Fast and bioluminescent detection of antibiotic contaminants by on-demand transcription of RNA scaffold arrays

Cell-free biosensors have inspired low-cost and field-applicable methods to detect antibiotic contaminants. However, the satisfactory sensitivity of current cell-free biosensors is mostly achieved by sacrificing the rapidity, which prolongs turnaround time by hours. Additionally, the software-based result interpretation provides an obstacle for delivering these biosensors to untrained individuals. Here, we present a bioluminescence-based cell-free biosensor, termed enhanced Bioluminescence sensing of Ligand-Unleashed RNA Expression (eBLUE). The eBLUE leveraged antibiotic-responsive transcription factors to regulate the transcription of RNA arrays that can serve as scaffolds for reassembling and activating multiple luciferase fragments. This process converted target recognition into an amplified bioluminescence response, enabling smartphone-based quantification of tetracycline and erythromycin directly in milk within 15 min. Moreover, the detection threshold of eBLUE can be easily tuned according to the maximum residue limits (MRLs) established by government agencies. Owing to this tunable nature, the eBLUE was further repurposed as an on-demand semi-quantification platform, allowing for fast (∼20 min) and software-free identification of safe and MRL-exceeding milk samples only by glancing over the smartphone photographs. Overall, the sensitivity, rapidity and user-friendliness of eBLUE demonstrate its potentials for practical applications, especially in resource-limited and household settings.