基于功能核酸的重金属离子电化学生物传感器研究

发布时间：2018-08-03 09:39

【摘要】：重金属污染指由重金属或其化合物形成的环境污染,主要来源于工业污染,交通污染和生活垃圾污染等。生活中不少有机化合物能够通过自身物理的、化学的或生物的净化,使有害性降低或解除,但是重金属污染与有机化合物的污染不同,重金属污染很难在环境中降解。这些重金属通过水、空气、土壤等途径进入动植物体内,最终通过食物链进入人体内,在人体内能与蛋白质及各种酶产生剧烈的相互作用,使蛋白质或酶失去活性;也可能在人体的某些器官中富集,假如超过人体所能承受的限制,会造成人身体不适或中毒等。传统的重金属检测方法主要有原子吸收光谱(Atomic Absorption Spectroscopy,AAS)、电感耦合等离子体质谱(Inductively Coupled Plasma Mass Spectrometry,ICP-MS)、荧光法(fluorescence)等,尽管这些方法的灵敏度高,但通常需要大型仪器、复杂的样品前处理、专业的人工操作技能、耗时耗力。因此,研究高灵敏度、高特异性、操作简单的重金属离子检测方法受到高度关注,目前学者们对检测重金属离子进行了广泛的研究,期望对环境及食品中重金属离子能够快速高效的检测。功能核酸指能与特定目标高特异性结合、或者具有催化功能的核酸。本文运用具有催化活性的DNAzyme和基于T-Hg~(2+)-T配合物的双链DNA作为特异性识别元件,在电化学交流阻抗谱(Electrochemical Impedance Spectroscopy,EIS)、石英晶体微天平(Quartz Crystal Microbalance,QCM)和计时电量(Chronocoulometry)高灵敏的电化学平台上,成功构建了检测Cu~(2+)和Hg~(2+)的高灵敏生物传感器,为食品和环境中铜和汞的检测提供了快速、简便、经济的分析方法。研究内容如下:1.基于DNAzyme和电化学阻抗高灵敏检测Cu~(2+)生物传感器的研究设计及研究了一种基于DNAzyme和电化学阻抗(electrochemical impedance spectroscopy,EIS)的简单、无标记的检测水溶液中Cu~(2+)生物传感器。将带有巯基的Cu~(2+)特异识别的DNAzyme通过Au-S键自组装在金电极的表面,当Cu~(2+)存在时,DNAzyme链被切断,可以观察到由于电子传递而影响的电阻强度变化,阻抗信号的变化与Cu~(2+)浓度有关。此传感器显示出高灵敏度和选择性,检测限为5nM,远低于美国环境保护局(EPA)规定的Cu~(2+)浓度(~20nM)。同时其他金属离子,例如Ca~(2+),Mg~(2+),Mn~(2+),Cr3+,Pb~(2+),Zn~(2+),Co~(2+),和Ni~(2+)对Cu~(2+)的检测几乎没有影响。2.基于DNAzyme与石英晶体微天平检测水溶液中Cu~(2+)生物传感器的研究石英晶体微天平(Quartz Crystal Microbalance,QCM)检测法简便、极速、实时测定,是一种能够检测纳克级质量变化的超高灵敏技术,因此成为化学领域和分子生物学的有效手段之一。本章以Cu~(2+)特异识别的DNAzyme为元件,在QCM平台上设计了一种快速、经济、实时检测水溶液中Cu~(2+)的生物传感器。运用自组装法将双链DNA固定在晶片电极表面,在室温下能被Cu~(2+)在切割位点处切断。由于晶片表面的质量发生了变化,QCM可以很灵敏地检测到共振频率发生改变,△F值与Cu~(2+)浓度成正比,体系检测限为3nM。同时其他金属离子,如Co~(2+),Ni~(2+),Mg~(2+),Zn~(2+),Ca~(2+)、Mn~(2+)和Pb~(2+)对Cu~(2+)的检测没有影响。此生物传感器表现出很高的灵敏度和选择性,有望应用于食品和环境中Cu~(2+)的测定。3.基于DNA双链电荷转移的Hg~(2+)电化学生物传感器的研究基于DNA双链电荷转移原理,利用胸腺嘧啶(thymine)与Hg~(2+)的特异性识别和计时电量法构建了一种高灵敏检测水溶液中Hg~(2+)的电化学生物传感器。该传感器将含有1个T-T碱基错配对的DNA互补双链通过Au-S键自组装在金电极表面,运用计时电量法在含有亚甲基蓝的铁氰化钾溶液中进行测定。T-T错配阻断了DNA双链内部电荷转移,而Hg~(2+)通过T-Hg~(2+)-T配位作用与双链DNA特异性结合并形成DNA双链内部电荷转移通路,引起电极表面计时电量的变化。计时电量测定结果显示:在亚甲基蓝的还原峰电位(-380 mV)附近,计时电量随着溶液中的Hg~(2+)浓度的增大而增加,Hg~(2+)浓度在1.0 nM~104 nM范围内,计时电量的变化量与Hg~(2+)浓度的对数呈良好的线性关系,线性相关系数(R2)为0.995,检测限为0.5 nM(S/N=3)。干扰实验表明,该传感器对Hg~(2+)具有良好的特异性和选择性。
[Abstract]:Heavy metal pollution refers to the environmental pollution caused by heavy metals or their compounds, mainly from industrial pollution, traffic pollution and domestic waste pollution. Many organic compounds in life can reduce or remove harmful effects through their own physical, chemical or biological purification, but the pollution of heavy metals and organic compounds is not polluted. In the same way, heavy metal pollution is difficult to degrade in the environment. These heavy metals enter the animal and plant through water, air and soil, and eventually enter the body through the food chain. In the human body it can produce violent interaction with protein and various enzymes to make protein or enzyme inactive; it may also be enriched in some organs of the human body. More than the human body can bear restrictions, it makes adult physical discomfort or poisoning. Traditional methods of detection of heavy metals include Atomic Absorption Spectroscopy (AAS), inductively coupled plasma mass spectrometry (Inductively Coupled Plasma Mass Spectrometry, ICP-MS), fluorescence (fluorescence), etc., despite these methods It is highly sensitive, but usually requires large instruments, complex sample pretreatment, professional manual operation skills, time consuming and energy consumption. Therefore, high sensitivity, high specificity, simple operation of heavy metal ion detection methods have been highly concerned. At present, scholars have studied the detection of heavy metal ions widely and expect to be in the environment and food. Heavy metal ions can be detected quickly and efficiently. Functional nucleic acids refer to high specific binding to