Safety Detection Technology Of Agricultural Products Based On The Peroxidase-like Activity Of Magnetic Nanoparticals

In recent years, food safety issues have affected people's daily life and health and the food safety have been the fourth-largest social problems after the population, resources and environment problems. Therefore, it is particularly important to establish the food safety detection system and develop fast, reliable and sensitive detection technology. Immunoassay detection technology has been widely used in pesticide and veterinary drug residues detection because of its adventages of convenience, low cost, no need of large equipments. However, the traditional immunoassay detection technology is based on the activity of natural enzyme immune markers, thus limiting the development of it. The research is to explore the peroxidase-like activity of Fe₃O₄ magnetic nanoparticals (MNPs), and to develop new detection technology for clenbuterol (CL) and melamine based on Fe₃O₄ MNPs. The research will also provide scientific basis and technical support for the promotion and application of Fe₃O₄ MNPs. The major results are as follows:(1) The Fe₃O₄ MNPs were prepared by co-precipitation method. After modified by APTES, the Fe₃O₄ MNPs still possessed peroxidase-like activity. Taking TMB concentration 3.10mM, H₂O₂ concentration 0.835M, the temperature 40℃and pH 4 as the optimum reaction conditions. The Km value of Fe₃O₄ MNPs to TMB and H₂O₂ were 0.836 and 23.466.(2) The grain diameter of new prepared Au-Fe₃O₄ MNPs was about 50nm, and the Au-Fe₃O₄ MNPs had maximum absorption peak at 564nm. Taking the proportion of Au-Fe₃O₄ MNPs and antibody, the reaction time and temperature as variable, we did research of Au-Fe₃O₄ MNPs to CL antibody purification. Finally, we adopted proportion 16:1, reaction time 12h and temperature 20℃as the optimum purification conditions. After purification by Au-Fe₃O₄ MNPs, the anti-BSA antibody reduced significantly, LOD was 0.189mg/L; the IC50 of anti-CL antibody was 2.294mg/L and decreased 24.37%.(3) The research comfirmed the optimum coating antibody, coating antigen and Fe₃O₄ -antibody complex dilutability were 1:500, 1:3200 and 1:1600. We established the CL residue detection method based on Fe₃O₄ -MNPs and got the fitting equation: B/B0=1/[1+(x/2.178)0.743], (R2=0.9951). The linear range was 0.337-14.07 mg/L, the LOD (IC10) and IC50 were 0.113 mg/L and 2.178 mg/L. The detection method had good adding recovery rate (88-105%).(4) The research confirmed the optimum reaction conditions of Fe₃O₄ MNPs in melamine detection method, including TMB concentration 6.20mM, H₂O₂ concentration 0.334M, reaction temperature 30℃, pH 5 and reaction time 6min. We also established the standard curve of melamine detection: Y= -0.3087X+1.401, R2=0.9476. The adding recovery rate in milk and milk powder were 86-104% and 96-116%. Finally, we chose 8 kinds of inorganic salt and microelement to do research of the interference of them to the detection method, and the results showed that they did no effect to the detection method.