Duplex DNase-powered RNA walker for multiplex fluorescent detection of trace pesticide residues in tea samples

The bipyridine herbicide paraquat (PQ) and the benzimidazole fungicide carbendazim (CBZ) play a key role in weed and fungus control on farmlands to elevate crop output. Yet, excessive deployment of these chemicals risks leaving residues in the food chain, constituting a grave danger to public health. The presence of multiple pesticide residues often elevates the risk of severe toxicity. Since existing detection techniques mostly target single pesticide residue and cannot fully assess the toxic impacts of both, developing new methods capable of simultaneously detecting PQ and CBZ residues is highly desirable.

To address this, we present a Duplex DNase-assisted RNA walker for multiplex fluorescent detection of PQ and CBZ. High-affinity binding of targets to their aptamers displaces ssDNA from magnetic beads. The released ssDNA hybridizes with free T7 promoter templates to reconstitute split promoter sequences. Subsequently, T7 RNA polymerase-driven transcription amplifies short RNA strands, which hybridize with fluorophore-labeled reporter DNA on gold nanoparticles to form heteroduplexes. Duplex-specific DNase selectively cleaves the DNA within these heteroduplexes, releasing fluorophores and recycling RNA to achieve an efficient walking mechanism. This platform enables highly sensitive detection with limits of 2.52 nM for PQ and 3.50 nM for CBZ, alongside a broad linear range (3.89 nM ∼ 7.78 μM for PQ, and 5.23 nM ∼ 10.46 μM for CBZ). It demonstrates good selectivity against common interferents and successfully achieves accurate simultaneous detection of PQ and CBZ in tea samples.

The synergistic combination of Duplex DNase and T7 RNA polymerase-driven transcription significantly enhances the system's sensitivity and accuracy through efficient signal amplification. Consequently, this developed approach serves as a robust tool for environmental monitoring and food safety evaluation. It holds great promise for safeguarding ecosystem integrity and public health by facilitating the precise and simultaneous detection of hazardous pesticide residues.