant analytes could be extremely valuable for high-volume, high-speed population screening necessary to minimize the economic and societal impact of epidemic outbreaks. However, LFA performance has been mostly limited to colorimetric binary diagnostics, primarily providing yes/no testing for clinically relevant analytes present at high concentrations (µM-nM) and cannot satisfy sensitivity requirements for reliable virus detection (fM-aM) which is presently achieved only using laboratory techniques (ELISA, PCR). Though signal amplification through fluorescence has steadily gained traction to improve sensitivity by 1-2 orders of magnitude in comparison with standard colorimetric tests, the inherent autofluorescence of materials used to construct LFA test strips interferes with test performance and introduces variability. By enabling rapid, attomolar sensitivity, fluorescence-based LFAs could facilitate a revolutionary shift in the analytical capability of point-of-care tests to provide quantitative actionable data to inform health authorities to launch timely responses. The pathway to this development involves advancement of the fluorescence detection system and material properties of the reporters. This proposal aims to strategically address both of these directions by advancing a recently emerged LFA platform based on fluorescent 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 was recently demonstrated by Debina Diagnostics in detecting Ebola virus glycoprotein at the picograms level, using a custom-made optoelectronic reader (Axxin, Inc.) and off-the-shelf 200 nm FND produced by Adámas Nanotechnologies. The most striking attribute of FND envisioned to improve FND-based LFA sensitivity by 2-3 orders 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 an external magnetic field. Based on this unique property, the FND-related signal can be separated from background autofluorescence in the frequency domain through lock-in analysis which is widely used in signal processing to extract small periodic signals present below noise levels. Lock-in signal processing of FND can allow 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 this proposal, Adámas and Debina join their efforts to tailor FND material properties in order to eliminate non-specific retention 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 the lock-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).