基于电化学生物传感器对谷胱甘肽及相关酶活性的研究

发布时间：2018-01-21 13:11

  本文关键词： 谷胱甘肽 电化学阻抗 DNA 谷胱甘肽还原酶 检测　出处：《中南林业科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：电化学传感器由于其选择性好,检测耗时短,无污染等优点,在发酵工业,疾病预防,医药食品加工等相关领域具有广泛的应用前景。而纳米材料由于具有良好的比表面积和独特的导电性能,被应用到高度敏感的生物感应方法设计中,如石墨烯,量子点,金纳米粒子及介孔二氧化硅纳米颗粒等。本论文针对谷胱甘肽(GSH)研究了一系列成本低、快速检测、具有更宽检测范围的新型电化学生物传感器,其主要内容如下:(1)DNA门控开关功能化介孔二氧化硅纳米粒子电化学检测GSH采用介孔二氧化硅粒子和DNA门控系统设计一种基于正十二硫醇修饰丝网印刷电极(SPE)的直接快速检测GSH的电化学方法。用于电化学检测的T-Hg2+-T结构的DNA门控系统可特异性与GSH发生取代反应,门控系统被打开,释放出亚甲基蓝分子。然后,由于亚甲基蓝(MB)分子渗透到硫醇层堆积,被固定的十二硫醇不仅能避免蛋白的非特异性吸附也提高电流响应信号,其检测范围1.0 nmol·L-1～1.0mmol·L-1,检测下限为0.6nmol·L-1。该方法检测下限较低,不用对检测样品处理,操作简单。(2)Hg2+触发链式杂交反应构建电化学传感器检测谷胱甘肽是基于杂交链式反应信号放大,我们设计一个具有高度敏感性、简单的GSH电化学检测方法。通过对目标分子进行杂交链式反应在传感器表面形成dsDNA聚合物。指示剂可以有效地嵌入到双链DNA聚合物凹槽处,从而实现对GSH的检测,该方法的对数线性响应范围为1～500nmol·L-1,检测下限可达0.6nmol·L-1。该法技术已被证明是一种简单且高度敏感的对不同生物分子具有检测作用的信号转导途径。(3)电化学阻抗法快速检测GSH及谷胱甘肽还原酶(GR)是金电极经过GSH修饰后,其阻抗值显著增加,而阻抗的增加与GSH浓度密切相关。通过检测酶反应溶液中GSH的变化,我们可以间接地求得GR的活性。该传感器对GSH的检测范围为250μmol·L-1-16 mmol·L-1。检测酶活性范围在0.005-0.5 U。此外,通过与比色法检测得到的GR活性进行比较,结果表明电化学检测方法具有良好的可靠性。与其他检测方法相比,这种方法具有灵敏度高、耗时较少的优点。
[Abstract]:Because of its good selectivity, short detection time, no pollution and other advantages, electrochemical sensors in the fermentation industry, disease prevention. Because of its good specific surface area and unique electrical conductivity, nano-materials have been used in the design of highly sensitive biological sensing methods, such as graphene. Quantum dots, gold nanoparticles and mesoporous silica nanoparticles. In this paper, a series of low-cost and rapid detection of glutathione glutathione (GSH) have been studied. A novel electrochemical biosensor with a wider detection range. The main contents are as follows:. Electrochemical detection of DNA gated switch functionalized mesoporous silica nanoparticles GSH based on 12 mercaptan modified screen printing electrode was designed using mesoporous silica particles and DNA gating system. The T-Hg2-T DNA gating system for electrochemical detection can react specifically with GSH. The gated system is opened to release methylene blue molecules, which then penetrate into the mercaptan layer due to methylene blue MBs. The immobilized 12 mercaptan could not only avoid the non-specific adsorption of the protein, but also enhance the current response signal. The detection range was 1.0 nmol 路L ~ (-1) ~ (-1) ~ (-1) nmol 路L ~ (-1). The detection limit is 0.6 nmol 路L ~ (-1). Simple operation. Hg2 triggered chain hybridization construction of electrochemical sensor for detection of glutathione is based on the amplification of hybrid chain reaction signal, we design a highly sensitive one. Simple GSH electrochemical detection method. DsDNA polymer is formed on the sensor surface by hybrid chain reaction of the target molecule. The indicator can be effectively embedded in the groove of double-stranded DNA polymer. The logarithmic linear response range of the method is 1 ~ 500nmol 路L ~ (-1). The detection limit can reach 0.6 nmol 路L ~ (-1). This method has been proved to be a simple and highly sensitive signal transduction pathway for different biomolecules. Electrochemical impedance method for rapid detection of GSH and glutathione reductase (Glutathione reductase) is a gold electrode modified by GSH. The impedance value was significantly increased, and the increase of impedance was closely related to the concentration of GSH. The changes of GSH in enzymatic reaction solution were detected. The activity of gr can be obtained indirectly. The detection range of GSH by this sensor is 250 渭 mol 路L-1-16. Mmol 路L ~ (-1). The range of enzyme activity was 0.005-0.5 U. in addition. The comparison of gr activity with colorimetric method shows that the electrochemical detection method has good reliability, and compared with other detection methods, this method has the advantages of high sensitivity and less time consuming.
【学位授予单位】：中南林业科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TP212;Q503
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