基于核酸适配体识别-时间分辨荧光纳米探针的生物毒素检测方法研究

发布时间：2018-07-23 19:38

【摘要】：生物毒素引起的食品中毒事件在全球各地时有发生,不但威胁人类的健康,而且由于其具有极强的生理毒性,有可能被恐怖分子用于恐怖袭击和生物战,对社会的食品安全和人们的身体健康造成了巨大威胁。要从根本上解决这类食品安全问题,就必须对从农田到餐桌的食品供应链中各环节实施全程管理和监控。而目前传统的生物毒素检测方法主要包括高效液相色谱、质谱和免疫学检测法等,大多需要依赖抗体,检测设备昂贵、操作繁琐且非常耗时,很难满足对现代食品安全检测技术快速、灵敏、便捷的要求。因此,本论文立足于食品安全检测技术的研究,以金黄色葡萄球菌肠毒素和蓖麻毒素为检测对象,结合核酸适配体技术和时间分辨荧光分析技术,建立了一系列快速、准确、灵敏、便捷的新型分析方法用于生物毒素定量检测,并对其相关机理进行了探讨。一方面,通过一步溶剂热法合成表面氨基化的Fe3O4磁性纳米颗粒(即磁珠),采用X射线衍射(XRD)、透射电镜(TEM)和傅里叶转换-红外光谱(FT-IR)分别对其晶型成分、形貌和表面基团进行表征,证实该材料具备表面化学修饰的条件。首先,进行了基于磁性分离的特异性识别金黄色葡萄球菌肠毒素A(SEA)核酸适配体筛选技术的研究。将SEA进行磁珠固定化,并以此为筛选靶标,以空白磁珠为负筛靶标、金黄色葡萄球菌肠毒素B、C1(SEB、SEC1)磁珠为反筛靶标,采用负筛、反筛相结合的FLu Mag-SELEX技术进行十四轮体外筛选。通过荧光分析法检验了SEA适配体对靶标结合的亲和力和特异性,确定最佳适配体A15,其解离常数为48.57±6.52nmol/L,具有良好的特异性识别SEA能力。并将筛选获得的适配体用于实际样品牛奶中SEA的测定,方法检测限为8.7 ng/m L。其次,基于与上述相似的原理和方法,进行了特异性识别SEC1核酸适配体筛选技术的研究。将SEC1进行磁珠固定化作为筛选靶标,以空白磁珠为负筛靶标、SEA和SEB磁珠为反筛靶标,采用负筛、反筛相结合的FLu Mag-SELEX技术进行十四轮体外筛选,获得了SEC1的特异性结合适配体,解离常数为49.43±11.76 nmol/L,并以此建立了SEC1的荧光检测方法,该法检测限为6 ng/m L。另一方面,利用一步溶剂热法合成多种镧系掺杂的时间分辨荧光纳米探针。通过研究反应条件,获得荧光寿命长、发光性能好、水分散性好的纳米级荧光颗粒,并含有生物官能团,为用于生物探针标记提供了条件。经过TEM、时间分辨荧光光谱、XRD、FT-IR、紫外吸收光谱(UV)等表征手段证实成功制备新型镧系掺杂的时间分辨荧光纳米探针。在新型镧系掺杂时间分辨荧光纳米探针的应用中,首先将蓖麻毒素适配体为识别元件,构建一种生物毒素的高灵敏检测方法。采用一步溶剂热法制备Eu3+掺杂的KGd F4纳米颗粒标记适配体生成荧光探针,利用氧化石墨烯(GO)良好的水分散性和高效的猝灭性能,建立了一种基于适配体的新型时间分辨荧光分析法检测了均相饮用水中蓖麻毒素含量。在最优条件下,该方法的检测线性范围为0.05~50 ng/m L(R2=0.9975),检测限为0.008 ng/m L(3S/N),方法的精密度好。进行了蓖麻毒素(0.075~12.5 ng/m L)在饮用水中的加标试验,该方法的回收率为89.42%~107.1%。将本方法与商业化的ELISA试剂盒进行对比试验,比较两种方法测定同批蓖麻溶液的浓度,结果表明二者一致性良好,证实所建立的方法可应用于实际分析检测。在上述研究的基础上,将基于适配体特异性识别-时间分辨荧光纳米探针的技术应用于多组分生物毒素的同时高灵敏检测。以SEA、SEB和SEC1适配体为识别元件,选择具有特征荧光发射互不干扰的多色镧系掺杂纳米颗粒为检测信号、GO作为高效宽频荧光猝灭剂,建立了一种基于多色时间分辨荧光共振能量转移(TR-FRET)的同时检测三种金黄色葡萄球菌肠毒素的分析方法。确定了建立该检测方法的关键条件为多色荧光生物探针的比例为Eu3+:Tb3+:Dy3+=2:1:3.5、氧化石墨烯浓度为0.25 mg/m L,并在该条件下实现了SEA、SEB和SEC1的同时检测,检测线性范围分别为0.08~10 ng/m L、0.10~9 ng/m L和0.05~5 ng/m L,检测限分别为0.062 ng/m L、0.069 ng/m L、0.040 ng/m L(3S/N)。将三种肠毒素以三个浓度(0.5 ng/m L、1.0 ng/m L、5.0 ng/m L)加入未稀释的牛奶样品中进行加标实验,回收率的结果在92.76%~114.58%之间。结果表明,该方法具有特异性良好、灵敏度高和高通量分析的特点,尤其提高了对食品复杂基质中多组分检测的分析性能。
[Abstract]:Food poisoning caused by biological toxins occurs all over the world, not only threatens human health, but also because of its strong physiological toxicity, it is likely to be used by terrorists for terrorist attacks and biological warfare, which poses a great threat to food safety and health of the people. The whole problem must be managed and monitored throughout the chain of food supply chain from farmland to table. And the traditional methods of detection of biotoxin mainly include high performance liquid chromatography, mass spectrometry and immunoassay. Most of them need to rely on antibodies, which are expensive, complicated and time-consuming, so it is difficult to meet the modern food. The product safety detection technology is fast, sensitive and convenient. Therefore, this paper is based on the research of food safety detection technology, based on Staphylococcus aureus enterotoxin and ricin as the detection object, combined with nucleic acid aptamer technology and time resolved fluorescence analysis technology, a series of fast, accurate, sensitive and convenient analytical formulas are established. The method is used for quantitative detection of biotoxin and its mechanism is discussed. On one hand, one step solvothermal method is used to synthesize the surface aminated Fe3O4 magnetic nanoparticles (magnetic beads). X ray diffraction (XRD), transmission electron microscopy (TEM) and Fu Liye conversion - infrared spectroscopy (FT-IR) are used to determine the crystalline components, morphology and surface groups of the surface groups respectively. It is confirmed that the material has the condition of surface chemical modification. First, a magnetic separation specific identification of Staphylococcus aureus enterotoxin A (SEA) aptamer screening technology was carried out. SEA was immobilized on magnetic beads and used as a screening target, blank beads as negative screening targets, Staphylococcus aureus enterotoxin B, C1 (SE). B, SEC1), the magnetic bead is the screening target, using the negative sieve and the FLu Mag-SELEX technique of the screen phase combined with the FLu Mag-SELEX. The affinity and specificity of the SEA aptamers to the target binding are tested by fluorescence analysis, and the best