Biosensors & bioelectronicsSep 20, 2025

Improved dual-method SARS-CoV-2 detection in wastewater using CRISPR and confined catalytic hairpin assembly

CRISPR Gene Editing Weekly Brief ↗PubMed ↗DOI ↗

Updated Jan 3, 2026

Abstract

The biosensing platform achieved detection limits of 100 copies/μL for SARS-CoV-2 RNA in wastewater samples.

A novel biosensing platform combined magnetic covalent organic framework-assisted catalytic hairpin assembly with CRISPR/Cas12a for detecting viruses.
The system achieved a 270-fold increase in local reactant concentration compared to traditional methods.
Detection kinetics were 20 minutes faster than solution-phase catalytic hairpin assembly.
The assay produced colorimetric and photothermal signals inversely correlated with enzyme activity upon target recognition.
Detection limits were 100 and 120 copies/μL for the colorimetric mode, and 100 and 140 copies/μL for the photothermal mode, specifically for S and N genes.

Simplified

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.

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Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

PubMed

Improved dual-method SARS-CoV-2 detection in wastewater using CRISPR and confined catalytic hairpin assembly

Abstract

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Funding

Competing interests

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Abstract

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