Plasmon-enhanced Lateral Flow Assay for Multiplexed Detection of SARS-CoV-2 RNA and Antigens in Point-of-Care Settings

Lateral flow assays (LFAs) are amongst the simplest, fastest, and cheapest point-of-care (POC) and at-home biodiagnostic platforms and offer a broad potential for individualized and population-level screening for infectious diseases. Conventional LFAs rely on gold nanoparticles as labels to produce visually detectable color in the presence of the target biomarker. However, the sensitivity of LFAs relying on gold nanoparticles as labels is often insufficient to detect low concentrations of biomarkers, resulting in false negatives at the early stage of disease progression. This project seeks to explore the use of an ultrabright fluorescent label for improving the sensitivity of the LFA by more than 100-fold. The project also aims to design and build a personal handheld fluorimeter that will communicate wirelessly with a linked mobile device to measure the fluorescence signal from an LFA test strip. Harnessing the ultrabright nanolabel and the personal handheld fluorimeter, a novel diagnostic platform that simultaneously detects SARS-CoV-2 (the virus causing COVID-19) RNA and antigen is envisioned. Simultaneous detection of viral RNA and antigen can potentially improve diagnostic accuracy and accurately indicate the stage of illness. The project also involves the development of a new Nano-Biosensors Summer School activity intended to introduce middle school students to nanotechnology and biosensors, with the goal of stimulating interest in science/engineering while instilling confidence in pursuing these topics among under-represented groups. There is a pressing need for a new generation of biosensor technologies that encompass the combined characteristics of low-cost instrumentation, simple assay methods, high sensitivity, accurate quantitation, and rigorous validity. The ultimate goal of the project is to design and realize a multiplexed plasmon-enhanced fluorescence-based lateral flow assay (p-LFA) for the detection and quantification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen and RNA in resource-limited settings. Specific objectives include: (i) Design, realize and validate an ultrasensitive multiplexed p-LFA for simultaneous detection and quantification of SARS-CoV-2 RNA and antigen; (ii) Devise efficient sample processing methods for simultaneous detection of RNA and antigens in nasopharyngeal swabs and saliva from coronavirus disease 2019 (COVID-19) patients; and (iii) Build and test a smart phone-interfaced portable p-LFA reader for deploying the proposed biosensing technology in point-of-care (POC) and resource-limited settings. The project represents a transformative advance in that it seeks to establish a novel biodiagnostic platform with sensitivity exceeding that of the standard laboratory tests, a simple assay workflow compatible with self-testing at the POC and patient home, rapid sample-to-answer, and an inexpensive/portable instrument. If successful, the proposed amplification-free assay enables spatially-multiplexed detection and quantification of SARS-CoV-2 RNA and antigens on the same LFA strip, which shortens the sample-to-answer time and potentially differentiates individuals in the infectious stage of the illness from those who have passed it. Beyond COVID, simultaneous detection of RNA and antigen is broadly applicable for highly specific and sensitive diagnosis of various infectious diseases, including Ebola, Influenza, and Human Immunodeficiency Virus. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.