An Easy-To-Use and Field-Deployable Cell-Free Bioreporter for Environmental Detection of Tetracyclines and Macrolides.

The widespread use of antibiotics in agriculture, livestock production, and clinical medicine has resulted in their continuous release into the environment, posing substantial ecological and public health risks. Although chromatographic approaches remain the gold standard for antibiotic detection due to their high accuracy and sensitivity, they require expensive instrumentation, labor-intensive workflows, and trained personnel, limiting their suitability for rapid or on-site monitoring. Here, we report a synthetic biology-based cell-free bioreporter that integrates antibiotic-responsive transcription factors, RNA polymerase, and engineered DNA transcription templates to generate a visible fluorescent readout upon exposure to tetracycline or macrolide antibiotics through an in vitro transcription reaction. This system eliminates biocontainment concerns inherent to whole-cell biosensors and demonstrates high sensitivity, specificity, and accuracy when applied to real water samples. In addition, the biosensor can be freeze-dried without substantial loss of activity, enabling long-term storage and convenient deployment outside laboratory settings. Together, these features establish a portable and robust assay for antibiotic monitoring in environmental applications.