| Food safety problem has always been the focus of attention, and foodborne pathogensis which can cause public health and safety problems is one of the biggest risks to food safety. The detection of foodborne pathogens is important. The detection technologies for foodborne pathogens are mainly divided into traditional detection technology and modern rapid detection technology, rapid technology received a great deal of attention because of its advantages include:short in time, simple, low cost and accuracy. Indirect agglutination test is a kind of serological reaction, it can quickly and easily achieve rapid detection of pathogens and is widely used in bacteriological diagnosis. It is also famous in a variety of inspection bodies for its simple in operating, agglutination intuitive, easy-sensitized particles save, fast in detection, without the aid of high-end instrument-aided detection, detection conditions requiring low etc. In indirect agglutination test, carriers are not only the necessary of indirect agglutination test but also the key factor of aggregation. It currently has many types, mainly such as red blood cells of animals or human, the activated carbon particle, polystyrene latex particles and gelatin carrier. Erythrocytes as carrier has the uniform in size, and the type of antigen adsorbed is mainly polysaccharide antigen, but relatively poor for the adsorption in antigen or protein. After erythrocytes sensitized it will easily hemolysis、brittle、polluted and etc. Red blood cells has poor stability and short in saving time, and generally must be used within2-3days. On the same time, Polystyrene latex is a synthetic carrier which is about0.8μm in size and its surface with a negative charge, mainly binding protein by means of physical adsorption, but the stability of this binding means is more poor, and antibody or antigen is easy to fall off from the surface of polystyrene latex, and the phenomenon of self-curing affected by various physical and chemical factors is easy to happen because of its physical and chemical property is more vulnerable, and lead to misjudgment of aggregation result. Both of the conventional carriers only have one single color, and the color of agglutination block is also one kind which depends on color of carrier. Therefore, it is necessary to select a carrier which has high performance, good stability and bright color. Silica nanoparticles (SiNps) will be a excellent carrier because of its strong stability, good biocompatibility, and other unique advantages. SiNps are colorless, can’t produce an effective signal amplification as the carrier of indirect agglutination test. To overcome this disadvantage, the surface of the SiNps can be modified by dyes. And the surface of SiNps contains a large number of hydroxyl groups or unsaturated residue bonds is easy to modified. Therefore, the obtained Silica nanoparticles not only does not affect properties of themselves but also has a bright color, we can maximize the amplification of aggregated signal, and make aggregation phenomenon more obvious and intuitive.In summary, the present study will attempt to synthesize nanoscale SiNps via reverse microemulsion and microscale SiNps via semi-continuous Stober method. Then colored silica nanoparticles (colored-SiNps) can obtained by modifying reactive dye onto the surface in the way of chemical modification. In this process, the silane coupling agent acted as a medium which can covalently bound to the surface of the microspheres. Reactive dyes can give the microspheres bright color and signal amplification of aggregation, leading to the aggregated phenomenon more eye-catching and intuitive. Colored-SiNps were modified with antibody by bioconjugated techniques, the obtained product had immunological activity. So we build a new agglutination technique based on colored-SiNps as a carrier for rapid detection of pathogenic microorganisms in food. We will also study the performance of the new technology agglutination for the detection of pathogens and viruses, and optimized the conditions for aggregation.1Study on the performance of new aggregation test based on colored-SiNpsIn order to explore an excellent carrier for indirect agglutination test, this study attempted to synthesis SiNPs by reverse microemulsion. To obtain Red-SiNps we modified SiNps with C.I. reactive red136and optimized the reaction conditions, Field emission scanning electron microscope SU-70was used to characterize morphology and particle size of the samples. The size and the potential of Red-SiNps before and after the antibody modification were characterized by laser particles size analyzer Nano2S, and obtained IgG-red-SiNps with immune activity. In the end, in order to study the performance of new aggregation test, we established an aggregation test based on IgG-red-SiNps which modified with antibodies of three different bacteria and viruses. Aggregation test were separately performed on the U-shaped plate, V-shaped plate and cove slide. The digital camera was used to recorded the aggregated results. The results showed:The particle size of Red-SiNps was uniform, size distribution of particle was narrow, performance was stable, dye modification was easy to and color of particles was rich. Meanwhile, The amount of dye coupled by APTES was much larger than APS, and the reaction time of coupling dye by APS was only8hours. The results of aggregation tests in the U-shaped plate, V-shaped plate and cove slide showed that Red-SiNps coupled with red dye gave the SiNps bright color, increased agglutination signal and made the phenomenon more intuitive and eye-catching. Meanwhile, The agglutination test illustrated the new aggregation technology based colored-SiNps had an excellent performance include good specificity, fast and high accuracy. And the aggregation test can not only detect bacteria such as S.pullorum&S.gallinarum and E. sakazakii in these reactors, but also detect viruses such as EDSV. All of these aggregation test were performed well. But the deficiencies were still exist, the aggregation happen on the U-shaped plate and V-shaped plate cost long time, it took20-30mins to get aggregated results, even so the time