Fluorescence Sensing Technology For Rapid Detection Of OTA And AFB1 Toxins Based On CRISPR-Cas12a

The food safety problem caused by mycotoxins has become the focus of attention all over the world.Among them,Ochratoxin A（OTA）and Aflatoxin B1（AFB1）are particularly serious pollution problems,which have teratogenic,nephrotoxic,carcinogenic,hepatotoxic,mutagenic and immunosuppressive effects on animals and humans.CRISPR system is a unique immune defense mechanism of bacteria,which can effectively fragment and degrade exogenous nucleic acid molecules specifically when combined with the corresponding Cas protein.CRISPR-Cas12a has unique targeting and accessory cutting activity,which provides a new idea for the development of biosensors targeting nucleic acids as substrates.Therefore,this study intends to use OTA and AFB1 as the detection targets to construct a CRISPR-Cas12a-mediated biosensing analysis technology.The research results obtained are as follows:1.CRISPR-Cas12a-mediated dual signal amplification upconversion sensing technology for sensitive detection of OTA.Firstly,upconversion nanoparticles（UCNP）were successfully prepared by solvothermal method,based on which upconversion nano-magnetic particle probes（UCNP-DNA-Fe₃O₄）were constructed to expand the selectivity of CRISPR-Cas12a cleavage substrates.Secondly,the OTA aptamer is used as the signal conversion element to construct a dsDNA（OTA probe）containing the aptamer sequence.When OTA is present in the detection system,the OTA aptamer competes with the OTA toxin to release the OTA aptamer.The complementary strand（Activator strain）,the Activator strain is subsequently recognized by the CRISPR-Cas12a complex and elicits the ability of CRISPR-Cas12a to degrade any ss DNA.In this chapter,traditional fluorescent probes（6’FAM-TTATT-BHQ1,FQ）and UCNP-DNA-Fe₃O₄ probes were used as substrates for Cas12a,respectively.The experimental results show that the content of OTA toxin is proportional to Activator strain and fluorescence value.The method based on the linear range of UCNP-DNA-Fe₃O₄ probe was 5～100 ng/mL,and the detection limit was 0.83 ng/mL;based on the linear range of FQ probe,it was 5～80 ng/mL,and the detection limit was 1.56 ng/mL.The UCNP-DNA-Fe₃O₄ probe established in this paper makes up for the defect that traditional FQ probes cannot be applied in complex environments.This method can realize the detection of OTA in actual corn flour samples,which provides a certain reference for the detection of mycotoxins based on CRISPR-Cas12a biosensing analysis technology.2.CRISPR-Cas12a-mediated universal biosensing technology for sensitive detection of OTA and AFB1.Here,in this chapter,a dsDNA containing PAM（Protospacer Adjacent Motif）sequence was designed to specifically activate CRISPR-Cas12a to trigger trans-cleavage.On this basis,magnetic nanoparticles were introduced,and a general biosensing platform（MB-ss DNA-OTA（or AFB1）aptamer-dsDNA）was constructed for different aptamers.When there is no target toxin in the reaction system,dsDNA is separated by magnetic separation technology,and the fluorescence does not change after adding the reporter solution（CRISPR-Cas12a-FQ）.After adding the corresponding single toxin,the specific binding of the toxin and the aptamer leads to the change of the spatial structure of the aptamer to release the dsDNA,and the supernatant containing the dsDNA after magnetic separation initiates CRISPR-Cas12a to initiate trans cleavage.The experimental results show that the detection range of this method for OTA is 0.01～100 ng/mL,and the detection limit is 0.03 ng/mL;the range of AFB1 is 0.01～50 ng/mL,and the detection limit is 0.05 ng/mL.This method can realize the detection of OTA and AFB1 in actual corn flour samples,and the recovery rate is97.20%～109.00%.Compared with the previous experiment,This chapter establishes a general biosensing technique,which overcomes the limitations and lower detection limit of CRISPR-Cas12a biosensing analysis technology in single target detection,making it suitable for food safety,and provides a solution for multi-target detection in the field of food safety.