Development Of Magnetic Nanoparticle Immunoassay For Detection Of Triazophos Pesticides

Food Safety is important to peoples’livinghood and courtrys’economy, and attentaioned by all all over the world. Pesticide residue, one of the critical hazard factors of food safety, is a significant aspect. Presently, all of the advanced countries and China have formulated strict limited standard of pesticide residues to regulate the utilizing of pesticide and to protect beneficial of countries with making the standards as techniqual barriers of trading. Triazophos is a organophosphorus insecticide、acaricide、nematicides of broad spectrum and mainly used for protecting fruit trees, cotton, grain crop, etc.This kind of pesticide have mildly toxicity to mammalian, characterized reducing the activity and affecting the central nervous function by inhibition the activity of cholinesterase. the contaminant of triazophos was paid great anttention. Till now, the determination techniques and methods have been deep studied. As the instrument analysis technologies such as GC, HPLC, GC/MS and HPLC/MS were researched deep and well, but the rapid testing techniques and efficient sample prepare technology are in serious shortage. And it is badly in needed of the devcorresponding researches.The sensitivity of the traditional ELISA does not accord with the demand of trace testing due to the titer and affinity of the antibody. Nano-magnetic particles was a new type of polymer materials that it refered to combining organic polymer and inorganic magnetic material together by using the appropriate methods and forming the magnetic particle with the special structure. In this thesis the concept of molecular design was used for thesynthesis of the nano-particles owning the characteristics of the signal amplification that could increase contact rate of antigen and antibody and thus to improve the sensitivity of ELISA. Based on the designed optimization of nano-particles by the view of the molecular, studies on the method of preparing and optimization of the combination of nano-particles and antibodies, and their stability were conduced. The sensitivity of the ELISA of nano-particles-antibody complex and control group was compared. The results could provid a scientific basis for further establishing Nano-zoom ELISA method.Firstly, the research background and progress of triazophos-. nano-technology and nano-materials were summarized, and the classification, preparation and application of magnetic nanoparticles were introduced as well as the application of magnetic immune microspheres, and the significance of the thesis was illustrates.Then, the Fe3O4nano-microspheres were synthesized、optimized、modified and characterization.Making use of method of coprecipitation, compared the different particle sizes of synthetic nano-particles in the different reaction conditions:molar ratio of Fe2+/Fe3+, time, temperatures and stirriing speed. The results turned out that the appropriate quantity and condition of response is molar ratio of Fe2+and Fe3+is0.6, temperature is70℃, time is60minutes, stirriing speeds is1200rpm. The chemical modification of Fe3O4, magnetic nano-particles were synthesed, which surface modified with-COOH. According to these conditions, the average size of synthetic nano-particles is10-15nm.The polymer magnetic microsphere adsorption of triazophos antibody was determined using Pierce(?) BCA Protein Assay Kit, and the triazophos antibody immune magnetic microspheres were successfully prepared using the method of EDC/NHS. Experimental results showed that the polymer magnetic microspheres had good adsorption effect of triazophos antibody, and the effect of triazophos antibody’s concentration and adsorption time on magnetic microspheres’adsorption efficiency were inspected. With the increase of triazophos antibody’s concentration, the adsorption of protein molecules increased gradually, and the saturated adsorption quantity was reached; suitable adsorption time was60min.In the end, the application of antibody-complex was discussed. The immune magnetic beads were applied to the ELISA reaction for the determination. By comparing the reactivity of antibody-particles complex instead of the antibody with the traditional ELISA, the research found that using antibodies-particles could improve ELISA responsiveness and confirmed that the nano-particles could improve the ELISA. Secondly, the method immune magnetic beads ELISA developed were applied for the determination of samples(n=5) for the affix recovery test and precision experiment, the results showed that the method could meet the demand since the recoveries between83.1%and115.9%and the RSD less than10.0%were satisfactory. Finally, The samples of apple and cabbage were determined by immune magnetic beads ELISA and GC-MS in the affix recovery test. with two methods of testing, a regression equation was figured out, the apple samples:y=0.9551x+11.129, R2=0.9799; cabbage samples:y=0.9503x+6.6975, R2=0.9769.The results showed that two kinds of methods have good correlation.GC-MS, however, due to its more expensive instrument, in domestic large scale popularization and application still exist certain difficulties.But because the immune magnetic beads ELISA has immune analysis method of detection with low cost, high flux and pretreatment is relatively simple and fast, make the immune magnetic beads ELISA analysis method still has a good application prospect.