A Novel Fluorescence Immunoassay Technique For Simultaneous High-sensitivity Detection Of Fumonisin B1 And Zearalenone In Maize

Zearalenone(ZEN)and Fumonisin B1(FB1),are mycotoxins that has been identified as a contaminant in grains,cacao beans,and meat products worldwide.The mycotoxins may induce acute to chronic effects.Acute effects are characterized by the rapid onset of poisoning or death,while chronic effects resulting in chronic pathological reactions of visceral function and physiological process damage,such as carcinogenesis,teratogenesis and mutagenesis.Food contaminated with fungi is a major cause of mycotoxin poisoning and cannot be sterilized by heat treatment in normal cooking methods.Currently accepted testing methods for food samples rely upon the use of Immunological technique followed by High Performance Liquid Chromatography(HPLC)and Enzyme-linked immunosorbent assay(ELISA).High performance liquid chromatography is widely used in chemistry,industry,medicine,food detection and other scopes owning to its high quantitative accuracy and sensitivity.Whereas,the limitations of HPLC,such as the complexity and cost of the equipment,it less capable of satisfying the need for field rapid detection.ELISA has a barely satisfactory effect in field detection in view of its high specificity,simple operation,and the ability to detect a variety of targets at the same time.Nevertheless,the deficiencies of ELISA are also obvious,such as relatively low sensitivity and the enzyme activity is easily affected by external conditions.Fluorescence-linked immunosorbent assay(FLISA)retains the superiorities of ELISA and promotes the sensitivity and stability on this basis.Here,I developed a novel FLISA for rapid simultaneous detection of ZEN and FB1 in food.The luminescent MOF was assembled by clicking chemistry to amplify the fluorescence signal,and then it was further connected to the secondary antibody.Thus,an immunoassay that directly generates fluorescent signals was developed and compared with ELISA and traditional FLISA,whose sensitivity is superior to ELISA and the traditional FLISA in the same group of corn samples.Rapid and recent progress in nanotechnology,nanotechnology has the potentialto play an important role in facilitating the application of fluorescent immunoassays.In recent twenty years,new fluorescent nanomaterials such as have emerged rapidly semiconductor quantum dots,carbon dots,carbon nanoparticles,carbon nanotubes,graphene-based nanomaterials,phosphors and metal–organic framework(MOF).Among the reported fluorescent nanomaterials,MOFs have attracted noteworthy attention in detection in virtue of their structural tunability,surface modifiability and excellent biocompatibility.luminescence metal–organic framework(LMOFs)not only has these advantages of MOF,but also is easy to induce luminescence,which is a potential fluorescent chemical sensor.The luminescence of MOFs can be emitted by either organic linkers or metal ions.Linkers central luminescence also includes three subtypes:Linker emission,ligand-metal charge transfer(LMTC),and metal-ligand charge transfer(MLCT).This paper explored the method of rapid on-site detection of commonmycotoxin contaminants(ZEN,FB1)in food.The main experimental contents and results are as follows:The first part:The competitive ELISA method was used to detect ZEN and FB1,and the checkerboard method was used to determine the best dilution ratio of antigen and antibody.A series of experimental conditions were optimized,and the standard curve was established.The results show that ZEN has a good linear relationship in the concentration range of 3.9?2000 ng·m L^-1,FB1 has a good linear relationship in the concentration range of 40?4000 ng·m L^-1,through calculation,The detection limits of ZEN and FB1 were 3.228 ng·m L^-1and 22.787 ng·m L^-1,respectively.As fluorescence immunity is characterized by high specificity and sensitivity atthe same time,an immunofluorescence method based on fluorescein isothiocyanate(FITC)was established to detect mycotoxins ZEN and FB1 in maize according to partial optimization results of the above experiments(such as coating conditions and coating solution),and FTIC could be directly stimulated by ultraviolet light.The concentration of antigen and antibody,the concentration of FITC-labeled secondary antibody and buffer solution were optimized.Under the optimized conditions,The fluorescence intensity showed a good logarithmic linear relationship with ZEN and FB1in the linear range of 0.2 ng·m L^-1 to 250 ng·m L^-1 and 2 ng·m L^-1 to 2500 ng·m L^-1,respectively.The ZEN linear equation is y=-143.85576 Logx+489.57469,R₂=0.9936,and the detection limit is 0.124 ng·m L^-1.The linear equation of FB1 is y=-124.46914Logx+636.1813,R₂=0.9887,and the detection limit is 2.103 ng·m L^-1.The average recovery rate was 91%?116%and RSD was less than 8.0%,which can be used in the detection of actual corn samples.The second part:We used the luminescent material Ui O-66-NH₂,which is formed by self-assembly with zirconiumm(Zr)as the metal center and NH₂·H₂BDC as the organic ligand.It has the advantages of simple preparation method and good stability.Ui O-66-NH₂ has inherent fluorescence due to its organic ligand luminescence,and the maximum emission wavelength is 450 nm.In order to achieve the conjugation of antibodies to Ui O-66-NH₂,click chemistry was used as a strategy to amplify the fluorescence signal of LMOF and conjugation of antibodies.Click chemistry uses readily available chemical materials to connect through fast,efficient,reliable,and selective modular chemical reactions.Based on this,a novel fluorescence immunosorbent assay based on metal organic framework and click chemistry(CLICK-FLISA)was developed for detection of zearalenone and fumonavin B1.The proposed fluoroimmunoassay method for Zearalenone(ZEN)and Fumonisin FB1(FB1)detection based on UIO-66-NH₂ assemblies probe afforded a linear response range of0.02?5 ng·m L^-1 for ZEN,0.4?250 ng·m L^-1 for FB1,and a very low detection limit(0.016 ng·m L^-1 for ZEN,0.394 ng·m L^-1 for FB1).The satisfying results demonstrate promising applications of on-site quick testing in practical sample analysis.Moreover,the amino functionalization may also provide a modification strategy to design luminescent sensors for other food contaminants.