| In recent years, the food safety issues have become the focus of concern gradually, the detection of veterinary drug residues in food has got more and more attention. Estrogenic substances are often added into animal feed in animal breeding process, which are used to improve the yield and quality of animal products, shorten the growth cycle of animals, and improve production efficiency. 17?-estradiol(17?-E2), one of the most active environmental estrogens, widely exists in the environment and animal products. If long-term intake 17?-E2, will enrichment in the human body through food chain, cause serious harm to human body.Currently, the methods of detecting 17?-E2 in samples include instrumental analysis and immunological methods. The instrumental analysis requires expensive equipment, preliminary procedures of sample purification and so on. Immunological methods need antibodies, but the antibody is hard to be screened, not easy to save, prone to denaturing. Thus a fast, simple, without the antibody, and efficient detection method is particularly necessary. The specific recognition sites on the molecular imprinting polymer(MIP) can identify the template molecules. It is similar to the identification between antigen and antibody.Two kinds of new methods were established to achieve 17?-E2 rapid detection: molecular imprinting biomimetic enzyme-linked immunosorbent assay test strips rapid detection methods, and molecular imprinting biomimetic immunosorbent rapid detection fluorescence analysis method respectively. Compared with the traditional method, both methods had a series of advantages such as simple, rapid, inexpensive, easy to operate, and so on. The paper mainly consisted of four parts as follows: In the first chapter, the research progress of 17?-E2 detection method, the principle, steps and the classification of molecular imprinting technique were summarized. The research progress of molecular imprinting technique based on test strip and enzyme- linked immunosorbent assay(ELISA) plate, and application in the environmental estrogens detection were emphasized.In the second chapter, a new 17?-E2 molecular imprinting biomimetic enzyme-linked immunosorbent assay test strip rapid detection method was established. Prepare the MIP on the surface of test strip, and the MIP has specific recognition sites. A direct competition reaction between 17?-E2 and enzyme-labeled 17?-E2 of the sites, if added the enzyme substrate, the results can be detected according to the color depth. The selection of test strip type, the optimization of synthesis condition, the improvement of sensitivity, the investigation of method performance and the elimination of matrix effects were mainly introduced. The results showed that the MIP's optimum synthetic conditions as follows: 12 m L acetonitrile was chosen as the solvent, the addition amount of template molecules 17?-E2 was 0.1 g, the functional monomer 3-aminopropyltriethoxysilane(APTES) was 1014 ?L, the cross-linker tetraethoxysilane(TEOS) was 963 ?L, the catalyst(0.1 mol·L-1 acetic acid) was 100 ?L, the MIP had a good recognition ability. Based on the MIP, the limit of detection(LOD) of this method was 0.25 ?g·L-1.In the third chapter, a new 17?-E2 molecular imprinting biomimetic immunosorbent assay based on ELISA plate rapid detection of fluorescence analysis method was established. Prepare the MIP on the surface of 96-well ELISA plate, and the MIP has specific recognition sites. A direct competition reaction of the sites between 17?-E2 and fluorescence-labeled 17?-E2 can be detected according to the intensity of fluorescence. The optimization of synthesis condition, the investigation of method performance and the elimination of matrix effects were mainly introduced. The results showed that the MIP's optimum synthetic conditions as follows: 10 m L anhydrous ethanol was chosen as the solvent, the addition amount of template molecules 17?-E2 was 0.1 g, the functional monomer APTES was 676 ?L, the cross-linker TEOS was 642 ?L, the catalyst(0.1 mol·L-1 acetic acid) was 100 ?L. The MIP had a good recognition ability, and the least non-specific adsorption. Based on the MIP, the limit of detection(LOD) of this method was 0.22 ?g·L-1, the sensitivity was 1.79 ?g·L-1.In the fourth chapter, the 17?-E2 molecular imprinting biomimetic immunosorbent assay based on test strip and ELISA plate rapid detection methods were summarized. These two methods are used to test strip and 96-well ELISA plate as the carrier success in synthesis of MIP, and established two kinds of methods for rapid detection of 17?-E2. The two methods compared with the traditional detection methods, have a lot of advantages such as fast, high sensitivity, simple operation, without using antibodies and so on. |
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