Recombinant Cell-Free Extracts for Biocatalytic Cascades: Fertilizers to Diagnostics

The ongoing COVID-19 pandemic has highlighted the need for timely, cost-effective, and accurate diagnostics. It has also revealed how shortages of plastic consumables, buffers, and PPE can limit traditional assays. Sample collection and centralized testing with RT-qPCR has dramatically improved the time to result, but it remains too slow to mitigate the spread of infection. To help address these issues, we have developed a frugal SARS-CoV-2 diagnostic enabled by cell-free synthetic biology circuitry coupled to a paper-based resonant sensor that can be safely and discretely scanned in a closed envelope and has the potential for at-home use and distributed contact-free testing. Lysed samples undergo isothermal reverse transcription recombinase polymerase amplification (RT-RPA) in a sample well which is placed onto a lyophilized, paper-based cell-free expression system. The cell-free system contains plasmids that harbor toehold switches selective to specific targets on the amplified RPA products. Complementary viral RNA amplicons complex with the toehold switches to activate translation of subtilisin, a strong protease reporter. The protease then degrades gelatin switches coated on the paper, which overlay a screen-printed paper-based resonant (LC) sensor. The sensor has a resonant frequency which shifts due to changes in permittivity caused by gelatin degradation. This clear frequency change can be measured rapidly through a closed envelope using an embedded portable vector network analyzer. We envision the technology can be deployed with autonomous scanners positioned at local drive-up lanes, hospitals, or workplaces and would be capable of signaling within 10 seconds if the closed sample envelope contains a positive or negative sample. This workflow of individualized administration followed by rapid, local scanning would eliminate the current problems of test site back-log, lack of PPE, and reagent scarcity and would be well-suited for deployment against a variety of existing and emerging pathogens for outbreak tracking and pandemic preparedness.