aptamer A15 is determined, and its dissociation constant is 48.57 + 6.52nmol/L. It has a good specific identification of SEA ability. And the sieves will be screened. The selected aptamers are used for the determination of SEA in the actual sample milk. The detection limit is 8.7 ng/m L.. Based on the similar principles and methods, the specific identification of SEC1 nucleic acid aptamer screening technology is carried out. SEC1 is used as a magnetic bead immobilization target, blank beads as negative sieve target, SEA and SEB magnetic beads. The screening target is screened by negative sieves and FLu Mag-SELEX combined with screen. The specific binding ligand of SEC1 is obtained, and the dissociation constant is 49.43 + 11.76 nmol/L, and the fluorescence detection method of SEC1 is established. The detection limit of the method is 6 ng/m L. on the other hand, one step solvothermal method is used to synthesize a variety of lanthanide doping. By studying the reaction conditions, the nanofluorescence particles with long fluorescence lifetime, good luminescence performance and good water dispersibility are obtained by studying the reaction conditions, and the biological functional groups are contained, which provide the conditions for the biological probe labeling. After TEM, the time resolved fluorescence spectra, XRD, FT-IR, UV and other characterization methods are proved to be successful. A novel lanthanide doped time resolved fluorescent nano probe was prepared. In the application of a new lanthanide doped time resolved fluorescent nano probe, a highly sensitive detection method for a biological toxin was constructed by using ricin aptamers as identification elements. One step solvothermal method was used to prepare Eu3+ doped KGd F4 nanoparticles. A novel time resolved fluorimetric method based on aptamers was established to detect the content of ricin in homogeneous drinking water by a novel time resolved fluorescence analysis method based on the good dispersibility and high efficiency of GO. Under the optimal conditions, the linear range of the method was 0.05~50 ng/m L (R2=0.9975), and the detection limit was 0.008 ng/m L. 3S/N), the precision of the method is good. The addition test of ricin (0.075~12.5 ng/m L) in drinking water was carried out. The recovery rate of the method was 89.42%~107.1%., the method was compared with the commercial ELISA kit, and the two methods were compared to determine the concentration of the same batch of castor solution. The results showed that the consistency of the two kinds of castor solution was good and proved to be established. The method can be applied to practical analysis and detection. On the basis of the above research, the technology based on the aptamer specific identification time resolved fluorescent nano probe is applied to the simultaneous high sensitivity detection of multi component biotoxin. SEA, SEB and SEC1 aptamers are used as identification elements to select the polychromatic lanthanide doped with characteristic fluorescence emission without interference. A method for the simultaneous detection of three Staphylococcus aureus enterotoxin based on polychromatic time resolved fluorescence resonance energy transfer (TR-FRET) was established by GO as a high efficiency and wide frequency fluorescence quenching agent. The key condition for establishing this detection method was that the proportion of polychromatic fluorescence probe was Eu3+: Tb3+ Dy3+=2:1:3.5, the concentration of graphene oxide is 0.25 mg/m L, and the simultaneous detection of SEA, SEB and SEC1 is realized under this condition. The detection linear range is 0.08~10 ng/m L, 0.10~9 ng/m L and SEA, respectively. The detection limit is 0.069. Three concentrations (0.5, 1, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5, 5 .0 ng/m L) was added to the samples of non diluted milk, and the results of recovery were between 92.76%~114.58%. The results showed that the method had the characteristics of good specificity, high sensitivity and high throughput analysis, and especially improved the analysis performance of multi component detection in the food complex matrix.
【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：O657.3;TS207.3

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