Point-of-care (POC) testing involves the rapid detection of analytes near patients and enables quick medical decisions that can improve disease diagnosis, monitoring, and management. Cell free systems have been particularly successful as POC diagnostics by utilizing gene-circuit based sensors outside the physiological constraints of cells, which accelerates the product development cycle and enables easy storage, distribution, and use. However, most demonstrations to date rely on fluorescent reporter proteins which require multi-well plate reader assays, leaving the technology for all practical purposes stuck in the laboratory. In line with this promise of cell free gene-circuit sensors, we have developed a cheap and accessible detection method at the University of Toronto.
Our inventors have demonstrated the use of a glucose meter as a convenient and universal reader for cell free gene circuit sensors. The overall system can be housed in a portable container and includes several capabilities (Fig 1). High temperature sample lysis exposes cellular or viral RNA. Nucleic Acid Sequence-Based Amplification (NASBA) or other isothermal amplification methods enable highly sensitive detection of genetic material indicative of disease without temperature cycling. Recognition of the diagnostic markers by a gene circuit leads to the production of reporter proteins that convert various substrates to glucose. Finally, the relative concentration of glucose, measured by an off-the-shelf meter, is interpreted using a purpose-built smartphone web application to relay the presence or absence of a disease.
Figure 1. Workflow of a portable cell free gene-circuit sensor with a glucose meter readout.
- Low-cost, point-of-care diagnostic for cell free gene circuit sensors
- Sensitive and selective detection capabilities
- Low attomolar (10-18 M) range detection of typhoid and COVID-19 RNAs
- Selective CoV-2 identification compared to similar viruses
- Established infrastructure - global network of glucose meter manufacturing and consumable distribution
- Simple or multiplexed diagnosis
- Diverse analyte detection
- Bacteria, virus, small molecule, mRNA
INTELLECTUAL PROPERTY STATUS
Proof-of-concept studies have demonstrated the ability to detect infectious diseases caused by both bacterial (typhoid, paratyphoid A/B; Fig 2) and viral (SARS-CoV-2) species, in addition to small molecule analytes and drug resistance genes. A portable prototype has been built and tested for complete point-of-care sample processing.
Figure 2. Typhoid detection by use of a glucose meter in concert with a cell free gene circuit system. Elevated glucose levels are indicative of the causative source of typhoid, Salmonella typhi (S. typhi). Similarly strong results were achieved for SARS-CoV-2 detection.