Versatile and tunable dual-layer signal amplifier enables ultrasensitive cellular sensors for chemicals

Whole-cell biosensing is a compelling technology in in situ detection of pollutants, toxins, valuable products and biomarkers. However, these systems often suffer from insufficient sensitivity and signal output required for detecting trace-level targets. To solve this problem, we developed a versatile protease-activated transcription (PAT) signal amplification system aimed to one-step and generally optimize the detecting sensitivity of broad types of biosensors. Through rational modulating the degradation tag-assistant background control and IPTG mediated-signal gain programs, the PAT system was quickly integrated with four exemplary biosensing circuits targeting metal and organic compounds, improving their signal output by 7-37-fold and detection limits by 5-502-fold, which achieved or approached the lowest detection values reported in extensively engineered biosensors. Notably, the detection limits of natural arsenic and mercury sensors were one-step improved to 2 nM and 1.4 nM, respectively, both well below the WHO/EPA safety thresholds of 10 nM and 134 nM. These results demonstrated the great potential of the PAT-amplified approach for rapid optimization of target sensing pathways to meet application-required sensitivity. Overall, PAT signal amplification technology is highly efficient and adaptable, which can be further extended to other intracellular reactions that require signal amplification.