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  本文關鍵詞：基于核酸鏈雜交反應用于腫瘤標記物EBNA-1的電化學免疫傳感器的研究　出處：《重慶醫(yī)科大學》2015年碩士論文　論文類型：學位論文

  更多相關文章： EB病毒核抗原-1 核酸雜交鏈反應 石墨烯-多壁碳納米管納米復合物 阿霉素 電化學免疫傳感器

【摘要】：目的：EB病毒(Epstein-Barr virus, EBV)即人類皰疹病毒4型,目前全球感染該病毒的人口大于90%。EB病毒在兒童時期常常成隱性感染,到了成人當免疫系統(tǒng)低下時便容易出現(xiàn)癥狀。EB病毒的檢測與多種疾病有關如：致死性傳染性單核細胞增多癥、Burkitt淋巴瘤、霍奇金病、鼻咽癌、胃癌、唾液腺腫瘤和近來發(fā)現(xiàn)的出現(xiàn)在免疫抑制患者身上的平滑肌腫瘤等相關。在上述疾病中,EB核抗原-1(EBNA-1)是最早期表達蛋白之一,且是持續(xù)表達的蛋白。EBNA-1不僅是EB病毒有關的腫瘤標志物而且能夠區(qū)分其它病原微生物感染。因此,本研究構建了一種準確、高靈敏和快速檢測EBNA-1電化學免疫傳感器新方法。方法：1.石墨烯-多壁碳納米管-殼聚糖(GS-MWCNTs-Chit)納米復合物的制備。利用一步合成法將石墨烯、多壁碳納米管均勻分散到殼聚糖中。在納米復合物上電沉積金納米顆粒。使用電鏡和電化學方法對合成的納米復合物進行表征。2.多壁碳納米管上連接DNA和多克隆抗(DNA-MWCNTs-Ab2)。DNA引發(fā)鏈和多克隆抗體通過原位合成法連接到多壁碳納米管上。發(fā)卡結構的兩類核酸發(fā)生核酸鏈雜交反應形成納米線結構。通過原子力顯微鏡和電化學方法對其進行表征。3.以玻碳電極作為反應基板,5mM鐵氰化鉀溶液為基礎反應液,在對基礎反應液pH值、石墨烯與多壁碳納米管的比例、核酸鏈雜交反應時間和抗原抗體孵育時間等實驗條件優(yōu)化后,利用差分脈沖伏安法對不同濃度的EBV核抗原1進行電化學檢測,并繪制標準曲線,對此EBV核抗原1(EBNA-1)電化學免疫傳感器的穩(wěn)定性、靈敏度和選擇性進行評估。結果：1.成功合成GS-MWCNTs納米復合物。通過一步原位合成法合成GS-MWCNTs納米復合物,從掃描電子顯微鏡下可以看到GS-MWCNTs的形態(tài)學特征。通過原子力顯微鏡(AFM)可以觀察到MWCNTs和DNA-MWCNTs-Ab2的三維圖像,表明信號放大復合物制備成功。2.所制備的免疫傳感器,在最佳條件下,對目標物EBNA-1進行檢測,在0.05-6.4 ng mL-1范圍內(nèi)信號與目標物濃度呈線性相關性,R2=0.99249,最低檢測限為0.7 pg mL-1 (S/N=3),線性方程為Y=24.81X-19.11。3.該傳感器性能良好：批間差異小于4.2%,批內(nèi)差異小于4.0%,選擇性好,最大干擾差異為4.3%。穩(wěn)定性好,儲存27天后,相應電流僅下降了8.1%。結論：通過將石墨烯、多壁碳納米管、金納米顆粒層層修飾到玻碳電極表面,來增大電極的表面積、提高電子傳遞能力及增加單克隆抗體的負載量,制備的免疫傳感器可用于快速、超靈敏檢測EBNA-1。本研究結合了免疫反應的特性與核酸雜交鏈反應放大系統(tǒng),同時利用了納米材料包括石墨烯、多壁碳納米管、金納米顆粒促進了電子傳遞,從而增加了免疫傳感器的靈敏性具有較寬的檢測范圍。此種電化學免疫傳感器具有特異性強、重復性好和靈敏度高等特點,為臨床定量檢測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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R446.6

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