纳米磁珠对单增李斯特菌低场磁共振检测的影响研究

发布时间：2018-06-29 15:26

本文选题：单增李斯特菌 + 低场磁共振技术　；参考：《上海师范大学》2016年硕士论文

【摘要】：食源性疾病是指通过摄食而进入人体的有毒有害物质(包括生物性病原体)等致病因子所造成的疾病。一般可分为感染性和中毒性。近年来,食源性疾患的发病率居各类疾病总发病率的前列,是当前世界上最突出的卫生问题。近年来,随着磁性纳米技术的不断发展,磁性纳米材料以其独特的理化性质,在各个领域都有着非常广泛地应用,如定向载药、生物分子的分离与富集、疾病的临床治疗与诊断、各种生物分子的检测以及生物传感器等方面。单增李斯特菌(Listeria monocytogenes)是全世界普遍公认一种重要的人畜共患食源性致病菌,其广泛存在于各种乳制品、蔬菜、肉类等食品中,特别是即食食品中,对人的健康安全造成了极大的威胁。人类受感染后可导致胃肠炎、败血症、脑膜炎、流产,尤其是孕妇、婴儿、老年人及免疫力低下者更易感染。本论文采用生物功能化磁性纳米粒子与低场磁共振技术结合而建立了一种单增李斯特菌的快速检测方法,探究不同纳米材料的低场磁共振技术在单增李斯特菌快速检测中的影响。第一部分成功合成了生物功能化的磁性纳米材料。首先通过高温热解法合成了油溶性Fe/Fe3O4和Fe3O4磁性纳米粒子,Fe/Fe3O4磁性纳米粒子粒径约为12.22nm,Fe3O4磁性纳米粒子粒径约为11.29nm。其次利用反相微乳液法对磁性纳米粒子进行表面修饰,制备出二氧化硅包裹,修饰氨基的Fe/Fe3O4和Fe3O4磁性纳米粒子,修饰之后,纳米粒子粒径均一、生物相容性好。接着使用EDC?HCL和NHS活化已经氨基功能化的Fe/Fe3O4和Fe3O4,从而制备得到特异性免疫化的纳米材料。第二部分探究了纳米磁珠对单增李斯特菌低场磁共振检测的影响。首先优化单增李斯特菌的培养条件,单增李斯特菌在李斯特氏菌增菌肉汤中生长最迅速、旺盛。其次使用不同纳米材料成功构建了一种单增李斯特菌的快速、灵敏检测方法,靶标菌可以与免疫功能化磁珠的抗体特异性结合,引起实验体系中横向弛豫时间(T2)的变化,NMR可以敏感的检测到这一变化。接着研究不同纳米材料在低场磁共振检测方法的影响,不同纳米材料都对靶标菌(L.monocytogenes)有很强的特异性。经比较实验,生物功能化的Fe/Fe3O4纳米粒子和Fe3O4纳米粒子进行核磁共振实验时最优稳定剂都为2%脱脂牛奶;反应时,Fe/Fe3O4纳米粒子的最优浓度为0.16 mg/m L,Fe3O4纳米粒子的反应最适浓度为0.2mg/m L;Fe/Fe3O4纳米粒子的最佳孵育时间为30min,而Fe3O4纳米粒子的最佳孵育时间为40min,前者所用时间稍短;不同的纳米材料,快速检测单增李斯特菌的线性检测范围均为1~103CFU/mL,检测范围一致,表明检测的灵敏度与纳米材料本身并没有很大关联。不同的纳米材料用于实际样品的检测均显示该检测方法可以应用于食品样品的检测并对于L.monocytogenes有很强的特异性。所以经比较可知不同纳米材料在低场磁共振技术快速检测L.monocytogenes中效果相似,在各项实验参数中并没有很大差别。本文构建了一种特异性强的单增李斯特菌快速检测方法,并且探究纳米磁珠对单增李斯特菌低场磁共振检测的影响,进而增加该方法的稳定性,对今后食源性致病菌的快速检测有重要意义。
[Abstract]:Food borne diseases are diseases caused by pathogenic factors such as toxic and harmful substances (including biological pathogens) which enter the human body through feeding. Generally, it can be divided into infectious and toxic. In recent years, the incidence of food borne diseases is the forefront of the total incidence of all kinds of diseases. It is the most prominent health problem in the former world. With the development of magnetic nanotechnology, magnetic nanomaterials have been widely used in various fields for their unique physical and chemical properties, such as directional drug loading, separation and enrichment of biomolecules, clinical treatment and diagnosis of diseases, detection of various biomolecules and biosensors. Single increment List Rand (Listeria monocytog) Enes) is widely recognized as an important food borne pathogenic bacteria in the world. It is widely found in all kinds of dairy products, vegetables, meat and other foods, especially in instant food, which poses a great threat to the health and safety of human beings. After human infection, it can cause gastroenteritis, septicemia, meningitis, abortion, especially in pregnant women, infants, and old age. In this paper, a rapid detection method of single increasing Lester bacteria was established by combining biofunctionalized magnetic nanoparticles with low field magnetic resonance technology to explore the effect of low field magnetic resonance (MRI) on the rapid detection of single increasing Lester bacteria in different nanomaterials. Oil soluble