Lateral flow cell-free transcriptional assay for contaminant detection

Cell-free transcriptional biosensors are emerging as a powerful technology, offering enhanced capabilities for detecting chemical contaminants in settings where traditional analytical techniques fall short of societal needs. However, the sensitivity of these biosensors for many critical chemical contaminants often remains insufficient to meet regulatory detection limits, while maintaining portability and ensuring selectivity. In this work, an in vitro transcription (IVT)-based lateral flow assay (LFA) is reported, which is 1,000 times more sensitive than reported IVT portable detection methods. Through combining the amplification power of IVT with nucleic acid LFA-based avidity effects, we develop rapid, ultrasensitive turn-on sensors. Our sensors meet regulatory detection limits for lead, a particularly challenging metal ion contaminant, in untreated spring water samples collected from a basin that exemplifies the environmental monitoring challenges. Additionally, we show that by optimizing the IVT conditions, we can tune the limit of detection to produce an on-off signal reaching the WHO regulatory threshold and others. Finally, we evaluate the selectivity of different metal-responsive repressors in the context of in vitro biosensing and discuss how this platform can identify potentially hazardous samples with an easy-to-use and point-of-care assay.