Stage-aware quantification of the SARS-CoV-2 3CLpro biomarker via CsPbBr3@COF-LZU1@AuNP electrochemiluminescence and Cas13a amplification.

Direct, activity-based quantification of the SARS-CoV-2 main protease (3CLpro) remains challenging in complex matrices. Here we report a water-compatible electrochemiluminescent (ECL) biosensor that integrates a CsPbBr3@COF-LZU1 emitter, a peptide-DNA conformational switch, and CRISPR/Cas13a-assisted amplification to convert protease activity into a robust optical "turn-on" signal. The covalent organic framework physically stabilizes perovskite nanocrystals in aqueous media and, together with a sparse Au nanoparticle layer, supports assembly of ferrocene-terminated reporters that impose an ultralow baseline via near-field/redox quenching. Target-specific cleavage unlocks an initiator that drives entropy-mediated T7 promoter formation and transcription, producing RNA activators that switch on Cas13a collateral cleavage; removal or distancing of ferrocene from the emitter restores photon output. Under optimized conditions the sensor exhibits a broad log-linear response from 10 to 108 aM with an ultralow detection limit of 4.31 aM, high analytical selectivity against common interferents, tight fabrication-to-fabrication precision (inter-electrode RSD ∼3%), and practical robustness (≈97% signal retention over 12 h, ≥90% after 7 days at 4 °C, and ∼92% after 120 ECL cycles). Stepwise ECL, cyclic voltammetry, and impedance analysis confirm layer-by-layer assembly and the intended mechanism of ferrocene-mediated quenching and Cas13a-driven recovery. Applied directly to minimally processed pharyngeal swab eluates, the platform resolves cohort-level differences across disease stages and captures the expected attenuation of 3CLpro activity in late-stage specimens, supporting stage-aware quantification in real clinical samples. The modular design-reprogrammable at the protease-cleavable motif, promoter template, and crRNA-points to a general route for sensitive, selective, and water-stable ECL assays of enzymatic activity with translational potential.