Studies On Binding-Induced DNA Assembly And Multicolor Immunoassay For Rapid Detection Of Mycotoxins In Agro-Products

Mycotoxins are the toxic secondary metabolites produced by fungi,which can be carcinogenic,teratogenic,and mutagenic.The mycotoxins are intractable to control due to the contamination of any process of“farm to table”.Because of the agricultural climatic difference,the multiple contamination of multiple mycotoxins happens frequently.The combined toxicity caused by multiple mycotoxins is high,which poses serious health hazards to human.On-site rapid detection technology is the important mean for the whole-process monitor of mycotoxins.Current rapid detection methods,such as ELISA and immunochromategraphic strip,require complicated separation and wash steps and most of them are only for the single mycotoxin detection,which cannot meet the requirement the convenient pretreatment and homogeneous detection of multiple mycotoxins.Thus,it is urgent to develop the rapid,simple and convenient detection methods.To solve the above problems,we developed three rapid,simple and convenient immunoassays.The main contents and innovations are as follows:1.FRET-based?fluorescence resonance energy transform?immunoassay was developed for aflatoxin B₁ detection.This immunoassay did not require any separation process,which was simple and easy to operate,and can meet the requirement of the rapid and convenient detection of aflatoxin B₁.Combining the FRET,fluorescence-quenching property of gold nanoparticles,with competitive immunoassay,the quantitation of aflatoxin B₁ was achieved by the measurement of the fluorescence intensity.The gold nanoparticle served as the fluorescence quencher and the scaffold of antibody labeling in the assay.The carboxyl fluorescein immobilized on the antigen was quenched due to the specific recognition between antigen and antibody.A linear range of 10.0 to 60.0 nmol/L and a detection limit of8.6 nmol/L were achieved.This assay is homogeneous and the whole process could be completed in a single vessel without any washing or separation steps,which simplified the detection procedure and can be widely applied in the on-site detection of aflatoxin B₁.2.The fluorescence assay based on binding-induced DNA assembly was developed for the detection of aflatoxin B₁,largely decreasing the fluorescence background.This is the first application of BINDA in the small molecule determination.Integrating BINDA,fluorescence-quenching property of gold nanoparticle,with competitive immunoassay,we developed BINDA-based fluorescence assay.Recognition probe and signal probe were designed to achieve the application of BINDA in the detection of small molecules.We obtained a LOD of 2.3 nmol/L and a linear range of 5.0 to 50.0 nmol/L.Analysis of real rice samples showed recovery of86.2%to 101.6%for aflatoxin B1 determination.We estimated the distance between the fluorophore and the quencher theoretically.The results indicated that the distance was largely decreased,reducing 38%fluorescence background and enhancing the sensitivity,compared to the conventional competitive fluorescence assay.3.The multicolor immunochromatographic synchronous assay for the determination of multiple mycotoxins contamination was developed to realize the simultaneous detection of three mycotoxins.We fabricated the multicolor immunochromatographic strip for the simultaneous detection of three mycotoxins.Different monoclonal antibodies were conjugated with varying dyed microsphere.Aflatoxin B₁ antigen,zearalenone antigen and T-2 toxin antigen were immobilized on three test lines,respectively.The simultaneous detection of three mycotoxins was achieved using the competitive immunoreaction and the visible detection limit were 1.6,64.2 and 12.6 nmol/L,respectively.This strip demonstrated good specificity without immune cross reactivity between these three mycotoxins.In the analysis of maize and feed samples,the results were consistent with that of HPLC-MS/MS method.Semi-quantitation of three mycotoxins was easily achieved by identifying the color of the test lines with ease-operation and cost-saving.