| The rapid detection of pathogenic microorganisms, their toxins and other harmful substances is of the first important to monitor, prevent, control, and timely diagnosis the contamination of food and the protection of their infection implement. And it is also the needs of the relevant inspection and quarantine departments and research focus of researchers. In a variety of rapid detection techniques, agglutination test is widely used in the fields of food safety, veterinary and medical fields, because of its advantages of simple operation, no special expensive equipment needed and the results easy to determine. Indirect agglutination test is a kind of agglutination test, and it more sensitive and their test results are more easy to determine compared with traditional agglutination test, because of addition reporter. the reporter used in indirect agglutination test have a major impact on agglutination test, the conventional reporter for indirect agglutination test is sheep red blood cells and polystyrene latex microspheres, the former has a better antibody binding capacity and more vibrant red which will make the determination of the test results more easy. However, the stability of sheep red blood cells are poor, after being processed and labeled, the red blood cells are prone to hemolysis, are also easily contaminated,and they are difficult to be save for a long time; Polystyrene latex microspheres are economic, easy to get, more stable than red blood cells, but more easily swelled. Polystyrene latex microspheres are colorless, their binding ability and aggregation properties are poor, as the reporter, the agglutination test results are not easy observed; furthermore, with these two repoter, only one antigen can be detected one time. Therefore, to find a more ideal reporter is of first important to improve indirect agglutination test.With the development of nanotechnology and biotechnology, the combination of nanoparticles with bioassay methods for pathogen detection have attracted more and more attention. Using nano-materials to modify and bio-labele antibodies, to prepare immune nanospheres, has been widely applied to the detection of pathogens. This paper prepared colored silica microspheres (CSN) using reverse microemulsion, and then using chemical crosslinking method to modify surface of CSN the antibody to get immune colored silica nanoparticles (ICSN), and using ICSN as reporter to development a new aggregation technology for the rapid detection of pathogens. With this new technology, we can simultaneously, rapidly, sensitively and stablely detect two or more kinds of food-borne pathogens in one test. This paper includes the following sections:1Study of synthesis of monodisperse immune color silica nanospheres (ICSN)Monodisperse colored silica nanoparticles (CSN) were synthesized by modified reverse micro emulision reaction. CSN formed by hydrolysis and condensation reaction of TEOS in the presence of a silane coupling and organic dyes, with n-hexane as solvent, the generated silica nanoparticles is modified dy organic dyes and show bright color. In this study, the effect of reaction conditions on the particle size and monodispersion of CSN was systematically studied. Field emission scanning electron microscope SU-70and laser particle size analyzer Nano2S were used to characterize morphology and particle size of the samples. The results showed that under the other conditions remain unchanged, with the concentration of TEOS and ammonia increases, the resulting particle size of the CSN increases slightly; the particle size and dispersion of CSN were significantly influenced by the water volume and temperature of reaction. On the other hand, the addition of silane coupling agent KH-550into the reaction system, could effectively reduce the agglomeration and increase dyeing effect of CSN, so as to provide an effective approach for the preparation of monodisperse CSN. Reaction mechanism for CSN was also discussed in the paper. According to the effect of reaction conditions on the synthesis of CSN, we could find out the optimum synthesis condition for the CSN of different particles size. Under the optimum reaction conditions, CSN was synthesized, then they was combinated with pathogenic antibody to prepare the immune colored silica nanoparticles (ICSN), which could be used for the rapid detection of foodborne pathogens.2The new agglutination test for rapid detection of Salmonella pullorum and Salmonella gallinarumThe aim of this work is to propose a new agglutination test for the rapid detection of Salmonella pullorum and Salmonella gallinarum (S. pullorum and S. gallinarum). In this study, three kinds of organic reactive dye and antibody were used to modify silica nanoparticles to prepare the immune colored silica nanoparticles (ICSN), ICSN were used to build new agglutination test for the rapidly detecting S. pullorum and S. gallinarum. The colored silica nanoparticles (CSN) were synthesized by reverse microemulsion method. The morphology and degree of dispersion of CSN were characterized by SEM. The agglutination test was used to characterize the detection effect of S. pullorum and S. gallinarum. The results showed that the new agglutination test was very sensitive, the aggregation results were prominently, intuitive and easy to distinguish by naked eye, moreover, consumption of antibody in new agglutination test is only1/500of that consumed in traditional test, the linear range for S. pullorum and S. gallinarum was from102to109cfu·mL-1. It was very stable and repeatable, after being stored at4℃for28days, the aggregation effect showed no significant difference; It also showed good specificity and accuracy. The new agglutination test was simple, fast, accurate, sensitive and economic. This new agglutination test could not only be used for rapidly detecting S. pullorum and S. gallinarum, but also provide a basis model for rapid detection of other pathogenic bacterium.3Study of a new agglutination technology for rapid detection of Enterobacter sakazakiiTo explore a new technology for the rapid detection of Enterobacter sakazakii (E. sakazakii), in this study, we first synthesis immune colored silica nanoparticles (ICSN) by using both organic reactive dyes and antibody as modifier, and then use ICSN to build the new agglutination technique for rapid detection of E. sakazakii. Color Silica Microspheres (CSN) was prepared by microemulsion technique. Scanning electron microscopy was used to character the morphology and degree of dispersion of CSN. The agglutination test was used to characterize the detection effect of E. sakazakii. The results showed that the new agglutination test was very sensitive, the aggregation results were prominently, intuitive and easy to distinguish by naked eyes, the linear range for E. sakazakii was from102cfu·mL-1to109cfu·mL-1; It was very stable and repeatable; it also showed good specificity and accuracy. The new agglutination test based on ICSN was sensitive, fast, stable, simple, accurate and economic. This new agglutination test could not only be used for rapidly detecting E. sakazakii, but also provide a basis model for rapid detection of other pathogenic bacterium.4Study of new agglutination test for simultaneously rapid detection of multiple pathogensIn this chapter, we attempts to prepare two kinds of immune colored silica nanoparticles,(ICSN), using prepared red and orange colored silica nanoparticles (CSN) as a carrier. These two CSN was first modified with activating carboxy, then modified with S. pullorum and S. gallinarum antibodies and E. sakazakii antibodies respectively, at last, these two ICSN were mixed to get the mixed solution which have specific immune activity to both two pathogens. Then ICSN were used to detect mixed bacteria solution of S. pullorum&S. gallinarum and E. sakazakii. The results show that the new agglutination test has fast detection speed, can appear clear agglutination only within20s-30s, and the two kinds of pathogens could been quickly detected by display color of aggregation block, we constructed a new kinds of agglutination test which could simultaneously rapid detect two pathogen. This new agglutination test is more intuitive, sensitive, economical, accurate, stable, simple, and have a good prospect in food safety, medicine, veterinary science, environment and other fields... |
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