Rapid Isothermal Detection of Heavy Metals via Transcription-Factor-Gated DNA Strand Synthesis

Conventional methods for monitoring toxic heavy metals typically require sophisticated laboratory instrumentation and leave a critical gap for rapid, on-site detection. Herein we present Transcription-factor-Occluded Nick Extension or TONE, a rapid, isothermal biosensing platform for heavy metal detection that exploits allosteric transcription factor (aTF) regulation to gate DNA strand-displacement amplification. In this approach, operator sequences modified with deoxyinosine (dI) substitutions are employed. When bound by an aTF, the dI sites are shielded from cleavage by endonuclease V. Upon exposure to target heavy metals, the aTF dissociates, permitting the enzyme to nick the DNA and trigger a strand-displacement reaction. The amplified DNA is then detected via an instrument-free lateral flow assay, delivering a visual readout within 25 min at room temperature. We demonstrate the utility of TONE using the TetR aTF and further adapt the assay for copper and lead detection through the transcription factors CsoR and CadC, respectively, reliably detecting concentrations as low as 40 and 80 nM, respectively. TONE offers a sensitive, instrument-free assay that operates at room temperature, with the potential for field deployment, for use in environmental monitoring and other applications requiring rapid heavy metal analysis.