A label-free fluorescence sensor for OTA detection based on enzymatic recycling -mediated precise regulation of DNA-templated silver nanocluster

Ochratoxin A (OTA) is a widespread mycotoxin in food chains, yet conventional aptamer-based assays remain challenging because of the background signals and matrix interference. Here, we introduced a target-triggered reporter synthesis mechanism in which OTA binding unlocks a TdT- Nt.AlwI enzymatic cycle that generates polyT strands exclusively in the presence of the target. These generated polyT products interact specifically with polyA-recognition motifs on DNA-templated silver nanoclusters (DNA-AgNCs), enabling fluorescence transduction for OTA detection. This “zero-to-one” generative logic gate ensures that signal molecules are produced exclusively after recognition, intrinsically suppressing blank background. Moreover, the enzymatic cycle allows a single OTA molecule to trigger the generation of multiple polyT strands, imparting signal amplification without external labels or immobilization. Furthermore, integrating target-initiated nucleic acid synthesis with orthogonal optical transduction enables the construction of a label-free assay that intrinsically decreases background noise from signal generation, thereby ensuring high specificity, low interference, and quantitative reliability for target detection. The assay shows a linear response from 10 pM to 50 nM, achieves a detection limit of 0.097 ng mL −1 , and yields 86.4–94.1 % recoveries with<8.1 % RSD in spiked soybean extracts. By suppressing baseline noise at the mechanism level and separating amplification from readout, this platform enables sensitive, low false-positive OTA quantification and is readily adaptable to other small molecules by exchanging the aptamer, advancing portable food-safety screening.