New Rolling Circle Transcription Based on Allosteric Transcription Factors (aTFs) for Trace Detection of Water Contaminants.

Antibiotic and heavy metal contamination in water poses a severe global public health risk, yet conventional detection methods remain constrained by reliance on complex laboratory instrumentation. Although cell-free biosensors utilizing allosteric transcription factors (aTFs) offer promising alternatives, their limited sensitivity impedes reliable detection of small-molecule contaminants. To address this limitation, we developed allosteric transcription factors (aTFs)-regulated rolling circle transcription assay (ARCTA) for ultrasensitive detection of water contaminants. The ARCTA system employs a circular DNA template comprising a double-stranded region with a T7 promoter and aTFs binding sequence, alongside a single-stranded domain transcribable into fluorescent RNA. Target-specific recognition by aTFs enables rapid, efficient transcription and amplification of fluorescent RNA, achieving an exceptionally low detection threshold. Applied to tetracycline, oxytetracycline, erythromycin, clarithromycin, Hg2+, Pb2+, Cd2+, and As3+ in water samples, ARCTA demonstrated detection limits of 1.25 nM, 0.93 nM, 0.28 nM, 0.45 nM, 0.034 nM, 0.006 nM, 0.13 nM, and 0.016 nM, respectively, and all had a broad dynamic ranges.Notably, ARCTA by designing distinct circular DNA templates to transcribe wavelength-specific fluorescent RNAs, enabling the simultaneous detection of multiple targets. In addition, the system also exhibited robust performance in real-world applications, underscoring its practicality. These results highlight ARCTA's potential as a transformative tool for ultrasensitive monitoring of diverse small-molecule contaminants.