Mitigating the deleterious impact of saliva on cell-free expression sensors for point-of-use diagnostics

Point-of-use sensors that detect biomarkers in noninvasive biofluids, such as saliva, could provide individuals with valuable insights into their health and performance status at home or in the field. Cell-free biosensors have the potential to meet these requirements, as these sensors can detect a wide variety of analytes and can be stored at room temperature after freeze-drying. However, saliva inhibits the function of cell-free biosensors, in part due to the presence of interfering proteins and nucleases. Here we show simple strategies to mitigate the inhibitory effects of saliva on the function of cell-free expression systems, including the use of nuclease inhibitors and pretreatment of saliva with centrifugal filtration or heat. We also show successful implementation of our mitigation strategies to detect cortisol spiked into pooled human saliva using a cell-free biosensor. Combined, our results show that simple approaches can be used to enable the function of cell-free biosensors in saliva.