is much shorter than aggregation test based on other carriers. While the results of the aggregation test on the cove slide could be achieved within20-30s, and it was faster and more intuitive than the aggregation test on U-shaped and V-shaped plate.2Simultaneously detect two pathogens byagglutination test based on Colored-SiNps as a carrierAimed to explore a new kind of agglutination test which can simply, sensitive, simultaneously detect pathogenic bacteria, an agglutination test based on colored-SiNps had been established. Colored-SiNps were used as agglutination test carrie, monodisperse red-SiNps and blue-SiNps were prepared by reverse microemulsion with C.I. Reactive red136and C.I. Reactive Blue14. Then the red-SiNps was sensitized with antibody against E. sakazakii and enoted as IgG-red-SiNps; the blue-SiNps were coated with antibody against S. pullorum&S. Gallinarum and denoted as IgG-blue-SiNps. The agglutination tests based on IgG-colored-SiNps were sensitive and specifici and the analysis time was less than3min. The mixture solutions of IgG-red-SiNps and IgG-blue-SiNps could agglutinate with E. sakazakii and S. pullorum&S. gallinarum separately on a glass slide. The agglutination test could detect both E. sakazakii and S. pullorum&S. gallinarum simultaneously with obvious agglutination phenomenon. The E. sakazakii and S. pullorum&S. gallinarum could be detected in a range from103to109CFU/mL. The mixture solution of IgG-red-SiNps and IgG-blue-SiNps could detect S. pullorum&S. gallinarum and E. sakazakii in the infected food sample too. The colored-SiNps were suitable for being used as agglutination test carrier. The new agglutination test took advantage of the signal amplification of colored-SiNps and specificity of antibody. This easy and rapid new agglutination test might be useful for simultaneously screening different pathogenic microorganisms of foods and feeds in the field.3Study the detection of egg drop syndrome virus with agglutination test based on Yellow-SiNpsThis paper has been implemented to simultaneously detect one kind or two kinds of pathogenic bacteria, in order to expand the range of application, we developed an agglutination test based on SiNps as carrier to detect virus. We selected egg drop syndrome virus (EDSV) as target and detected it with our new aggregation test. And in this study the yellow silica nanoparticles (Yellow-SiNps) were synthesized by reverse microemulsion method, scanning electron microscopy and laser particle size analyzer were used to characterize their morphology, dispersion and stability. The result showed that Yellow-SiNps had uniform particle size, good stability and color stability. The Yellow-SiNps were modified with antibodies to EDSV, and used as the carrier of agglutination test. The established a new aggregation test was used for the rapid detection of EDSV. The results showed that the process of detection was simple, the phenomenon of agglutination test was obvious, intuitive and easy to distinguish by naked eye, specific. The detected limit for the target virus was among103～109cfu·mL-1, IgG-Yellow-SiNps were very stable and repeatable, after being stored at4℃for60days, the aggregation test result did not have any difference. The application of agglutination test based on SiNps as carrier to detect virus also showed good specificity and accuracy. Therefore, the new agglutination test could not only be used for rapidly detecting pathogenic bacteria, but also be used for rapid detection of virus.4Preparation of micron monodisperse silica nanoparticlesThe agglutination techniques based on nanoscale silica particles of prior study have shown that the nanoscale silica particles as the carrier of indirect agglutination test had an excellent performance not only in simultaneously detecting two kinds of bacteria but also in detecting virus. In order to study the effect of particle size on the agglutination test and to achieve aggregated signal amplification, we explored a kind of microscale colored silica nanoparticles (colored-SiNps) and established a new agglutination test based on microscale colored-SiNps for rapid detection of foodborne pathogens. Therefore, we synthesized microscale SiNps by semi-continuous Stober method, and modified the surface with C.I. active red136. 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 microscale SiNps has a narrow size distribution, ruled morphology and good monodispersity. And Red-SiNps had bright red and the reaction mechanism for it was also discussed. We also successfully developed a new agglutination test based microscale SiNps as the carrier.5Study on the detection of E. sakazakii with agglutination test based on microscale red silica nanoparticlesIn order to explore a rapid, accurate simple technology for detection of foodborne pathogen, in this paper E. sakazakii was detected by agglutination test based on microscale red silica nanoparticles (Red-SiNps) as a carrier. In this experiment Red-SiNps were synthesized by Stober method and modified with C.I. Reactive Red136. And then Red-SiNps were modified by amino and carboxyl and antibody against E. sakazakii to prepare IgG-red silica nanoparticles (IgG-Red-SiNps). E. sakazakii was detected by agglutination test, in which IgG-Red-SiNps were used as carrier. Color of Red-SiNps was characterized by UV-vis spectroscopic, the surface potential of Red-SiNps was characterized by Nano2s laser granularity potential instrument, specificity, sensitivity, stability results of agglutination test were recorded with digital camera. Results showed that color of Red-SiNps was bright, surface charge of Red-SiNps was negative, which allowed Red-SiNps was dispersed evenly. Results of new agglutination test based on IgG-Red-SiNps as the carrier were striking and easy to judge, the detection limits of agglutination test for target bacteria was103cfu/mL. Agglutination reaction was no cross and good specific. At4℃, IgG-Red-SiNps could be preserved for nearly30days, with good stability. Agglutination test based on Red-SiNps as the carrier has many advantages:good specificity, stability, fast, simple and economical. |
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