| Veterinary drug residues not only seriously affect the quality of food, result in residues harmfulness to health of animal and human, and threaten the environment, but also seriously influence China’s import and export trade. Excessive residues cause our country export trade of agricultural loss to be huge in recent years. Misapplication or illegal use of veterinary medicine was an important factor. Thus, developing a rapid, specific and simple operations detection method of veterinary drug residue was an important guarantee of food safety monitoring capacity. In Food Safety Law which was just published in 2015, it has officially defined the status and role of rapid testing technology that can be able to qua the basis of executing the law.In this work, the veterinary drug residues: diethylstilbestrol and estradiol were chosen as the detecting targets. Firstly, the antigens and antibodies of these targets were prepared. After optimizing the antibody coupling conditions, a two-in-one immunochromatography assay(ICA) strip was constructed for simultaneous detection of diethylstilbestrol and estradiol.. This two-in-one ICA strip would provide a theoretical basis and technical support for the residue monitoring in the field of food safety.and In order to improve the detection sensitivity and simplify the pre-treatment of complex sample, a bifunctional magnetic nanoparticle was prepared, which could be used in rapidly separating and enriching trace targets in complicated samples, and as an energy acceptor. On this basis, an ECL resonance energy transfer technology for sensitive detection of trace estradiol residues was established. This project could be utilized in environment pollutants monitoring, food safety and medical diagnosis, and had an important theoretical significance and broad application prospects.1 Simultaneous detection of diethylstilbestrol and estrodiol residues with a single ICA stripTwo monoclonal antibodies raised against those two estrogens were conjugated to colloidal gold particles and were applied to the conjugate pads of the test strip. The DES-BSA/E2-BSA conjugates were immobilized onto a nitrocellulose membrane at two detection zones to form T1 and T2, respectively. The ICA strip had a visual detection limit of DES at 200 ng/mL in PBS, 30 ng/g in milk powder, 25ng/g in liquid milk and 25 ng/g in shrimp tissue respectively, and the results could be judged within 7-10 min. The visual detection limit of E2 was 500 ng/m L in PBS, 75 ng/g in milk powder, 65ng/g in liquid milk and 60 ng/g in shrimp tissue respectively, and the results could be judged within 3-4 min.The method was compared with the HPLC-MS/MS method by testing 37 milk and shrimp tissue samples from local markets, and no statistical significant difference was obtained between the two methods.This novel ICA strip could provide technical support for high-throughput and rapid detection. If it was further developed, multi-residue detection object in a single ICA strip would be achieved, which made it play a more important role in food safety and other fields.2 Synthesis of dual-function composite nanoparticlesMonodisperse oleyl amine-coated ferroferric oxide nanoparticles were prepared by heating pyrolysis method and ferric oleate as precursor. These magnetic nanoparticles dispersed well in organic solutions, but scarcely dispersed in an aqueous solution, which limited their further application. Through a phase transfer method, the magnetic nanoparticlescan could be dispersed in an aqueous solution., The thickness of Au shell could be controlled by the the amount of mercaptopropionic acid, which realized the spectral band regulation of the composite nano particles(Fe3O4 @ Au:550-575nm). Ethylene glycol and diethylene glycol were applied as solvent, and magnetic nanoparticled were prepared by solvothermal method. The TEM results indicated that the ratio between ethylene glycol and diethylene glycolcould affect the size of nanoparticles,. and they were too larger to prepare Fe3O4@Au. Then smaller magnetic nanoparticles were prepared by precipitation method, and were subsequently used to prepare Fe3O4@Au. Furthermore, they could be well dispersed in an aqueous solution. The VSM results indicated that after coated with Au, Fe3O4@Au still had well magnetism(Fe3O4:49.83 emu/g; and Fe3O4@Au:39.22 emu/g), and could be utilized to pre-treat food samples.3 Construction signal amplification ECL system by nanotubesA novel signal amplification system of electrogenerated chemiluminescence(ECL) was fabricated with CdS quantum dots as ECL nanoemitters, multi-walled carbon nanotubes as signal amplifying material. Water-soluble CdS quantum dots were prepared and characterized by fluorescence spectra(PL), ultraviolet-visible(UV-vis), transmission electron microscopy(TEM). GCE electrode was gradually modified by multiwalled carbon nanotubes(MWCNTs), CdS QD and chitosan(CS). The mechanism of based on quantum dot ECL was studied. The multi-walled carbon nanotubes(MWCNTs) and chitosan(CS) on CdS quantum dots ECL were discussed. Two kinds of carbon nanotubes on ECL signal were investigated. Optimizing pH and the concentration of the quantum dots. The signal amplification of Cd S MWCNTs ECL system was achieved for up to 10 times. This simple and efficient modified method laid the foundation for the further research of electrochemiluminescence quantum dot.4 Energy research ECL immunoassays resonanceA novel ECL resonance energy transfer immunoassay methods was established. Bifunctional magnetic composite nanoparticles Fe3O4@Au were applied as ECL energy acceptor, CdS quantum dot were applied as energy donors. The composite nanoparticles Fe3O4@Au were coupled to estradiol antibody. By indirect competitive immunoassay method, a sensitive ECL-RET immune sensor technology for estradiol was constructed. The ECL signal linearly depended on the logarithmic value of E2 concentration. The calibration curve was A = 472.2lgC + 9523, the relative standard deviation(R2) :0.9936. The working range was 2 × 10-10 ~ 2 × 10-4mg/mL. The lowest limit of detection was 3.2 × 10-12 mg/m L. Dual-function Fe3O4@Au nanoparticles were not only applied as probes, but also be able to simplify sample preparation, and enrich targets efficient. The novel resonance energy transfer immunoassay strategy were implemented, showing wide applications in the fields of high sensitivity detection of trace contaminants. |
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