研究建立了免疫磁珠和荧光量子点标记的抗体检测大肠杆菌O157∶H7的方法。采用溶剂热法制备了Fe3O4纳米颗粒,并用SiO2、羧基和大肠杆菌O157∶H7抗体依次包覆制得免疫磁珠。游离的大肠杆菌O157∶H7首先与免疫磁珠结合,然后由量子点标记的抗体与大肠杆菌结合,形成一个三明治结构;随后分析磁分离采集的磁珠的荧光强度(激发/发射波长为370 nm/472 nm)。SiO2壳结构的引入,减少了传统免疫磁珠的非特异性吸附,提高了对目标菌的选择性,同时有效的阻止了三明治结构中量子点因电子转移至Fe3O4而引起的荧光猝灭,保证了分析方法的可行性。动态范围为10~108 CFU/mL,检出限为10 CFU/mL。牛肉、牛奶和蜂蜜样品中大肠杆菌O157∶H7的平均加样回收率分别为95%~104%、90%~94%和90%~110%;相对标准偏差分别为2.1%~3.8%、3.2%~7.8%和5.8%~6.3%。
1. Research background and content
Escherichia coli O157∶H7 is a major foodborne pathogen, which often leads to severe diseases such as hemolytic uremic syndrome, bloody diarrhea and even death. Infection with very low doses of live E. coli O157∶H7 can cause disease and further outbreaks. Rapid and sensitive detection methods can help clinical management and prevent transmission. In this paper, immunomagnetic beads and fluorescent quantum dots were used to detect E. coli O157∶H7.
2.Methods
The principle of reaction is shown in Fig.1. Using FeCl3·6H2O as the iron source, anhydrous sodium acetate as the precipitant, and polyethylene glycol 2000 as the surfactant, reacted in a mixed solution of ethylene glycol and diethylene glycol as the reducing agent at 200 ℃ for 12 h to prepare the monomer Fe3O4 nanoparticles with good dispersibility and uniform particle size. Fe3O4 was dispersed in deionized water, and then ammonia water and isopropyl alcohol solution were added uniformly, followed by the addition of ethyl teosilicate drop by drop. After 20 h of continuous reaction at 35 ℃ under the protection of nitrogen, Fe3O4@SiO2 was obtained. Then, coreshell immunomagnetic beads Fe3O4@SiO2@Ab1 were obtained by carboxyl binding antibodies against E. coli O157∶H7, and then the fluorescent probe Qds@Ab2 was obtained by using fluorescent quantum dots to label the resistance regime of E. coli O157∶H7. Subsequently the free E. coli O157∶H7 was first combined with the immune magnetic beads, and then the antibody labeled by quantum dots was combined with E. coli O157∶H7 to form a sandwich structure. Finally, the complex of “immune nano magnetic beads - bacteria - immune quantum dots” was detected by fluorescence (excitation/emission wavelength was 370/472 nm).
3.Conclusion
The introduction of silica shell structure reduces the non-specific adsorption of traditional immune magnetic beads, improves the selectivity of target bacteria, and effectively prevents the fluorescence quenching caused by electron transfer to ferric oxide in the sandwich structure of quantum dots, ensuring the feasibility of the analysis method. The dynamic range of the immunoassay was 10-108 CFU/mL, and the detection limit was 10 CFU/mL. The average recoveries of E. coli O157∶H7 in beef, milk and honey samples were 95%-104%, 90%-94% and 90%-110% respectively; and the relative standard deviations were 2.1%-3.8%, 3.2%-7.8% and 5.8%-6.3% respectively.
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