Development Of Rapid And Visual Detection Based On Gold Nanoparticles And ELISA For Melamine In Milk Products

Food safety is contemporarily a major issue of common concern which is directly relatedto the life and health of the population as well as the national economy in the world. The foodsafety issue, especially the dairy industry in China is facing serious challenge. The Sanlu MilkPowder Incident happened in2008, which involved illegal adulteration of melamine in thepowder, render the issue of food safety coming into the focus of the public’s attention. It is ofgreat social benefit and practical value not only for maintaining the health of the people andlegal interest of consumers but also for facilitating the development of dairy industry to studyand establish a rapid and convenient detection of melamine, thus solve the problem of on siteand rapid detection, which is difficult for the regular detection method, and eventuallyguarantee the quality of dairy products. In this study, using melamine as target, two rapiddetection were established based on the immunological principal and Au nanoparticlechromogenic technology respectively.1. The modification and characterization of melamine hapten, the synthesis andcharacterization of antigen.First, A derivative of mealine,2-chloro-4,6-diamino-1,3,5-triazine was chose to conductthe carboxylation,3-Mercaptopropionic acid was used to substitute the chlorine on themelamine. The product was characterized by1H-NMR,13C-NMR and MS. By the activeester method, carboxyl melamine was coupled with carrier protein BSA and OVArespectively, and then the melamine antigen (MEL-BSA and MEL-OVA) was prepared. Theresult of subsequent SDS-PAGE and identification of MS indicated the successful preparationof the melamine antigen.2. The preparation of polyclonal antibody and the establishment of ELISA.The acquired melamine antigen was used to conduct the immunization of laboratoryanimals,6-week-old Balb/C mice was chose. A small dose of medium-range immunity were carried out with the mixture of complete antigen with equal volume Freund adjuvant (FA) andimmunization. During the immunization, interval was about two weeks, the mice of differentimmune dose was divided into two groups with6in each group. After the forth immunization,the antiserum was collected by cutting tails and the titers were determined by indirect ELISA,of which the result suggested that the antiserum titer of B group were all equal to or higherthan1:20000, and the antiserum possess a relative high specificity and affinity to the targetmolecule. Mice in group B were further immunized subsequently, the blood was collectedfrom the orbit after7days immunization, and polyclonal antigen was purified byimmunoaffinity chromatography. The linear regression equation obtained by the indirectELISA was Y=-46.8376+27.4957X,(R2=0.9938) with a detection range of0.270～41.02mg/L, the calculated IC50was3.33mg/L, and detection limit was0.117mg/L. Through across reaction of atrazine and their analogues, the specificity of the antigen was tested to behigh. The accuracy experiment showed the repeatability of our method is satisfactory, and theaverage recovery result verified the feasibility of our method to detect the melamine in milkand milk powder, which laid a solid foundation for the ELISA.3. A rapid and visual detection based on gold nanoparticles of melamine in milkproducts.An efficient, reliable and low cost colorimetric method based on Au NPs to detectmelamine in milk was established, and a rapid detection kit was developed. Melamine canlead to a fast aggregation of gold nanoparticles through hydrogen bond, result in a change ofthe optical properties of the Au NPs. To be specific, the color of Au nanoparticles turned fromred to purple along with the increment of melamine concentration. That is the mechanism ofour gold nanoparticles based detection of melamine. We prepared Au NPs of different sizesby NaBH4and sodium citrate, through the synergic characterization of naked eye, visiblespectrometer, and TEM. The sensitivity, stability and kinetics of aggregation of the methodwere subsequently observed. We concluded that the the Au NPs with the diameter of5nm isthe most suitable as probe. Based on the above, we test properties of different aspects of theAu NPs probe, the result showed that the Au NPs probe is of better selectivity andanti-interference. The optimized pretreatment of the sample can not only meet the demandedrapid detection of melamine, but also facilitate the simplified operation, in turn improve thedetection efficiency. At last, we develop a rapid visual detection of melamine in milksuccessfully. 4. Research and development of a melamine kit for rapid and visual detection based onAu-NPs.After the method was constructed, we developed a melamine kit base on Au NPs. Firstly,the Au NPs with the diameter of5nm was prepared using sodium borohydride method, and astandard card it was made by the colorimetric reaction of Au NPs at different concentration ofmelamine with Au NPs. This method in some extent overcome some traditional deficits,which was more facile and rapid, without the use of instrument, the total examine time was10min. By distinguishing the standard card, we achieve the qualitative and semi-quantitativedetection of melamine, of which the detection interval of1-120mg/L meet the internationalstandard. The maximum absorption peak appeared to red shift when combine with UV-Vis,the shown favorable linear range was0.2-20mg/mL, and the detection limit was0.2mg/mL;simultaneously the stability test verified that the period of validity of the kit under roomtemperature was about1year; the testing result of the study was close to that of theHPLC-MS. Therefore, in this study, a rapid and on site screening, as well as the qualitativeand semi-quantitative detection of melamine in dairy were established, and a domesticmelamine detection kit with independent intellectual property right was developed.