Study On The Application Of New Immunochromatographic Technique For Detection Of 17?-estradiol Residue In Food

17?-estradiol(E2),derived from cholesterol,is a type of natural fat-soluble steroid hormone,which could regulate the function of the reproductive system in humans and many other animals and maintain potential hormone metabolism.In addition to promoting the growth and development of animals,E2 can also make animals single sex,improve lean meat ratio and shorten the fattening period,so it has been widely used in animal husbandry and aquaculture industries.However,the abuse of E2 may form residues in animals,and also penetrate into water and soil,and eventually enter the human body through the accumulation effect of the food chain.Studies have shown that excessive intake of E2 from the outside can cause hormone disorder in the human body,which can promote early puberty in children,even affect female fertility and lead to the occurrence of ovarian cancer and other diseases,as well as increase the risk of prostate cancer in men.Therefore,it is very important to establish rapid detection method to detect E2 residue in food samples.Two different lateral flow immunoassays(LFIA)are respectively constructed,including Fe₃O₄ magnetic nanoparticles(MNPs)based indirect probe LFIA and graphite-like carbon nitride-laden gold nanomaterials(g-C₃N₄@Au)based signal amplification LFIA,realizing the fast detection of E2 in several kinds of food samples.The research contents and main results of this subject are as follows:1.The preparation and performance evaluation of anti-E2 monoclonal antibody(mAb).The ascites,contained anti-E2 mAb,were prepared by injecting the hybridoma cells into the abdominal cavity of mice,then purified by classical method of caprylic acid-ammonium sulfate.SDS-PAGE gel electrophoresis proved that the purity of mAb was high.And the results of indirect ELISA and indirect competitive ELISA showed that the titer of the mAb was 1:64000 and the IC₅₀ value was 2.41 ng ml^-1,respectively.The mAb can be used as a core immune reagent to establish an immunochromatographic analysis method.2.The construction and performance evaluation of immunochromatographic method based on MNPs labeled indirect probe.Firstly,carboxyl-modifed MNPs were synthesized using a one-step hydrothermal method.MNPs labeled polyclonal goat anti-mouse Ig G antibody(GAMA)as an indirect probe,and the free anti-E2 mAb(detection antibody)directly capture antigens.In this way,the detection antibody and signal probe could be regulated respectively to produce enough detection signals and greatly reduce the use of detection antibody.The results indicate that only need 10 ng mAb per test can achieve detection,and the cost was low.The visual detection limit(v LOD)for the standard solution of E2 was 0.2 ng ml^-1,which was 5-fold decrease compared with the traditional LFIA strip.Three other sex hormones with similar structure were used as interferences to determine the specificity of the indirect probe strip,and the results showed that the developed strip showed good specificity for E2.Finally,the indirect probe strip was used to detect E2 in four real food samples including milk,chicken,fish and shrimp,the v LOD were 0.4 ng mL^-1,1 ng g^-1,1 ng g^-1 and 1 ng mL^-1,respectively.Although the detection performance of the strip decreased under the interference of the complex matrix of food samples,it can still reach the concentration of E2 in the standard detection method stipulated in China.3.The construction and performance evaluation of immunochromatographic method based on g-C₃N₄@Au signal amplification label.The g-C₃N₄ nanomaterial was synthesized by high-temperature calcining of urea in a tubular furnace,and g-C₃N₄ nanosheet was obtained by ultrasonic stripping.Then,the g-C₃N₄@Au was formed by reducing chloroauric acid on the surface of carbon nitride nanosheet using sodium citrate.After optimizing the amount of reducing agent found that when adding 300?L sodium citrate with concentration of 7.74 mg mL^-1,the synthesized g-C₃N₄@Au composite has bright color,and the Au NPs loaded on the surface of g-C₃N₄ nanosheets were evenly distributed with the same morphology and size.The g-C₃N₄@Au nanocomposites were conjugated with anti-E2 mAb to prepare g-C₃N₄@Au-mAb probe.Due to g-C₃N₄ nanosheet could load a lot of gold nanoparticles to amplify the signal of gold nanoparticles,only need coupling small amounts of mAb can produce significant signal.After optimizing the key experimental parameters,the optimal mAb labeling concentration was 4?g mL^-1 and the probe volume was 2?L.After that,the analytical performance of the strip based on g-C₃N₄@Au was evaluated,and the results showed that the v LOD was 0.5 ng ml^-1,and the sensitivity enhance 3-fold than traditional colloidal gold strip.In addition,the results of the interference experiment showed that this method also had good specificity for the detection of E2.Finally,the indirect probe strip was used to detect E2 in four real food samples including fish,pork,shrimp and chicken,and the v LOD were 0.5 ng mL^-1,1 ng mL^-1,1 ng mL^-1 and 0.5 ng mL^-1,respectively.It is possible that the concentration of heme,fat and salt ions in the pork samples may have influenced the analytical performance of these two samples.