Synthesis Of Gold-Magnetic Composite Particles And Fluorescent Carbon Dots And Their Application In The Detection Of Ochratoxin A

Ochratoxin A(OTA)is a secondary metabolite produced by fungi,and it is also a highly toxic pollutant with strong carcinogenic and toxic effects.It usually occurs in grain crops.It poses a huge threat to human health problems,and has attracted the attention of researchers for many years,and a variety of detection methods have been developed.A variety of advanced nanomaterials are applied in the detection scheme to achieve the detection of residual toxins in food.Magnetic nanoparticles(MNPs)have been developed as enrichment and screening tools in food safety control.It is a superparamagnetic nanoparticle with the advantages of high efficiency,time saving,and convenient separation,and is a commonly used carrier material for point-of-care detection(POCT).In this study,a fluorescent immunoassay system based on gold-coated magnetic nanoparticles was constructed,and two fluorescent materials,fluorescent protein and fluorescent carbon dots,were used as the output signal of the system,and were applied to the determination of OTA.The specific research is as follows:1.Construction of an OTA detection system based on MNPs@Au NPs and fluorescent fusion proteinsMagnetic nanoparticles(MNPs)were prepared by solvothermal method,and gold nanoparticles(Au NPs)were modified on the surface of magnetic nanoparticles by tyrosine reduction method to form gold-coated magnetic composite particles(MNPs@Au NPs).It has good stability and can adsorb protein macromolecules stably and efficiently.The anti-OTA nanobody VHH28 was fused and expressed with two red fluorescent proteins(m Cherry,Tag RFP)by means of genetic engineering,and the antigen binding of the fusion proteins was analyzed.The ability and fluorescence performance,Tag RFP and gold-coated magnetic composite particles were selected.Then an immunoassay method(MNPs@Au NPs Fluorescence Protein Immunoassay,MAPIA)based on MNPs@Au NPs and fluorescent proteins was established,and the linear curve for detecting the concentration of OTA was fitted.The limit of detection(LOD)was0.58 ng/m L.A high degree of selectivity for the target has also been demonstrated in cross-reaction experiments with a variety of different toxins.2.Establishment and application of OTA detection system based on MNPs@Au NPs and fluorescent carbon dotsGreen fluorescent carbon dots with uniform size and good water solubility were prepared by microwave method,and successfully coated with polyethyleneimine(PEI)to aminate them,and covalently coupled with ochratoxin to form fluorescent probe CDs-PEI-OTA.CTB-VHH28(fusion protein of cholera toxin B subunit and antiochratoxin nanobody)was coated on the surface of gold magnetic nanoparticles,and a fluorescence detection system based on magnetic separation was established.By optimizing the detection system,the MNPs@Au NPs Fluorescence Immunoassay(MAFIA)showed high sensitivity(LOD = 0.12 ng/m L)and comparable intra-assay recovery(74.60-101.30%),inter-assay recoveries(69.55-96.30%),and demonstrated high selectivity for the target.Compared with Ultra Performance Liquid Chromatography(UPLC),the detection results are consistent.These results demonstrate that this method can be used for the detection of OTA in cereal foods.In addition,this method also has the advantages of simple sample pretreatment,simple detection procedure and time saving,which provides a new and effective way for the rapid and accurate determination of trace OTA in complex samples.