1. Towards detection of SARS-CoV-2 RNA in human saliva: A paper-based cell-free toehold switch biosensor with a visual bioluminescent output.
- Author
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Hunt, J. Porter, Zhao, Emily Long, Free, Tyler J., Soltani, Mehran, Warr, Chandler A., Benedict, Alex B., Takahashi, Melissa K., Griffitts, Joel S., Pitt, William G., and Bundy, Bradley C.
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SARS-CoV-2 , *COVID-19 pandemic , *SALIVA , *RNA , *BIOSENSORS , *COVID-19 , *LUCIFERASES - Abstract
Illustration of the assembly, distribution, and point-of-care use of a rapidly-deployable, cell-free COVID-19 biosensor: 1) Assemble: Assembling CFPS reagents by mixing E. coli lysate, murine RNase Inhibitor (mRI), energy sources, cofactors, and toehold switch riboregulator plasmid. 2) Print: aliquoting CFPS reagents onto paper substrates housed in a plastic test cassette. 3) Dehydrate: lyophilizing CFPS reagents on paper substrates. 4) Distribute. 5) Saliva sample: applying saliva samples onto cassette without pretreatments. 6) Reaction: bioluminescent protein expression in presence of target RNA (+), or ribosome detachment in absence of target RNA (-). 7) Visual result: bioluminescent output in the presence of target RNA and NanoLuc luciferase expression. [Display omitted] • Progress towards a rapid, low-cost, point-of-care Covid-19 biosensor. • Engineered toehold switches respond to saliva enriched with SARS-CoV-2 RNA. • mRI enables robust paper-based cell-free protein synthesis in the presence of saliva. • Plastic cassette houses paper-based diagnostic test for simple distribution and use. The COVID-19 pandemic has illustrated the global demand for rapid, low-cost, widely distributable and point-of-care nucleic acid diagnostic technologies. Such technologies could help disrupt transmission, sustain economies and preserve health and lives during widespread infection. In contrast, conventional nucleic acid diagnostic procedures require trained personnel, complex laboratories, expensive equipment, and protracted processing times. In this work, lyophilized cell-free protein synthesis (CFPS) and toehold switch riboregulators are employed to develop a promising paper-based nucleic acid diagnostic platform activated simply by the addition of saliva. First, to facilitate distribution and deployment, an economical paper support matrix is identified and a mass-producible test cassette designed with integral saliva sample receptacles. Next, CFPS is optimized in the presence of saliva using murine RNase inhibitor. Finally, original toehold switch riboregulators are engineered to express the bioluminescent reporter NanoLuc in response to SARS-CoV-2 RNA sequences present in saliva samples. The biosensor generates a visible signal in as few as seven minutes following administration of 15 μL saliva enriched with high concentrations of SARS-CoV-2 RNA sequences. The estimated cost of this test is less than 0.50 USD, which could make this platform readily accessible to both the developed and developing world. While additional research is needed to decrease the limit of detection, this work represents important progress toward developing a diagnostic technology that is rapid, low-cost, distributable and deployable at the point-of-care by a layperson. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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