糖修饰荧光多聚对苯乙炔检测致病菌的研究

发布时间：2018-02-11 21:47

本文关键词： 多聚对苯乙炔糖修饰食源性致病菌大肠杆菌荧光　出处：《中国农业科学院》2016年博士论文　论文类型：学位论文

【摘要】：近年来,全球食源性疾病的发病率在逐步升高,已成为主要的社会公共安全问题。食源性致病菌如大肠杆菌(E. coli)的来源有许多,因此我们需要建立一种快速的检测技术来确保食品的安全性,以避免食源性疾病。传统的检测方法存在许多缺陷,如检测速度比较慢。因此,建立一种成本低,特异性强,灵敏度高的快速检测方法是十分必要的。本论文介绍了糖修饰的多聚对苯乙炔的合成方法及功能。糖修饰的荧光多聚物是一种新兴的快速检测技术,由于其具有识别和信号传导的独特性质,在筛查和检测细菌方面具有良好的应用前景。另外,因为在一条高分子单链上有很多的配体,使其可产生多重相互作用。尽管近几年PPEs应用广泛,但是还存在着应用对象范围有限、合成成本高等一些缺陷,而其中中间体设计和生产上的简单化和经济可行性是PPEs成为有效生物传感的主要限制因素。因此,可通过设计合成一种具有高效荧光且成本低廉的PPE多聚物来解决上述问题。本论文的主要目的即在PPE作为信号传导单元的基础上通过侧链糖基修饰实现识别分子与目标分子的快速结合,从而达到快速检测的目的。实验进展可分为三部分：探针的合成,光物理特性的测定和性能评估。合成工作是从乙氨基甘露糖和氨基半乳糖开始的,这样可引入胺基官能团而不影响羟基的功能。糖由于对细胞表面凝集素具有高结合力,可作为识别基团连接到多聚链上。N-Cbz-aminoethanol具有商业化、晶体状且去保护可一步实现(如第二章所述)等优点,因此被选择作为最适糖基受体。此技术简单,步骤少,经验少的化学家也可以轻松掌握,且产物总收率很高。第三章详细介绍了PPE和1,4-二甲氧基苯的合成。采用多步反应合成单体diethynyldimethoxybenzene和diidodipropanoic acid,期间产生大量的中间体。通过钯催化剂的Sonogashira聚合反应将其相连并进一步用甘露糖修饰以减少非特异性相互作用的影响。通过紫外-可见光谱和荧光光谱来研究其荧光特性和对刀豆蛋白A(Con A,可与碳水化合物相连接的蛋白)的响应.利用荧光共聚焦显微镜研究细胞成像以及检测灵敏性,实验表明多聚物发出的绿色荧光可被甘露糖结合凝集素猝灭。我们合成的中间体化合物可由廉价易得的化工原料通过几步反应而合成,并且这些中间体可以转化成高效的识别基团。第四章详细介绍了聚合物的光物理特性,这些特性是荧光基团性能和是否适用于生物传感器领域的关键指标。该聚合物表现出绿色荧光,吸收波长为393nm,发射波长为466.5nm。虽然它的量子产率不是很高(10.8%),但具有较大的斯托克斯位移,使它成为理想的传感器分子。通过研究糖修饰PPE的荧光猝灭过程,可知聚合物的荧光被淬灭是由于和Con A形成了基态复合物。Con A是一种能够与糖(甘露糖)特异性结合的凝集素,在细胞信号传导、细胞表面识别和病原体的对接过程中起着关键作用。在此研究中,我们选择Con A,是由于它的毒性低并且可以用来检测配体-蛋白质的相互作用。第五章详细介绍了聚合物的检测机制。肠道内大肠杆菌通过与聚合物的多价相互作用得到荧光细菌聚合物。随后,其荧光强度在逐渐降低。为了确定最佳结合时间,需要测定一定时间间隔内聚合物细菌悬浮液的荧光强度。实验表明,培养30分钟之内该体系可达到结合的最佳水平,其对细菌亲和力的检测限达到103cfu/ml。该体系的一个主要优点就是,它能够在30分钟内进行检测,克服了传统检测方法检测时间需要数天的缺陷。本文揭示了糖修饰PPE可成为良好的生物传感分子。分子末端的羧酸基团还可进一步与其他目标分子相结合。同时也证明了甘露糖修饰的聚合物可以检测FimH型大肠杆菌,这种菌能够表达甘露糖专一凝集素。多聚物表面的糖基保留了其与甘露糖结合凝集素相结合的能力。
[Abstract]:In recent years, the global incidence of foodborne illness rises gradually, has become a social problem of public safety. The main foodborne pathogenic bacteria such as Escherichia coli (E. coli) there are many sources, so we need to establish a rapid detection technology to ensure food safety, to prevent foodborne illness. Traditional detection methods exist many defects, such as the detection speed is slow. Therefore, the establishment of a low cost, high specificity, rapid detection method with high sensitivity is necessary. This paper introduces the synthesis method of sugar modified poly phenylenevinylene and fluorescence function. Sugar modified polymer is a new technique for rapid detection because of its unique character, recognition and signal transduction, has good application prospect in screening and detection of bacteria. In addition, because there are a lot of ligands in a single polymer chain, so that it can produce Multiple interactions. Although in recent years, PPEs is widely used, but there exists the application object of limited scope, high cost of synthesis of some defects, which is simple and economic feasibility of the production of intermediates and PPEs design is become the main limiting factors. Because of this biosensor, through the design and synthesis of a high fluorescence and low cost PPE polymer to solve the above problems. The main purpose of this paper is on the basis of PPE as signal transmission unit through the side chain glycosylation with rapid recognition molecules and target molecules, so as to achieve the purpose of rapid detection. Experimental progress can be divided into three parts: probe synthesis, determination and performance evaluation of optical properties. The synthesis work began from ethylamino mannose and