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

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.