Cell-Free Biosensing for Environmental and Performance Monitoring

Cell-free expression systems are an exciting platform for the detection of environmental hazards and human performance biomarkers. Once lyophilized onto paper, cell-free biosensors can be transported to austere locations and rehydrated onsite for field sensing applications. Here, we show the development of a cell-free sensor that detects bile acids in both environmental samples and human biofluids. Secondary bile acids, including deoxycholic acid (DCA) and lithocholic acid (LCA), are indicators of fecal pollution in water and biomarkers for certain disease states. Importantly, a cell-free bile acid sensor could potentially detect fecal contamination in water in less than two hours, an improvement over traditional approaches with fecal coliform cultures that require greater than 24 hours of incubation. Our bile acid sensor uses BreR, a TetR-like repressor from Vibrio cholorae, and was successfully transferred from whole cells to cell-free with an approximately ten-fold increase in DCA sensitivity. Further characterization of the sensor in cell-free showed a strong response to several secondary bile acids. Therefore, we next explored the ability of this sensor to detect bile acids in human feces and wastewater samples. To further broaden the application space of our sensor, we also established strategies to mitigate the inhibitory effects of human blood serum on the functionality of the sensor for biomarker detection in blood. Ongoing work includes optimization of the sensor in a lyophilized paper-based assay. Additionally, we have shown that several biosensors can be transferred from whole cells to cell-free systems with an improvement in analyte sensitivity, this seems to be a common feature we are currently exploring. In summary, our bile acid sensor highlights the utility of cell-free systems for rapid onsite sensing of biomarkers and analytes for a variety of different applications.