基于核酸链杂交反应用于肿瘤标记物EBNA-1的电化学免疫传感器的研究

发布时间：2018-01-15 23:30

本文关键词：基于核酸链杂交反应用于肿瘤标记物EBNA-1的电化学免疫传感器的研究　出处：《重庆医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的：EB病毒(Epstein-Barr virus, EBV)即人类疱疹病毒4型,目前全球感染该病毒的人口大于90%。EB病毒在儿童时期常常成隐性感染,到了成人当免疫系统低下时便容易出现症状。EB病毒的检测与多种疾病有关如：致死性传染性单核细胞增多症、Burkitt淋巴瘤、霍奇金病、鼻咽癌、胃癌、唾液腺肿瘤和近来发现的出现在免疫抑制患者身上的平滑肌肿瘤等相关。在上述疾病中,EB核抗原-1(EBNA-1)是最早期表达蛋白之一,且是持续表达的蛋白。EBNA-1不仅是EB病毒有关的肿瘤标志物而且能够区分其它病原微生物感染。因此,本研究构建了一种准确、高灵敏和快速检测EBNA-1电化学免疫传感器新方法。方法：1.石墨烯-多壁碳纳米管-壳聚糖(GS-MWCNTs-Chit)纳米复合物的制备。利用一步合成法将石墨烯、多壁碳纳米管均匀分散到壳聚糖中。在纳米复合物上电沉积金纳米颗粒。使用电镜和电化学方法对合成的纳米复合物进行表征。2.多壁碳纳米管上连接DNA和多克隆抗(DNA-MWCNTs-Ab2)。DNA引发链和多克隆抗体通过原位合成法连接到多壁碳纳米管上。发卡结构的两类核酸发生核酸链杂交反应形成纳米线结构。通过原子力显微镜和电化学方法对其进行表征。3.以玻碳电极作为反应基板,5mM铁氰化钾溶液为基础反应液,在对基础反应液pH值、石墨烯与多壁碳纳米管的比例、核酸链杂交反应时间和抗原抗体孵育时间等实验条件优化后,利用差分脉冲伏安法对不同浓度的EBV核抗原1进行电化学检测,并绘制标准曲线,对此EBV核抗原1(EBNA-1)电化学免疫传感器的稳定性、灵敏度和选择性进行评估。结果：1.成功合成GS-MWCNTs纳米复合物。通过一步原位合成法合成GS-MWCNTs纳米复合物,从扫描电子显微镜下可以看到GS-MWCNTs的形态学特征。通过原子力显微镜(AFM)可以观察到MWCNTs和DNA-MWCNTs-Ab2的三维图像,表明信号放大复合物制备成功。2.所制备的免疫传感器,在最佳条件下,对目标物EBNA-1进行检测,在0.05-6.4 ng mL-1范围内信号与目标物浓度呈线性相关性,R2=0.99249,最低检测限为0.7 pg mL-1 (S/N=3),线性方程为Y=24.81X-19.11。3.该传感器性能良好：批间差异小于4.2%,批内差异小于4.0%,选择性好,最大干扰差异为4.3%。稳定性好,储存27天后,相应电流仅下降了8.1%。结论：通过将石墨烯、多壁碳纳米管、金纳米颗粒层层修饰到玻碳电极表面,来增大电极的表面积、提高电子传递能力及增加单克隆抗体的负载量,制备的免疫传感器可用于快速、超灵敏检测EBNA-1。本研究结合了免疫反应的特性与核酸杂交链反应放大系统,同时利用了纳米材料包括石墨烯、多壁碳纳米管、金纳米颗粒促进了电子传递,从而增加了免疫传感器的灵敏性具有较宽的检测范围。此种电化学免疫传感器具有特异性强、重复性好和灵敏度高等特点,为临床定量检测EBNA-1提供了有效的方法。
[Abstract]:Objective to investigate Epstein-Barr virus (EBV) of human herpesvirus 4. Currently, the number of people worldwide infected with the virus is greater than 90. Epstein-Barr virus often becomes a recessive infection in childhood. Detection of Epstein-Barr virus is associated with many diseases such as fatal infectious mononucleosis Burkitt lymphoma Hodgkin's disease and nasopharyngeal carcinoma. Gastric cancer, salivary gland tumors and recently discovered smooth muscle tumors in immunosuppressive patients are associated with EBNA-1, one of the earliest expressed proteins. EBNA-1 is not only a tumor marker related to Epstein-Barr virus but also can distinguish other pathogenic microorganism infection. High sensitivity and rapid detection of EBNA-1 electrochemical immunosensor. Method: 1. Graphene multiwalled carbon nanotube-chitosan GS-MWCNTs-Chits-Chits-GS-MWCNTs-Chits-GS-MWCNTs-Chits-graphene. Preparation of nanocomposites. Graphene was prepared by one-step synthesis. Multiwalled carbon nanotubes were uniformly dispersed into chitosan. Gold nanoparticles were electrodeposited on nanocomposites. The synthesized nanocomposites were characterized by electron microscopy and electrochemical methods. DNA was attached to multi-walled carbon nanotubes. And polyclonal antibodies (. DNA-MWCNTs-Ab2). DNA initiation chains and polyclonal antibodies are connected to multiwalled carbon nanotubes by in situ synthesis. Two types of nucleic acids with hairpin structure undergo nucleic acid chain hybridization to form nanowires. Atomic force microscopy and electrochemical methods. The glassy carbon electrode was used as the reaction substrate. 5 mm potassium ferricyanide solution was used as the base reaction solution. The pH value of the base reaction solution, the ratio of graphene to multi-walled carbon nanotubes, the reaction time of nucleic acid chain hybridization and the incubation time of antigen and antibody were optimized. Differential pulse voltammetry was used to detect EBV nuclear antigen 1 at different concentrations, and the standard curve was drawn. The stability of the electrochemical immunosensor for this EBV nuclear antigen 1a EBNA-1 was studied. The sensitivity and selectivity were evaluated. Results: 1. GS-MWCNTs nanocomposites were synthesized successfully. GS-MWCNTs nanocomposites were synthesized by one step in situ synthesis. The morphological features of GS-MWCNTs can be seen from the scanning electron microscope. 3D images of MWCNTs and DNA-MWCNTs-Ab2 can be observed. The results showed that the signal amplification complex was successfully prepared. 2. The immunosensor was prepared and the target EBNA-1 was detected under the optimum conditions. In the range of 0.05-6.4 ng mL-1, there was a linear correlation between the signal and the concentration of the target. The lowest detection limit was 0.7 PG mL-1 / S / N ~ (3 +) and the linear equation was (24.81X-19.11.3). The sensor had good performance: the difference between batches was less than 4.2%. The intra-batch difference was less than 4.0, the selectivity was good, the maximum interference difference was 4.3.The stability was good. After 27 days of storage, the corresponding current decreased only 8.1. Conclusion: graphene, multi-walled carbon nanotubes were added. Gold nanoparticles were modified on the surface of glassy carbon electrode to increase the surface area of the electrode, improve the electron transfer ability and increase the load of monoclonal antibody. The immunosensor prepared can be used for rapid development. Hypersensitive detection of EBNA-1. This study combined the characteristics of immune reaction with nucleic acid hybridization chain reaction amplification system, and used nano-materials, including graphene, multi-walled carbon nanotubes. Gold nanoparticles promote electron transfer, thus increasing the sensitivity of the immunosensor with a wide range of detection. This electrochemical immunosensor has the characteristics of strong specificity, good reproducibility and high sensitivity. It provides an effective method for clinical quantitative detection of EBNA-1.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.6

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