New Methods For Rapid Detection Of Mycotoxins In Food Based On DNA Nanotechnology

Mycotoxins are a class of toxic secondary metabolites produced by fungi,which have serious teratogenic,carcinogenic and mutagenic effects on humans and animals.Mycotoxins can contaminate any part of the "farm-to-table" process,with many susceptible sources and great difficulty in prevention and control.The existing detection methods achieve accurate detection of mycotoxins,however,these methods are limited by the expensive instrument and complex operation,resulting the susceptible result of interference.Therefore,the construction of new method for rapid and sensitive detection of mycotoxins is important to prevent mycotoxin-contaminated products from entering the foodchain and causing human health hazards.Because of its excellent biocompatibility,high programmability and ease of modification,DNA nanotechnology,which used DNA molecules as unit,is playing an increasingly important role in environmental monitoring,food safety,drug analysis and disease diagnosis.In this thesis,considering the actual status of the problem of mycotoxin detection in food,we constructed mycotoxin detection methods for the detection and analysis of mycotoxins in agricultural products by combining functional nucleic acid and isothermal amplification technologies with portable spectroscopic instruments as signal acquisition means.The main research works are as follows:(1)A dual enzyme cascade amplification-mediated homogeneous fluorescent biosensing method was developed for the detection of ochratoxin A(OTA)in soybean.In this strategy,the aptamer acts as a recognition unit,recognizing the OTA and inducing the release of the complementary chain.The exposed 3’-OH end(complementary strand)is continuously cycled to generate and release free poly thymidine(Poly T)strands by the synergistic action of terminal deoxynucleo tidyltransferase(TdT)and endonuclease Nt.AlwI.The DNA-AgNCs were used as fluorescent tags,and the generated free Poly T could induce structural changes of the DNA-AgNCs to reduce the fluorescence intensity when the target OTA was present,thus achieving a highly sensitive fluorescence detection of OTA with a low detection limit of 0.097 ng mL-1.This strategy was successfully used for the detection of OTA in soybean samples.(2)An aptamer recognition-triggered enzyme-free catalytic hairpin assembly(CHA)-assisted signal amplification by naked-eye and surfaceenhanced Raman scattering(SERS)dual-mode biosensing strategy was developed for the rapid and sensitive detection of aflatoxin B1(AFB1)in wheat samples.In this study,AFB1 aptamer was used as the recognition unit for signal conversion between AFB1 and DNA.Ag+-labeled hairpin DNA was used as the unit for CHA signal amplification,and when target AFB1 was present,the CHA reaction was initiated and continuously cycled to release free Ag+to induce AuNPs agglomeration,thus achieving naked-eye detection of AFB1.Meanwhile,the CHA double-chain product was used as a methylene blue loading element,which combined with the advantage of SERS detection,realizing the rapid and sensitive detection of AFB1.Benefitting from the efficient CHA-assisted signal amplification,high sensitivity was achieved with detection limits of 1.6 pg mL-1(SERS detection)and 152 pg mL-1(naked-eye detection),respectively.The strategy was successfully applied to real samples and provided a new idea for highly sensitive and dual-mode detection of mycotoxins.(3)A polystyrene microsphere(PS)mediated Raman reporter controlled release low background interference SERS aptamer sensor was designed and developed for the highly sensitive simultaneous detection of multiple mycotoxins.In this strategy,PS microspheres were used as nanocontainers to prepare novel Raman labels using their properties of swelling in organic solvents.Single-chain binding protein-modified magnetic beads(MNPs@SSB)were used as non-target binding sites to distinguish free aptamers and binded aptamers.The binding of PS-Apt to the target mycotoxin hinders the binding between PS-Apt and MNPs@SSB when the target mycotoxin is present.After tetrahydrofuran treatment,a reduced Raman reporter is released from the PS microspheres,resulting in a decrease of the SERS signal with increasing concentration of the target mycotoxins.Due to the high loading capacity of PS microspheres and the excellent signal conversion and amplification properties of aptamer-assisted SERS detection,the proposed strategy was used for the simultaneous detection of AFB1,OTA and zearalenone(ZEN),providing a new idea for the multiplex detection of mycotoxins.(4)A multicomponent nuclease(MNAzyme)-based lateral flow aptamer sensor was designed and developed for the portable detection of OTA in agricultural products.Pregnancy test strips(PTS)was chosen as a portable signal readout device.In the presence of OTA,specific recognition of the OTA with the aptamer leads to the release of the complementary chain(H1).Subsequently,three components of the MNAzyme were introduced,where the substrate was modified with magnetic beads and human chorionic gonadotropin(hCG)on either side of the cleavage site(rA-rU),respectively.With H1 as the trigger,the complete structure of the MNAzyme is formed,then the substrate chain is cleaved and free hCG is released in the presence of the cofactor Mg2+.hCG flows through the sample collection area of the PTS strips and a red band is observed on the T-line,thus enabling portable detection of OTA.Thanks to the cyclic cleavage of MNAzyme,one H1 can trigger multiple cleavage reactions of MNAzyme,releasing a large amount of free hCG,which breaks the 1:1 relationship between the trigger chain and free hCG in the existing method and improving the sensitivity of the assay.This study has established four sensing and detection methods based on DNA nanotechnology for the rapid and sensitive detection of mycotoxins in agricultural products.This research provides new ideas for the development of accurate and rapid detection technologies for food contaminants.