A MnO2 nanosheet-mediated CRISPR/Cas12a system for the detection of organophosphorus pesticides in environmental water

Abstract

Nowadays, easy, convenient, and sensitive sensing strategies are still critical for organophosphorus pesticides in environmental water samples. Herein, a novel organophosphorus pesticide (OP) assay based on acetylcholinesterase (AChE) and a MnO₂ nanosheet-mediated CRISPR/Cas12a reaction is reported. The single-strand DNA (ssDNA) activator of CRISPR/Cas12a was simply adsorbed on the MnO₂ nanosheets as the nanoswitches of the assay. In the absence of target OPs, AChE hydrolyzed acetylcholine (ATCh) to thiocholine (TCh), which reduced the MnO₂ nanosheets to Mn²⁺, resulting in the release of the activator followed by activation of the CRISPR/Cas12a system. The activated Cas12a thereafter nonspecifically cleaved the FAM/BHQ1-labeled ssDNA (FQ-reporter), producing a fluorescence signal. Upon the addition of target OPs, the hydrolysis of ATCh by AChE was inhibited owing to OPs combining with AChE, and thus effective quantification of OPs could be achieved by measuring the fluorescence changes of the system. As a proof of concept, dichlorvos (DDVP) was chosen as a model OP analyte to address the feasibility of the proposed method. Attributed to the excellent trans-cleavage activity of Cas12a, the fluorescent biosensor exhibits a satisfactory limit of detection (LOD) for DDVP at 0.135 ng mL⁻¹. In addition, the excellent recoveries for the detection of DDVP in environmental water samples demonstrate the applicability of the proposed assay in real sample research.