赭曲霉毒素A的荧光生物传感检测新方法研究

发布时间：2018-06-11 16:05

本文选题：量子点 + 纳米金　；参考：《聊城大学》2017年硕士论文

【摘要】：真菌毒素引起的食品安全问题已成为世界各国关注的焦点,目前已报道的检测方法有很多种但各自都有其优缺点,因此,发展一种高选择性、高灵敏度的检测方法具有重要的理论和现实意义。随着科技的发展,纳米材料由于其独特的结构及特性在众多的领域研究中受到越来越多的青睐。其在生命科学领域中的应用已经成为该领域研究的主流。纳米材料与生物传感器相结合,主要包括生命医药、化学化工、纳米生物学等多个学科,具有很高的研究价值。本论文以核酸适配体的特异性识别能力为基础,将纳米技术与生物传感技术结合,利用纳米材料在光学、化学及生物学方面表现出的特殊性质,以一种真菌毒素-赭曲霉毒素A为研究对象,建立了三种测定赭曲霉毒素A的新方法,实现了对赭曲霉毒素A的高灵敏、高选择、高效率的识别与检测。全文分为以下三部分:一、基于量子点荧光共振能量转移检测赭曲霉毒素A基于量子点与荧光猝灭基团之间构成的荧光共振能量转移体系,以量子点标记赭曲霉毒素A适配体与荧光猝灭基团标记的补体杂交构成荧光传感探针,当有赭曲霉毒素A存在时,由于其适配体与赭曲霉毒素A的高度亲和作用,使传感探针上结合的荧光猝灭剂减少,荧光增强,从而建立了一种检测赭曲霉毒素A的荧光分析方法。这种检测方法简单、快速、特异性强,在最优的条件下,该方法在0.2μmol/L~1.6μmol/L内有良好的线性关系,线性回归方程为:ΔF=92.2c+95.06,线性相关系数R=0.9955,方法的检出限为0.16μmol/L。二、基于纳米金猝灭荧光的适配体传感技术检测赭曲霉毒素A基于分散和聚沉状态下的纳米金的荧光猝灭效应的差异以及核酸适配体的特异性识别能力为基础,一种简单、快速、低成本的适配体传感技术检测赭曲霉毒素A。当赭曲霉毒素A存在时,可以与其适配体特异性结合,导致适配体构象发生改变,适配体脱离纳米金表面,纳米金抗盐能力减弱,荧光增强,从而建立了一种检测赭曲霉毒素A的荧光分析方法。实验过程中对盐浓度、反应时间等条件进行了优化。在最优的条件下,该方法在25 nmol/L~300 nmol/L范围内有良好的线性关系,线性回归方程为:ΔF=0.293c+4.263,线性相关系数R=0.9957,方法的检出限为22.7 nmol/L。三、基于抗体包被的荧光免疫分析新模式测定赭曲霉毒素A基于二抗与半抗原竞争结合固相抗体的免疫分析新模式,建立了测定赭曲霉毒素A的新型免疫荧光分析方法。本实验采用竞争型的免疫反应模式,IgG-FITC与半抗原OTA竞争结合固相抗体,随着半抗原OTA浓度的增加,与固相抗体结合的IgG-FITC浓度减少,荧光强度逐渐降低。在最优的实验条件下,该方法在1 nmol/L~100μmol/L范围内有良好的线性关系,线性回归方程为ΔF=43.68lgC+4.619,线性相关系数R=0.9918。方法的检出限为0.34 nmol/L。
[Abstract]:The food safety problems caused by mycotoxins have become the focus of attention in the world. At present, there are many kinds of detection methods reported, but each has its own advantages and disadvantages. Therefore, the development of a high selectivity, High sensitivity detection method has important theoretical and practical significance. With the development of science and technology, nanomaterials are more and more popular in many fields because of their unique structure and characteristics. Its application in the field of life science has become the mainstream of research in this field. The combination of nanomaterials and biosensors, including life medicine, chemical chemistry, nanobiology and so on, has high research value. Based on the specific recognition ability of aptamers of nucleic acids, this paper combines nanotechnology with biosensor technology and makes use of the special optical, chemical and biological properties of nanomaterials. Three new methods for the determination of ochratoxin A were established with a mycotoxin-ochratoxin A as the object of study. The identification and detection of ochratoxin A with high sensitivity, selectivity and efficiency were realized. The thesis is divided into three parts as follows: first, the fluorescence resonance energy transfer system between quantum dots and fluorescence quenching groups is used to detect ochratoxin A based on the fluorescence resonance energy transfer of quantum dots. A fluorescence sensing probe was constructed by hybridization of the aptamer of ochratoxin A with the complement labeled by fluorescence quenching group. When ochratoxin A was present, the aptamer was highly compatible with ochratoxin A. A fluorescence analysis method for the detection of ochratoxin A was established by reducing the binding fluorescence quenching agent and enhancing the fluorescence intensity on the sensing probe. The method is simple, rapid and specific. Under the optimal conditions, the method has a good linear relationship in the range of 0.2 渭 mol / L ~ (1.6 渭 mol / L). The linear regression equation is: 螖 F ~ (92. 2) c 95.06, the linear correlation coefficient is R ~ (0.9955), and the detection limit of the method is 0.16 渭 mol 路L ~ (-1). Secondly, the difference of fluorescence quenching effect of ochratoxin A based on the fluorescence quenching of gold nanoparticles and the specific recognition ability of aptamers of nucleic acid is based on the difference of fluorescence quenching effect of ochratoxin A based on the fluorescence quenching of nanocrystalline gold and the ability of specific recognition of aptamers of nucleic acid. Rapid and low cost aptamer sensing technology for detection of ochratoxin A. When ochratoxin A exists, it can bind specifically with its aptamer, which leads to the change of aptamer conformation, the aptamer breaks away from the surface of nano-gold, the salt resistance of nano-gold decreases and the fluorescence increases. A fluorescence analysis method for the detection of ochratoxin A was established. The conditions of salt concentration and reaction time were optimized. Under the optimal conditions, the method has a good linear relationship in the range of 25nmol / L and 300nmol / L, the linear regression equation is 螖 F _ (0.293c) 4.263, the linear correlation coefficient R _ (r) is 0.9957, the detection limit of the method is 22.7 nmol / L, the linear regression equation is: 螖 F _ (0.293c) 4.263, the detection limit of the method is 22.7 nmol / L. 3. A new immunofluorescence analysis method for the determination of ochratoxin A was established based on a new immunoassay model based on the second antibody and hapten competition combined with solid phase antibody. In this experiment, competitive immunoreaction model was used to compete with hapten OTA to bind solid phase antibody. With the increase of hapten OTA concentration, the concentration of IgG-FITC bound with solid phase antibody decreased, and the fluorescence intensity decreased gradually. Under the optimal experimental conditions, the method has a good linear relationship in the range of 1 nmol / L ~ 100 渭 mol / L, the linear regression equation is 螖 F _ (4) 43.68 lgC 4.619, and the linear correlation coefficient R _ (0) 9918. The detection limit of the method was 0.34 nmol / L.
【学位授予单位】：聊城大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3

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