Fe/Fe3O4 and Fe3O4 magnetic nanoparticles are synthesized by high temperature pyrolysis. The particle size of Fe/Fe3O4 magnetic nanoparticles is about 12.22nm, and the particle size of Fe3O4 magnetic nanoparticles is about 11.29nm.. Secondly, the surface modification of magnetic nanoparticles by reverse phase microemulsion is used to prepare silicon dioxide package. Coated, modified amino Fe/Fe3O4 and Fe3O4 magnetic nanoparticles, after modification, nanoparticles have uniform particle size and good biocompatibility. Then EDC HCL and NHS are used to activate the amino functional Fe/Fe3O4 and Fe3O4, and the specific immunized nanomaterials are prepared. The second part explores the low field magnetic flux of nanomagnetic beads to the single increase of Lester bacteria. The influence of resonance detection. First, the culture conditions of monosalmus monocytogenes were optimized. The growth of the single increase of Lester bacteria was the fastest and vigorous in the mushroom broth of Listeria bacteria. Secondly, a rapid and sensitive detection method of single increase of Lester bacteria was successfully constructed with different nanomaterials, and the target bacteria could be specific to the antibody specificity of the immunomagnetic beads. In combination, the changes in the transverse relaxation time (T2) in the experimental system can be caused by NMR. The effect of different nanomaterials on the low field MRI detection method is studied. The different nanomaterials have strong specificity to the target bacteria (L.monocytogenes). By comparative experiments, the biofunctionalized Fe/Fe3O4 nanoparticles and The optimal stabilizer for Fe3O4 nanoparticles was 2% skimmed milk, the optimum concentration of Fe/Fe3O4 nanoparticles was 0.16 mg/m L, the optimum concentration of Fe3O4 nanoparticles was 0.2mg/m L, the optimum incubation time of Fe/Fe3O4 nanoparticles was 30min, while the best incubation time of Fe3O4 nanoparticles was 40min, the former was the former. The time used is slightly shorter, and the linear detection range of the rapid detection of Lester bacteria is 1~103CFU/mL, and the detection range is the same. It shows that the sensitivity of the detection is not closely related to the nanomaterial itself. The detection of different nanomaterials for the actual samples shows that the detection method can be applied to food samples. The detection has a strong specificity for L.monocytogenes, so it is known that the effect of different nanomaterials in the rapid detection of L.monocytogenes in low field magnetic resonance technology is similar, and there is no big difference in the experimental parameters. The effect of beads on the detection of single increased Lester's low field MRI can further increase the stability of the method and is of great significance for the rapid detection of foodborne pathogens in the future.
【学位授予单位】：上海师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R446.5;TB383.1

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