RAPID: COVID-19 diagnostics for limited resource settings via improved sample preparation

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COVID-19 continues to be a global challenge. A critical aspect of the disease is that up to half of all people infected with the SARS-CoV-2 virus are asymptomatic, rendering screening and containment strategies based solely on clinical presentation impossible. Society cannot fully return to work and school unless both symptomatic and asymptomatic infected individuals can be screened regularly. There is therefore an urgent need for a universally accessible, rapid point-of-care (POC) diagnostic with accurate, reliable results that can be deployed in an affordable way on an unprecedented global scale. However, the diagnostics field has struggled for decades to make these complex tests compatible with POC settings. Great progress has been made developing nucleic-acid amplification assays that are ultrasensitive and rapid; yet the core technology for the initial RNA-extraction step remains largely unimproved since 1990. The RNA extraction step is the key bottleneck to developing a globally deployable COVID-19 diagnostic. This project will bring advances in interfacial engineering to COVID-19 diagnostic technology to decentralize molecular diagnostics from the laboratory, which is needed to reopen the US economy and to protect the vulnerable members of society. The ultimate impact of this project will be to improve the performance and availability of SARS-CoV-2 RNA testing so that these tests can be run at the POC by minimally trained users. There are two goals: (1) reduce the logistical burden associated with relying on supply-chains and centralized labs and (2) simplify the RNA extraction step to eliminate the need for complex equipment. Standard RNA extraction follows a complex, multi-step protocol based on solid-phase extraction (SPE). The protocol requires centrifugation, and it suffers from lowered assay performance due to the carryover of inhibitory buffers. This project will directly address both bottlenecks associated with RNA extraction by integrating an innovative approach: a two-phase wash (TPW) that reduces inhibitors while maintaining the RNA yield during the extraction step. The TPW technology integrates a wash buffer immiscible with water. TPW removes contaminants from the extraction column by leveraging the combination of solid-liquid and liquid-liquid interfacial properties and solubility of the inhibitory components. Extra purity obtained via TPW will improve assay sensitivity and reduce cost and will enable the use of lyophilized reagents and isothermal amplification, eliminating refrigeration requirements and reducing testing time (from hours to minutes). Finally, using TPW with a pressure-based RNA-extraction technology will eliminate the need for centrifugation and will improve the speed and accessibility of RNA extraction. Additionally, this project will leverage interfacial engineering via TPW to develop sample-preparation modules that can be used as stand-alone components and combined (plug-and-play) with other state-of-the-art amplification and readout technologies, such as those designed by industrial collaborators and other sensing/detection technologies currently under development in the RAPID program. The technologies developed in this project can be immediately adopted by commercial and pre-commercial diagnostic manufacturers.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.