CRISPR-integrated nanoconfined interparticle catalytic hairpin assembly for enhanced dual-mode SARS-CoV-2 detection in wastewater

Accurate monitoring of pathogenic viruses in wastewater is critical for early outbreak and risk assessment. This study presented a novel biosensing platform that combined an interparticle magnetic covalent organic framework (MCOF)-assisted mismatched catalytic hairpin assembly (iMMCHA) with CRISPR/Cas12a-activated colorimetric-photothermal dual-mode detection of SARS-CoV-2 RNA. The system strategically immobilized CHA reactants (H1 and mismatched H2) on separate MCOF nanoparticles, creating a spatially confined and collision-enhanced interparticle MCHA that achieved 270-fold higher local reactant concentration and 20-min faster kinetics than solution-phase CHA. Upon target recognition, the iMMCHA system generated dsDNA activators that triggered Cas12a-mediated cleavage of ssDNA linkers on magnetic bead-glucose oxidase conjugates. This cleavage event reduced the TMB-oxidizing activity of the magnetically isolated integrated enzyme system, producing inversely correlated colorimetric and photothermal signals. This iMMCHA-CRISPR dual-mode assay allowed for the rapid and sensitive detection of SARS-CoV-2 pseudovirus in sanitary wastewater samples, with detection limits of 100 and 120 copies/μL (colorimetric mode) and 100 and 140 copies/μL (photothermal mode) for S and N genes, respectively. This work established a powerful platform for aqueous environmental virus monitoring that combined the specificity of CRISPR with the signal enhancement and kinetics acceleration of nanoconfined interparticle CHA and the reliability of dual-mode detection.