Multiple gRNAs-assisted CRISPR/Cas12a-based portable aptasensor enabling glucometer readout for amplification-free and quantitative detection of malathion.

Background: The threat of toxic malathion residues to human health has always been a serious food safety issue. The CRISPR/Cas system represents an innovative detection technology for pesticide residues, but its application to malathion detection has not been reported yet. In addition, the multiple-guide RNA (gRNA) powered-CRISPR/Cas biosensor has the advantages of being fast, sensitive and does not require pre-amplification. However, the reported multiple-gRNA CRISPR/Cas-based biosensors are largely only used for the detection of nucleic acid targets, and there are still certain challenges in detecting non-nucleic acid targets.

Results: In this work, a multiplex-gRNA-assisted CRISPR/Cas12a-based portable aptasensor (MgCPA) is developed for amplification-free and quantitative detection of malathion using a glucometer. When target malathion is present in the MgCPA strategy, it specifically binds with aptamer and then activates the trans-cleavage activity of the multiplex-gRNA CRISPR/Cas12a. The activated multiple Cas12a/gRNA complexes cut invertase-HP probes on the electrode surface to obtain glucose signals with glucometer assistance. Under optimal conditions, the developed MgCPA strategy achieves satisfactory portable quantitative and sensitive detection of malathion down to 300 fM (S/N = 3) without pre-amplification. Moreover, the satisfactory selectivity, high reproducibility, and good stability of the proposed strategy are also obtained. Due to its excellent and robust shelf life, our developed MgCPA strategy can be practically applied in detecting malathion in orange, apple, cabbage, and spinach samples.

Significance: Amplification-free, sensitive, portable quantitative and selective detection of malathion in food samples is achieved by employing our developed MgCPA strategy. This strategy not only opens up a new path for the non-nucleic-acid target detection using amplification-free methods based on multiple-gRNA-assisted CRISPR/Cas12a, but also has broad application prospects in ensuring food safety.