基于银纳米粒子的蛋白质与小分子荧光分析方法研究

发布时间：2019-06-26 22:43

【摘要】：适配体是通过SELEX技术筛选得到的单链寡核苷酸,因其所具有的种种性质,如高稳定性,易合成以及能高特异性地结合多种目标分子(小分子,蛋白质甚至是整个细胞)等,所以,适配体受到了越来越多的关注。因为纳米材料具有的独特的光学、电学、催化特性的性质,使得它能够和生物分子相互作用,进而达到信号放大和目标识别的目的。两者结合起来所形成的适配体功能化纳米粒子,在生物分析和医疗诊断方面具有广泛的应用前景。在本文中,我们基于银纳米粒子对荧光的猝灭作用设计了一种简单且高灵敏的蛋白质检测分析方法。首先将修饰有荧光分子的适配体自组装到银纳米粒子表面,合成了银纳米粒子探针。固定在96孔板中的适配体,和银纳米粒子探针分别作为捕获单元和检测探针,可以完成对加入的目标蛋白PDGF-BB的特异性识别,三者形成三明治复合物。此时由于FRET(fluorescence resonance energy transfer,荧光共振能量转移)效应,荧光基团被银纳米粒子猝灭,信号处于“off”状态。当加入蚀刻剂硫代硫酸钠后,银纳米粒子被溶解,FRET效应被破坏,修饰在银纳米粒子表面的荧光分子便会脱离,并释放到孔板中,信号"turn-on"。结果表明,目标蛋白PDGF-BB的浓度与检测到的荧光信号的强度呈正比。该方法可以实现对目标蛋白的高灵敏和高特异性的检测。此方法具有以下优点：三明治结构能大大提高检测的特异性和灵敏度；蚀刻剂的引入使信号从“off”变为“on”。该方法还可以通过对适配体分子的合理设计,进一步扩展到其它蛋白或小分子的分析检测中,在分析领域具有良好的应用前景。基于纳米金属结构的LSPR(localized surface plasmon resonance,局部表面等离子体共振)的金属增强荧光(metal enhanced fluorescence, MEF)的发展,拓宽了荧光分析的应用范围。由于其独特的表面等离子体共振特性,银纳米粒子能够增强许多荧光分子信号,因此利用银纳米粒子的MEF效应,发展出很多应用。在本文中,基于银纳米粒子的MEF效应和FRET效应,我们建立一种小分子检测分析方法。首先,我们将荧光分子自组装至纳米银表面,然后在荧光分子外层包裹一层银壳,实现对荧光分子的荧光增强效果。然后,通过核酸杂交的方式将淬灭分子BHQ修饰的适配体连接至银壳上形成FRET传感器。此时荧光分子的荧光信号猝灭。而当加入目标物腺苷后,由于适配体与腺苷的特异性结合,BHQ脱离银壳表面,荧光信号得到恢复。实验结果显示,荧光的恢复程度与目标物腺苷的浓度呈线性关系。该方法充分利用了银纳米粒子的特性,灵敏度高,特异性强,且操作简单,拥有很大的潜力。
[Abstract]:Aptamer is a single-stranded oligodeoxynucleotides screened by SELEX. Because of its various properties, such as high stability, easy synthesis and high specific binding to a variety of target molecules (small molecules, proteins and even whole cells), aptamers have received more and more attention. Because of its unique optical, electrical and catalytic properties, nanomaterials can interact with biomolecules, and then achieve the purpose of signal amplification and target recognition. The functional nanoparticles formed by the combination of the two have a wide range of application prospects in biological analysis and medical diagnosis. In this paper, we designed a simple and highly sensitive protein detection and analysis method based on the quenching effect of silver nanoparticles on fluorescence. Firstly, the aptamer modified with fluorescent molecules was self-assembled on the surface of silver nanoparticles, and the silver nanoparticles probe was synthesized. The adaptor fixed in 96 well plate and silver nanoparticles probe can be used as capture unit and detection probe, respectively, to complete the specific recognition of the added target protein PDGF-BB, and the three can form sandwich complex. At this time, due to the fluorescence resonance energy transfer effect of FRET (fluorescence resonance energy transfer, the fluorescence group is quenched by silver nanoparticles and the signal is in the "off" state. When the etch agent sodium thiosulfate is added, the silver nanoparticles are dissolved and the FRET effect is destroyed. the fluorescent molecules modified on the surface of the silver nanoparticles will be detached and released into the pore plate, and the signal "turn-on" will be released. The results showed that the concentration of the target protein PDGF-BB was proportional to the intensity of the detected fluorescence signal. This method can be used to detect the target protein with high sensitivity and specificity. This method has the following advantages: sandwich structure can greatly improve the specificity and sensitivity of detection, and the introduction of etch agent can change the signal from "off" to "on". This method can also be further extended to the analysis and detection of other proteins or small molecules through the reasonable design of aptamer molecules, and has a good application prospect in the field of analysis. The development of metal enhanced fluorescence (metal enhanced fluorescence, MEF) based on local surface plasmon resonance (LSPR (localized surface plasmon resonance,) with nano-metal structure broadens the application range of fluorescence analysis. Because of its unique surface plasmon resonance characteristics, silver nanoparticles can enhance many fluorescent molecular signals, so many applications have been developed by using the MEF effect of silver nanoparticles. In this paper, based on the MEF effect and FRET effect of silver nanoparticles, we establish a small molecular detection and analysis method. First of all, we self-assemble the fluorescent molecules to the surface of nano-silver, and then wrap a layer of silver shell in the outer layer of the fluorescent molecules to achieve the fluorescence enhancement effect of the fluorescent molecules. Then, the quenched molecular BHQ modified aptamers were connected to the silver shell to form a FRET sensor by nucleic acid hybridization. At this time, the fluorescence signal quenching of fluorescent molecules. When the target adenosine was added, the fluorescence signal of BHQ was recovered because of the specific binding of aptamer to adenosine. The experimental results show that there is a linear relationship between the recovery degree of fluorescence and the concentration of adenosine. This method makes full use of the characteristics of silver nanoparticles, has high sensitivity, strong specificity, simple operation and great potential.
【学位授予单位】：南京大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TB383.1;O614.122

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