specific targets, or nucleic acids with catalytic functions. In this paper, a double stranded DNA with catalytic activity DNAzyme and T-Hg~ (2+) -T complexes as a specific identification element is used in electrochemistry AC impedance spectroscopy (Electrochemical Impedance Spectro). Scopy, EIS), quartz crystal microbalance (Quartz Crystal Microbalance, QCM) and high sensitive electrochemistry (Chronocoulometry) electrochemistry platform, a highly sensitive biosensor for detection of Cu~ (2+) and Hg~ (2+) was successfully constructed to provide rapid, simple and economical analysis methods for the detection of copper and mercury in food and environment. 1 Based on the high sensitivity detection of Cu~ (2+) biosensor based on DNAzyme and electrochemical impedance, a simple, unmarked Cu~ (2+) biosensor based on DNAzyme and electrochemical impedance spectroscopy (EIS) was used to detect the Cu~ (2+) biosensor in a aqueous solution. The bond is self assembled on the surface of the gold electrode. When Cu~ (2+) exists, the DNAzyme chain is cut off. The change of resistance intensity influenced by electron transfer is observed. The change of the impedance signal is related to the concentration of Cu~ (2+). The sensor shows high sensitivity and selectivity, and the detection limit is 5nM, far below the Cu~ (2+) concentration specified by the US Environmental Protection Agency (EPA). Degree (~20nM). At the same time other metal ions, such as Ca~ (2+), Mg~ (2+), Mn~ (2+), Cr3+, Pb~ (2+), Zn~ (2+), Zn~ (2+), and quartz crystal microbalance detection of biosensor in aqueous solution In this chapter, Cu~ (2+) specific DNAzyme as component, a fast, economical and real-time biosensor for detecting Cu~ (2+) in aqueous solution is designed on the QCM platform. The double stranded DNA is immobilized on the surface of the wafer electrode by the self assembly method. At room temperature, Cu~ (2+) can be cut off at the cutting site. Because the mass of the wafer surface changes, the QCM can be sensitive to detect the resonance frequency change. The delta F value is proportional to the Cu~ (2+) concentration, and the body system detection is limited to 3nM. and other metal ions, such as Co~ (2+), Ni~ (2). +), Mg~ (2+), Zn~ (2+), Ca~ (2+), Mn~ (2+) and Pb~ (2+) have no effect on the detection of Cu~ (2+). This biosensor shows high sensitivity and selectivity, and is expected to be applied to food and environment. An electrochemical biosensor for high sensitivity detection of Hg~ (2+) in aqueous solution is constructed by the specific identification of adenourine (thymine) and Hg~ (2+). The biosensor will contain 1 T-T base mismatched pairs of DNA complementary double chains assembled on the gold electrode surface through Au-S bonds, and the methylene blue contains iron cyanidation by the chronoelectric method. The determination of.T-T mismatch in the potassium solution blocks the internal charge transfer of DNA double strands, while Hg~ (2+) combines the T-Hg~ (2+) -T coordination with the dual chain DNA and forms a DNA double chain internal charge transfer pathway, resulting in the change of the chronoelectric charge on the electrode surface. Time measurement results show that the peak potential of the methylene blue is near the reduction peak potential (-380 mV). The time quantity increases with the concentration of Hg~ (2+) in the solution, and the concentration of Hg~ (2+) is within the range of 1 nM~104 nM. The variation of time quantity is in good linear relation with the logarithm of Hg~ (2+) concentration, and the linear correlation coefficient (R2) is 0.995 and the detection limit is 0.5 nM (S /N=3). And selectivity.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：O657.1;TP212

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