核酸探针电化学传感技术用于酶活性和腺苷检测的研究
发布时间：2018-11-05 11:19
【摘要】： 电化学生物传感器,具有易于实现微型化、集成化、操作简单等优点,在化学、生物学基础研究、临床检验、环境监测等方面发挥着越来越重要的作用。核酸探针能够与多种目标物特异性结合,形成多种灵活的分子识别和信号转换机制。本论文基于核酸探针的特点,针对酶和生物小分子的检测,发展简便、快速、灵敏的电化学传感器,具体完成了以下研究工作： 1.二茂铁核酸探针的合成及电化学表征 对琥珀酰亚胺偶联方法进行改进,尽量消除水对反应体系中活性中间体的影响,并采用高效液相色谱(HPLC)纯化,得到了二茂铁标记的核酸探针。电化学测量证明二茂铁已成功标记到3’为NH2的核酸探针上。相比于一般的采用透析分离纯化二茂铁核酸探针的方法,HPLC分离纯化的二茂铁核酸探针具有纯度高、信号强等优点。为下一步的工作打下了基础。 2.二茂铁核酸探针用于APE1酶活性的电化学检测 APEl酶是一种重要的DNA损伤修复蛋白,经典的放射性标记活性分析虽然灵敏度高,但存在操作复杂、费时、有放射性污染等缺陷。我们在带有AP位点的核酸探针上修饰二茂铁,并与固定在金电极表面的捕获探针杂交,形成带AP位点的二茂铁修饰的双链DNA。利用APE1酶对AP位点切割产生的信号变化实现了酶活性的电化学检测。该方法对APEl酶活性的检测范围是0.02-2 U／ml,检测下限为0.02 U／ml,具有操作简单、灵敏度高等优点,有望用于生物样品中DNA损伤修复的研究。 3.“裂开型”核酸适体结合金纳米颗粒信号放大用于腺苷的高灵敏检测 虽然核酸适体能够实现小分子的特异性识别,但由于结合常数一般相对较低,往往需要结合信号放大技术以提高检测灵敏度。我们构建了一种基于核酸适体和金纳米颗粒信号放大的检测小分子腺苷的电化学生物传感器。该传感器对腺苷检测的线性范围为0.1pM-1nM,检测下限为0.1 pM,且特异性好,有望拓展到其他小分子的检测中。
[Abstract]:Electrochemical biosensor, which is easy to realize miniaturization, integration, simple operation and so on, plays an increasingly important role in chemistry, biological basic research, clinical testing, environmental monitoring and so on. Nucleic acid probes can specifically bind to various targets and form a variety of flexible molecular recognition and signal conversion mechanisms. Based on the characteristics of nucleic acid probes, this paper develops a simple, rapid and sensitive electrochemical sensor for the detection of enzyme and biological small molecules. The following research work has been done: 1. The synthesis and electrochemical characterization of ferrocene nucleic acid probes improved the succinimide coupling method to eliminate the effect of water on the active intermediates in the reaction system and purified by high performance liquid chromatography (HPLC). Ferrocene labeled nucleic acid probes were obtained. Electrochemical measurements show that ferrocene has been successfully labeled into a 3 'NH2 nucleic acid probe. The ferrocene nucleic acid probe separated and purified by HPLC has the advantages of high purity and strong signal. It lays the foundation for the next work. 2. Ferrocene nucleic acid probe for electrochemical detection of APE1 enzyme activity APEl enzyme is an important DNA damage repair protein. Although the classical radiolabelling activity analysis is highly sensitive, its operation is complex and time-consuming. There are defects such as radioactive contamination. We modified ferrocene on a nucleic acid probe with AP site and hybridized with a trap probe fixed on the surface of gold electrode to form ferrocene modified double stranded DNA. with AP site. The electrochemical detection of enzyme activity was achieved by using APE1 enzyme to change the signal generated by AP site cleavage. The detection range of APEl enzyme activity is 0.02-2 U / ml, and the detection limit is 0.02 U / ml. This method has the advantages of simple operation and high sensitivity. It is expected to be used in the study of DNA damage repair in biological samples. 3. "split-type" aptamer combined with gold nanoparticles signal amplification for high sensitive detection of adenosine although nucleic acid aptamers can achieve specific recognition of small molecules, the binding constants are generally relatively low. Signal amplification is often needed to improve detection sensitivity. We have constructed an electrochemical biosensor for the detection of small molecule adenosine based on aptamer of nucleic acid and amplification of gold nanoparticles. The linear range of the sensor for adenosine detection is 0.1pM-1nM.The detection limit is 0.1 pM, and the specificity is good. It is expected to be extended to the detection of other small molecules.
【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341
本文编号：2311898
[Abstract]:Electrochemical biosensor, which is easy to realize miniaturization, integration, simple operation and so on, plays an increasingly important role in chemistry, biological basic research, clinical testing, environmental monitoring and so on. Nucleic acid probes can specifically bind to various targets and form a variety of flexible molecular recognition and signal conversion mechanisms. Based on the characteristics of nucleic acid probes, this paper develops a simple, rapid and sensitive electrochemical sensor for the detection of enzyme and biological small molecules. The following research work has been done: 1. The synthesis and electrochemical characterization of ferrocene nucleic acid probes improved the succinimide coupling method to eliminate the effect of water on the active intermediates in the reaction system and purified by high performance liquid chromatography (HPLC). Ferrocene labeled nucleic acid probes were obtained. Electrochemical measurements show that ferrocene has been successfully labeled into a 3 'NH2 nucleic acid probe. The ferrocene nucleic acid probe separated and purified by HPLC has the advantages of high purity and strong signal. It lays the foundation for the next work. 2. Ferrocene nucleic acid probe for electrochemical detection of APE1 enzyme activity APEl enzyme is an important DNA damage repair protein. Although the classical radiolabelling activity analysis is highly sensitive, its operation is complex and time-consuming. There are defects such as radioactive contamination. We modified ferrocene on a nucleic acid probe with AP site and hybridized with a trap probe fixed on the surface of gold electrode to form ferrocene modified double stranded DNA. with AP site. The electrochemical detection of enzyme activity was achieved by using APE1 enzyme to change the signal generated by AP site cleavage. The detection range of APEl enzyme activity is 0.02-2 U / ml, and the detection limit is 0.02 U / ml. This method has the advantages of simple operation and high sensitivity. It is expected to be used in the study of DNA damage repair in biological samples. 3. "split-type" aptamer combined with gold nanoparticles signal amplification for high sensitive detection of adenosine although nucleic acid aptamers can achieve specific recognition of small molecules, the binding constants are generally relatively low. Signal amplification is often needed to improve detection sensitivity. We have constructed an electrochemical biosensor for the detection of small molecule adenosine based on aptamer of nucleic acid and amplification of gold nanoparticles. The linear range of the sensor for adenosine detection is 0.1pM-1nM.The detection limit is 0.1 pM, and the specificity is good. It is expected to be extended to the detection of other small molecules.
【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341
【参考文献】
相关博士学位论文 前1条
1 刘斌;荧光分子探针技术在基因表达产物研究中的应用[D];湖南大学;2007年
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