Diagnostics provide critical medical information but are too often unavailable to those in need or too complex to deploy outside of centralized labs. We develop low-cost portable diagnostics that can be used to detect pathogens in low-resource settings. These assays make use of cell-free in vitro transcription–translation systems that are stably embedded in paper substrates and couple them with rapid isothermal nucleic acid amplification reactions. These features enable the tests to be implemented with limited equipment and provide results visible to the naked eye. A critical component of these assays are engineered nucleic acid sensors that detect target DNA and RNA species with high sensitivity and high specificity. We have been working to deploy these tests against pathogens such as the Zika virus, norovirus, the Ebola virus, and HIV.
For detection of the Zika virus, we implemented a rapid development strategy that coupled computational toehold switch design with screening protocols that could be implemented in a matter of hours. This approach enabled us to develop an assay to detect clinically relevant concentrations of Zika RNA using isothermal RNA amplification and paper-based cell-free reactions hosting Zika-specific toehold switches. Since this work, we have integrated viral concentration schemes, improved riboregulators, and updated enzymes to improve sensitivity, specificity, and speed of these assays.
- A. A. Green, P. A. Silver, J. J. Collins & P. Yin, “Toehold Switches: De-Novo-Designed Regulators of Gene Expression,” Cell 159, 925-939 (2014).
- K. Pardee, A. A. Green, T. Ferrante, D. E. Cameron, A. DaleyKeyser, P. Yin & J. J. Collins, “Paper-based Synthetic Gene Networks,” Cell 159, 940-954 (2014).
- K. Pardee*, A. A. Green*, M. K. Takahashi*, D. Braff*, G. Lambert*, J. W. Lee, T. Ferrante, D. Ma, N. Donghia, M. Fan, N. M. Daringer, I. Bosch, D. M. Dudley, D. H. O’Connor, L. Gehrke & J. J. Collins†, “Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components,” Cell 165, 1255-1266 (2016).
- D. Ma, L. Shen, K. Wu, C. W. Diehnelt & A. A. Green†, “Low-Cost Detection of Norovirus Using Paper-Based Cell-Free Systems and Synbody-Based Viral Enrichment,” Synthetic Biology 3 (1), ysy018 (2018).