Interfacial tripedal DNA walking coupled with oxygen-depletion/ECL-RET dual quenching for femtomolar detection of Tobacco Mosaic Virus RNA in plant extracts.

Sensitive quantification of Tobacco Mosaic Virus RNA (TRNA) is imperative for preventing large-scale agricultural losses. In this study, we engineered a sophisticated quenching-based electrochemiluminescence (ECL) biosensor by integrating a high-performance CdTe@ZnS nanoscaffold with a target-triggered tripedal DNA walker. To ensure a robust and stable initial luminophore density, multi-walled carbon nanotubes (MWCNTs) were utilized to anchor CdTe@ZnS core-shell quantum dots, where the ZnS shell effectively passivates surface defects and prevents cadmium leakage. Upon recognition of TRNA, a catalytic hairpin assembly (CHA) and toehold-mediated strand displacement (TMSD) cascade are initiated, driving the tripedal DNA walker to autonomously navigate the electrode surface. This movement recruits a multitude of Cu2O nanoparticles (quenchers) to the sensing interface, which efficiently suppresses the ECL emission through the consumption of the co-reactant (O2) and dual-pathway quenching. Benefiting from the synergistic amplification of the MWCNT carrier and the high walking efficiency of the multivalent DNA machine, the biosensor achieved a broad linear range from 80 fM to 100 nM, with a remarkable detection limit of 9.18 fM. The successful discrimination of TRNA from mismatched sequences and its performance in complex matrices underscore the potential of this platform for early-stage phytopathogen diagnostics.