galactosamine, which introduced amine groups without affecting hydroxyl function. Because of the sugar Cell surface lectin with high adhesion, can be used as the recognition group connected to the poly chain.N-Cbz-aminoethanol has commercial, crystal shape and to protect a step (as described in chapter second) etc., therefore is chosen as the most suitable glycosylacceptors. This technique is simple, less steps, less experienced chemist it can be easily mastered, and the yield is very high. The third chapter introduces the synthesis of PPE and 1,4- two methoxy benzene. The reaction of monomer diethynyldimethoxybenzene and diidodipropanoic during acid, produce a large number of intermediates. Through palladium catalyst Sonogashira polymerization reaction will affect its connected and further modified to use mannose to reduce nonspecific interactions. By UV Vis and fluorescence spectroscopy to study the fluorescence characteristics of concanavalin A (Con and A, can be connected with carbohydrate protein). Response. Using fluorescence confocal microscopy imaging of cells and the sensitivity experiments showed that green fluorescent polymers can be a mannose binding lectin quenching. We synthesized intermediate compounds by cheap chemical raw materials through several step reaction synthesis, and these intermediates can be transformed into the recognition group efficiently. The fourth chapter the photophysical properties of the polymer, these characteristics are the key indicators of the fluorophore performance and is applicable to the field of biosensors. The polymers exhibited green fluorescence, absorption wavelength is 393nm, emission wavelength is 466.5nm. although the quantum yield is not very high (10.8%), but with a large Stokes shift, make it become a sensor the ideal molecular. The fluorescence quenching process of sugar modified PPE, the fluorescent polymer is quenched because of Con and A forms The ground state complex.Con A is a sugar (Gan Lutang) lectin binding specificity, signal transduction in cells, plays a key role in the docking process of cell surface and identification of pathogens. In this study, we choose Con A, due to its low toxicity and can be used to detect the protein ligand interaction the fifth chapter introduces the detection mechanism of the polymer. The intestinal Escherichia coli by multivalent interaction with polymer polymer fluorescent bacteria. Then, the fluorescence intensity gradually reduced. In order to determine the best combination of time required, the fluorescence within a certain time interval of polymer bacterial suspension. Experimental results show that the training for 30 minutes the system can achieve the best level combination, the bacterial affinity detection limit is one of the major advantages of the 103cfu/ml. system is that it can in 30 minutes. For detection, overcomes the traditional detection method of time need several days. This paper reveals the defects of sugar modified PPE can be good molecular biological sensing. Molecular terminal carboxylic acid groups can be combined further with other target molecules. It also proved that mannose modified polymer can detect FimH type of Escherichia coli, the expression of mannose binding lectin this fungus can. Carbohydrate polymer surface retains its ability and mannose binding lectin combination.

【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R155.5

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