Self-validated SARS-CoV-2 detection via an automated DNA molecular machine-regulated ECL/SERS signal switching...

Rapid and ultrasensitive detection of SARS-CoV-2 nucleic acid residues in environmental and cold-chain scenarios is of great importance for early warning and transmission control. Herein, we report a dual-mode electrochemiluminescence (ECL) and surface-enhanced Raman scattering (SERS) biosensor based on a COF/CsPbBr₃/Au nanocomposite integrated with an inverted DNA tetrahedral molecular machine for highly reliable detection of the SARS-CoV-2 RdRp gene. The porous covalent organic framework (COF) provides a robust scaffold for in situ growth of CsPbBr₃ perovskite nanocrystals, endowing strong ECL activity, while uniformly anchored Au nanoparticles serve as efficient SERS hotspots and anchoring sites for thiolated DNA probes. Upon target recognition, strand displacement and Nt.BbvCI-assisted cleavage trigger the release of ferrocene tags, resulting in a synchronized enhancement of ECL signal and attenuation of SERS response, enabling ratiometric self-validation. The proposed biosensor exhibits a wide linear range from 0.01 fM to 1 pM, with an ultralow detection limit down to the attomolar level (3.2 aM). Excellent selectivity, reproducibility, and stability are demonstrated, even under complex environmental conditions and cold-chain storage temperatures. Importantly, the sensor enables accurate detection of RdRp-Covid spiked on food packaging materials and pork samples, highlighting its applicability for environmental surveillance and cold-chain monitoring. This work presents a robust dual-mode biosensing platform that combines signal amplification, anti-interference capability, and built-in cross-validation, offering a promising strategy for on-site environmental monitoring of viral contamination.