| Deoxynivalenol is a secondary metabolite resulted from harmful mold fusariumgraminearum, and it widely exists in moldy cereals. At a cellular level, the main toxiceffect of DON is due to the inhibition of protein and nucleic acid synthesis, viabinding to the ribosome and by activating cellular kinases involved in signaltransduction, which consequently results in the decrease of the cell proliferation.DON consumption may cause feed refusal, emesis, vomiting and disrupt the immunesystem in different animal species, as well as growth inhibition, and other symptoms,even teratogenic and embryonic toxicity. In addition, it is a potential carcinogen. As aserious threat to the health of the human and beast, it has attracted great attentions inrecent years.In this study, the advantages of nanotechnology, magnetic separation technology,enzyme-linked immunosorbent assay, and chemiluminescence detection werecombined skillfully: Firstly, the DON-OVA conjugate was synthesized and coupled tothe magnetic nanocomposites (Fe3O4@SiO2@Au) as the solid phase antigen. Then,the luninol-H2O2-HRP-phenols system was used as an enhanced chemilumincencedetection system. A rapid and sensitive chemiluminescence enzyme immunoassaymethod for determination of DON in cereals was developed. The reactionmechanisms were investigated, which would lay good groundwork for the rapidscreening of DON in cereals. The experiment was carried out from the followingthree aspects:1. Preparation and characterization of gold-coated Fe3O4/SiO2nanocompositesCo-precipitation process conditions of Fe3O4were optimized by response surfacemethodology. The optimum conditions were as follows: temperature72℃, pH9,and reaction time0.5h. Under the optimal conditions, the prepared Fe3O4magneticnanoparticle (NPs) has excellent suspension stability. To further improve its chemicalstability, a thin layer of silica was coated onto its surface using Stober method. Next,amino-functionalized silica NPs (Fe3O4/SiO2-NH2) were preparaed with 3-Aminopropyltriethoxysilane, and the amino groups on the surface are moreconducive to combine with gold nanoparticles to form Fe3O4@SiO2@Au. Moreover,the magnetic nanocomposites will be used for loading biomacromolecules.2. Study on the interaction between gold magnetic particles and OVAInteractions between gold magnetic particles and OVA were evaluated byadsorption theory. The influences of phase contact time, adsorbent dosage, and OVAinitial concentrations were investigated to optimize the conditions for maximumadsorption. The experimental data fitted very well the Langmuir isotherm model.Thermodynamic analysis showed that the adsorption was a favorable, spontaneousand endothermic process in nature. The process of adsorption kinetics of OVA bygold magnetic particles follows both the pseudo-first order and pseudo-second orderkinetic models. And the process controlling rate may be primarily controlled byexternal diffusion.3. Establishment of MECLIA based on gold magnetic particles for detecting DONDON-OVA artificial antigens were prepared by coupling agent N, N’-carbonyl-diimidazole, and coupled with gold magnetic particles for preparing the DON goldmagnetic particles. After that, MECLIA based on gold magnetic particles fordetecting DON was established on the basis of the existing laboratory research. Thesignals were dependent on DON concentrations in a linear range from0to20ng/mL,and the linear regression equation is y=-521951x+2883979(x is the logarithm ofconcentration of DON, y is the illumination value of the standard concentration), thecoefficient of determination is0.9986. The limit of detection (LOD) of this methodwas as low as0.031ng/mL (S/N=3). The intra-and inter-assay coefficients ofvariation were <10%. The content of DON from artificial wheat sample was testedwith the proposed. |
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