An oxidative cleavage-based HCR-CRISPR/Cas12a biosensor for highly sensitive detection of hypochlorous acid

Abstract

Hypochlorous acid (HClO) is a critical disinfectant in public health and water treatment, yet its excessive presence in environmental matrices poses significant risks to human health (e.g., respiratory irritation, tissue damage) and aquatic ecosystems (e.g., biodiversity loss). Herein, we developed an oxidative cleavage-based hybridization chain reaction (HCR)-CRISPR/Cas12a biosensor for highly sensitive detection of HClO. The sensor utilizes a dual-lock switch mechanism: HClO selectively cleaves a phosphorothioated (PS) DNA hairpin (Lock 1), releasing an initiator strand to trigger the HCR and generate PAM-containing long dsDNA. Simultaneously, the locked crRNA (L-crRNA), which is caged by a PS-modified silent chain (Lock 2), can be released when the PS-modified silent chain is degraded under HClO incubation, allowing the activated crRNA to dynamically regulate Cas12a–crRNA complex formation. Then, HCR-dsDNA activates Cas12a's trans-cleavage activity, enabling fluorescence signal readout via reporter DNA cleavage. The dual-lock strategy minimizes nonspecific activation in CRISPR/Cas12a systems, significantly enhancing sensitivity and specificity. Our work establishes a robust platform for environmental pollutant monitoring, with applications in water safety assessment and food quality control.