Abstract

Affinity Selection and Enumeration of SARS-CoV-2 Viral Particles from Saliva Samples using Integrated and Modular Microfluidic Systems for COVID-19 Diagnostics

Steve Soper, Foundation Distinguished Professor; Director, Center of BioModular Multi-scale System for Precision Medicine, Adjunct Professor, Ulsan National Institute of Science & Technology, The University of Kansas

Coronavirus disease 2019 (COVID-19) arises from the SARS-CoV-2 virus and has been found to be highly contagious. To mitigate spreading, testing has been deemed an important asset. Testing has predominately utilized RT-qPCR as well as serological-based tests. However, while new machines are rolling out for point-of-care testing (POCT), issues are present with these common testing systems, for example the need for reagents (e.g. enzymes, fluorescent reporters, antibodies), workflows that sometimes require specialized operators, and the inability to distinguish between infectious and non-infectious individuals, which is important in determining the need for quarantining. We report an innovative COVID-19 diagnostic test that directly addresses the aforementioned issues. The assay accepts a clinical sample and specifically selects SARS-CoV-2 particles from the sample using surface-immobilized DNA aptamers targeting the spike protein, releases photolytically the selected viral particles (VPs), and then counts the number of SARS-CoV-2 particles using a label-free approach. The workflow is simple and fully automated and also, no reagents are required once the assay is deployed for testing. The entire assay was carried out using microfluidic chips made from a plastic that were injection molded to allow for high scale production at low cost. The VP selection chip consisted of 1.5 million pillars that allowed for affinity loading up to 1010 SARS-CoV-2 particles at a recovery ~90%. Following selection, the VPs were released from the capture surface using a photocleavable linker by a blue-light LED (79% release efficiency) and subsequently counted using a nano-Coulter Counter (nCC). For high throughput single VP counting, 5 nCCs were placed in parallel and offered 100% detection efficiency for VPs travelling through a 200 nm pore. These two chips were interconnected through the use of a fluidic motherboard, also made from a plastic; the integrated system allowed for complete sample processing automation. The system’s valving employed plastic membranes made from an elastomeric COC. The entire assay could be completed in <20 min. In a 20 patient blinded study, the test correctly identified 10 non-infected individuals (clinical specificity = 100%) and in 5 COVID-19 patients, VPs were detected indicative of “active” disease, while 4/5 others were deemed infected by RT-qPCR, but those individuals had no VPs suggesting these patients were not contagious (clinical sensitivity = 95%).