A ratiometric dual-fluorescent paper-based synthetic biosensor for visual detection of tetracycline on-site.

The excessive use of tetracycline poses a threat to human health, making it essential to monitor and regulate its usage. While whole-cell biosensors offer a simple and cost-effective method, their utility is constrained by limitations in sensitivity, portability, and robustness, hindering real-time measurements within complex environmental contexts. In this study, a ratiometric i/cTetR synthetic biosensing test strip with an engineered modified dual-fluorescence reporting was developed for detecting Tet antibiotics in water and food. First, the standardized unidirectional promoter PtetR by tailoring and screening TetR transcription factor binding sites and verified by molecular docking, shortening the detection time. Secondly, decoupling the sensing and reporting modules enhances the biosensor's performance, eliminating genetic background leakage and tripling the output signal. Thirdly, a ratiometric dual fluorescence signal i/cTetR biosensing test strip was designed. Under the light box LED/UV light source, the dual signal output method significantly reduced false negative results and enhanced the anti-interference capability of the biosensor. The i/cTetR strips can detect Tet in tap water (5-1280 μg/mL) and milk (50-3200 μg/kg) within 45 min in high volume on-site without separation and purification. This study provides a standardized and universal sensing method for the field detection of antibiotic contaminants.