Study On Simultaneous Detection Of Multiple Mycotoxins Based On Novel Nanoprobes

Mycotoxins seriously harm human health and pose a serious threat to social economy and environment.Sensitive and reliable detection technology can effectively ensure food safety.Immune analysis method for combining immune response and the modern testing technology and establish the trace of the target detection technology,it has strong specificity,high sensitivity,convenient and quick,analysis of large capacity,low testing cost,and the advantages of safe and reliable,for the detection of food contaminants provides a good platform.As a trace target detection technology established by combining immune response and modern detection technology,immunoassay has the advantages of strong specificity,high sensitivity,convenient and fast,large analysis capacity,low detection cost,safety and reliability,and provides a good platform for detecting food contaminants.In immunoassay,the nanoprobe prepared by antibody or antigen-coupled nanomaterials is the most important detection element,which plays an important role in converting the information of analyte into easily detectable signals.With the progress of nanoscience,various advanced functional nanomaterials have been applied to develop novel nanoprobes and construct food safety detection technologies to improve the sensitivity and application range of detection technologies.In this paper,hazardous mycotoxins in food were used as the research object,and the new nanoprobes were prepared by using multifunctional nanomaterials.The catalytic,photothermal,fluorescent signal amplification,magnetic separation and fluorescence quenching properties were studied.Four detection techniques were constructed by introducing immunoassay to achieve rapid,highly sensitive and high-throughput detection of mycotoxins in food.The details are as follows:（1）In this study,Ti₃C₂T_x/Au NPs nanozyme with peroxidase-like activity were successfully prepared and conjugated with zenalenone（ZEN）monoclonal antibody（Zen-m Ab）to prepare an immunoprobe with nanozyme activity.The TMB-H₂O₂ colorimetric system was introduced,and the oxidation product（ox TMB）produced by the immunoprobe catalyzing the substrate TMB showed both a color reaction and a photothermal effect under808 nm laser irradiation.A novel colorimetric and photothermal dual-mode immunoassay was successfully constructed.This method uses ZEN as the detection target and the detection limit（LOD）of ZEN is 0.15 pg/m L in colorimetric mode,and the LOD of photothermal mode is 0.48 pg/m L.The detection results of this method and UPLC-MS/MS for analyzing real contaminated cereal samples have good consistency,indicating that this method can be applied to the sensitive detection of trace ZEN in real samples.The dual-mode analysis strategy provides a double guarantee for the accuracy of the detection results of a single target.The thermometer-based quantitative method proposed in this method shows great advantages in food safety determination under low resources conditions,and is expected to be applied to the detection of other contaminants in food.（2）Based on the near-infrared（NIR）photothermal conversion characteristics of Ti₃C₂T_x/Au NPs nanocomposites found in the previous chapter,the photothermal properties of Ti₃C₂T_x/Au NPs nanocomposites were studied in this chapter,and a novel photothermal probe was prepared by coupling Ti₃C₂T_x/Au NPs with ZEN-m Ab,which was applied in immunochromatography for the detection of ZEN.By analyzing the temperature change at the T line monitored by an infrared thermometer,thereby achieving sensitive detection of ZEN.The photothermal detection limit（p LOD）of ZEN is 0.0093μg/L at the linear detection range of 0.005-0.5μg/L.Furthermore,while ensuring the sensitivity of ZEN detection,AFB₁ was introduced as the second detection target to establish a multiple immunochromatographic assay（MLFIA）for simultaneous detection of ZEN and AFB₁.The p LOD of ZEN detected by this multiple immunochromatography is 0.0097μg/L,which is consistent with the sensitivity of single target detection.The linear range of AFB₁ is 0.01-2.5μg/L,and the p LOD is 0.065μg/L.The new photothermal probe has good photothermal performance under 808 nm laser irradiation,and effectively improves the sensitivity and detection range of the detection method.A portable infrared thermometer was applied to record temperature changes to achieve sensitive detection of analytes,and the results of testing real cereal samples were consistent with those of UPLC-MS/MS,indicating that this method can meet the needs of detecting actual samples contaminated by ZEN and AFB₁.This MLFIA constructed in this study has the advantages of short detection time,low detection equipment requirements,simple operation,accurate detection results,and provides new ideas for the analysis and detection of other trace food contaminants.（3）In this study,four fluoresceins with non-overlapping excitation and emission wavelengths were screened and used to modify oligonucleotide single strand（ss DNA）,respectively.Based on the strategy of fluorescence-labeled bio-barcode signal amplification,four fluorescence quenching probes were prepared by successively coupling with antibodies and Au NPs of four mycotoxins（ZEN,FB₁,OTA and AFB₁）,and a multiple fluorescence immunoassay was established.In one detection process,four fluorescence signals from multiple fluorescence quenching probes were simultaneously detected to achieve simultaneous quantitative determination of four mycotoxins.The detection ranges of ZEN,FB₁,OTA and AFB₁ are 0.005-11.11μg/L,0.41-100μg/L,0.005-3.70μg/L and 0.015-33.33μg/L,respectively.And the LODs are 0.004μg/L,0.483μg/L,0.006μg/L,and 0.013μg/L,respectively.In addition,the results of ZEN,FB₁,OTA and AFB₁ in real cereal samples determined by this method are consistent with those of UPLC-MS/MS,indicating that this method has good accuracy and reliability in detecting actual samples.This technique saves detection time and reduces repeated steps for the determination of multiple mycotoxins.At the same time,the multi-residue detection strategy based on biological barcode can be easily extended to the multiple detection of other toxins and small molecule hazards by changing the corresponding target probes.（4）In this study,Fe₃O₄ core-shell magnetic fluorescent nanomaterial（Mag TQD）with three-layer quantum dots（QD）shell was used as the element of fluorescent probe.The fluorescent capture probes（Mag TQD probes）were prepared by EDC/NHS activated ester method with four kinds of mycotoxin coating antigens,and the polydopamine nanospheres（PDANs）with dual-spectral overlapping fluorescence quenching performance were introduced.The PDANs probes specifically recognizing the target mycotoxin were prepared by coupling with four kinds of mycotoxin antibodies,and a multiple fluorescence quenching immunoassay method based on Mag TQD/PDANs quenching system was constructed.Under the optimum conditions,the detection ranges of AFB₁,ZEN,OTA and FB₁are 0.002-3.70μg/L,0.0006-3.7μg/L,0.002-11.11μg/L and 0.015-11.11μg/L,respectively.The LODs are 1.29 pg/m L,0.80 pg/m L,1.97 pg/m L and 13.89 pg/m L,respectively.The detection results of real contaminated cereal samples by this method are in good agreement with those by UPLC-MS/MS.Using this method,four fluorescence signals can be measured simultaneously in one detection process.The application of magnetic separation saves detection time and avoids repeated experimental operations.It is more suitable for high-throughput detection of various hazardous substances in food.