CRISPR/Cas12a-mediated electrochemiluminescent biosensor integrating Ag modified Co-doped metal-organic frameworks for dual detection of malathion and phorate.

Abstract

With growing concerns about food safety, the detection of pesticide residues in food has become increasingly important. The mixed organophosphorus pesticides (OPs) formulations are commonly employed to maximize crop production; however, the excessive application of OPs has posed severe threats to food safety and human health. Therefore, there is an urgent need to develop rapid and sensitive analytical methods that could simultaneously detect the multiple OPs in food samples. Currently, studies on high-performance electrochemiluminescence (ECL) sensors for two or more target sensing have been rarely reported. This work reports a CRISPR/Cas12a-mediated ECL biosensor for the dual detection of two organophosphorus pesticides, i.e., malathion and phorate. The composite of metal-organic framework material (Co-PTC) loaded with silver nanoparticles (AgNPs@Co-PTC) serves as the single-signal probe, in which Co-PTC was the ECL emitter and AgNPs function as co-reaction accelerators to amplify ECL signals effectively. The method obtains an off-ECL signal by incubating the biosensor with malathion and DNA labelled with black hole quencher 1 (BHQ1-DNA); subsequently, after converting phorate to activator DNA, the sideloading activity of CRISPR/Cas12a was activated, which resulted in an on-ECL signal. By integrating spherical nucleic acid switching strategy with CRISPR/Cas12a for signal amplification, the method achieved the detection limits of 0.108 pM for malathion and 1.01 pM for phorate (S/N = 3), with satisfactory recovery rates of 95.7%-106.4% in food samples (cabbage and lettuce). The established dual-detection mode eliminates the need for multiple signal reporters, simplifying the detection procedure and effectively avoiding cross-interference. Accordingly, the CRISPR/Cas12a-based ECL biosensors featured high selectivity and stability and offered a novel analytical strategy for food safety monitoring.