DNA电化学生物传感器的制备及对莠去津检测的研究
摘要
DNA电化学生物传感器是近几年发展起来的一种全新的生物传感器。它既具有DNA杂交反应的高度特异性,又具有灵敏度高、响应快、操作方便、无污染、价格低廉等特点,已被广泛应用于食品工业、疾病诊断、药物分析、环境监测等领域。DNA是生物体的基本遗传物质,一些化学物质易与其形成加合物。因DNA加合物能够反映化学污染物与DNA的作用情况,因此可以用作生物标志物来评价和预测污染物的潜在致癌性。结合上述两方面内容,DNA电化学生物传感器有望提供一种全新的检测技术,在污染物的检测方面具有广阔的应用前景。
在传感器的构建过程中,如何有效利用生物分子的固定技术及选择合适的固定材料决定着DNA电化学生物传感器的稳定性、选择性和灵敏性等主要性能。同时,选择合适的杂交指示剂也是DNA电化学生物传感器研制的关键技术之一。本论文设计了两种新型的DNA电化学生物传感器,并研究了除草剂莠去津与DNA二者之间的相互作用。研究内容主要分为以下三部分:
(1)用电化学氧化法使玻碳电极表面氧化生成羧基,利用偶联活化试剂将1.0代树状高分子(G1 PAMAM)固定在玻碳电极表面,并通过共价结合固定ssDNA,制备了一种新型的DNA电化学生物传感器。以亚甲基蓝为指示剂,采用循环伏安法,示差脉冲伏安法等电化学方法对DNA电化学生物传感器进行了表征。结果发现,通过亚甲基蓝与双链dsDNA作用的氧化还原电流的变化,可以识别和定量检测溶液中互补的ssDNA片段。
(2)采用紫外吸收光谱法、荧光光谱法以及电化学方法研究了莠去津与鲱鱼精DNA的相互作用,探讨了莠去津对DNA的损伤及其毒性作用机制。结果表明,莠去津与鲱鱼精DNA作用后,莠去津的紫外光谱呈现减色效应,并有轻微红移现象,而其荧光光谱强度明显增强;循环伏安法显示莠去津与DNA作用能引起莠去津还原电位正移,峰电流减小。以上实验结果表明,莠去津平面分子能够嵌插到DNA双螺旋链中,形成较稳定的加合物。
(3)将4.0代树状高分子(G4 PAMAM)与壳聚糖按一定比例混合后,利用壳聚糖的成膜特性,将G4 PAMAM固定在玻碳电极上,然后利用EDC的偶联活化作用将氨基基团与ssDNA 5′端的磷酸基团共价结合,选用[Co(phen)3]3+做指示剂,制备了一种新型的DNA电化学生物传感器。通过循环伏安法、示差脉冲伏安法和交流阻抗法对DNA的固定杂交情况进行了表征,并对溶液中的莠去津进行了检测。结果表明,此方法能增加修饰层上DNA探针的固定量,并使固定的ssDNA保持伸展状态,有利于杂交的进行,同时发现该传感器对莠去津具有较灵敏的响应。
DNA electrochemical biosensor is noted widely in recent years for rapid development. It has been used in many fields such as food industry, disease diagnosis, pharmaceutical analysis and environmental monitoring due to the high sensitivity, better specificity, rapid response, easy handling, no pollution and low cost. DNA is the basic genetic material of organism, the interaction of some chemicals and DNA make the formation of DNA adducts, which can evaluate and predict potential carcinogenicity of pollutants because of reflecting the interaction condition between chemical pollutants and DNA. So DNA electrochemical biosensor holds some promise of a novel detection technique, and it has a broad prospect of application in the pollutant monitoring field.
However, the method and material used to immobilize biomolecules is one of the crucial factors for improving the stability, selectivity and sensitivity of biosensor in the preparation, and the selection of appropriate indicator is also the key technology. Two new-typed DNA electrochemical biosensor was prepared, and the interaction between atrazine and Herring Sperm DNA was studied in this paper. The dissertation is divided into three main parts:
(1) A novel DNA electrochemical biosensor was prepared by immobilizing the first generation polyamidoamine (G1 PAMAM) dendrimer on the glassy carbon electrode (GCE) surface by coupled activation agent, which was treated via electrochemical oxidation, then ssDNA was also immobilized through covalently action. Using methylene blue as the indicator, the DNA electrochemical biosensor was characterized by cyclic voltammetry and differential pulse voltammetry. The results indicated that the complementary ssDNA segment in the solution could be recognized and detected through mensurating the oxidation-deoxidation peaks current of embed methylene blue in dsDNA.
(2) In order to explore the DNA damage effects induced by atrazine, ultraviolet absorption spectrometry, fluorescence spectrometry and cyclic voltammetry were used to study the the interaction between atrazine and Herring Sperm DNA. The results showed that a hypochromic effect and a slight red shift on the UV spectrogram of atrazine were observed after the interaction with Herring Sperm DNA, while the fluorescence increased obviously. Cyclic voltammetry indicated that the reductive potential of atrazine shifted positively with the peak current decreasing after the interaction with DNA. The above results proved that atrazine could be inserted into the double-helix of DNA and formed a stable DNA adduct.
(3) The mixture of the fourth generation polyamidoamine (G4 PAMAM) dendrimer and chitosan with certain proportion was immobilized on the glassy carbon electrode (GCE) surface by the film forming properties of chitosan, then the amino group and the 5′-phosphate group of ssDNA had a covalently action by coupled activation agent of EDC. Using [Co(phen)3]3+ as the indicator, a new DNA electrochemical biosensor was prepared. The immobilization and hybridization of ssDNA was characterized by cyclic voltammetry, differential pulse voltammetry and electrochemical impedance spectroscopy. Experiment results showed that the immobilized amount of DNA probe on modified layer increased, and the immobilized ssDNA kept a extension state, which was beneficial to the hybridization of DNA. Furthermore, this biosensor had a sensitive response for atrazine.
在传感器的构建过程中,如何有效利用生物分子的固定技术及选择合适的固定材料决定着DNA电化学生物传感器的稳定性、选择性和灵敏性等主要性能。同时,选择合适的杂交指示剂也是DNA电化学生物传感器研制的关键技术之一。本论文设计了两种新型的DNA电化学生物传感器,并研究了除草剂莠去津与DNA二者之间的相互作用。研究内容主要分为以下三部分:
(1)用电化学氧化法使玻碳电极表面氧化生成羧基,利用偶联活化试剂将1.0代树状高分子(G1 PAMAM)固定在玻碳电极表面,并通过共价结合固定ssDNA,制备了一种新型的DNA电化学生物传感器。以亚甲基蓝为指示剂,采用循环伏安法,示差脉冲伏安法等电化学方法对DNA电化学生物传感器进行了表征。结果发现,通过亚甲基蓝与双链dsDNA作用的氧化还原电流的变化,可以识别和定量检测溶液中互补的ssDNA片段。
(2)采用紫外吸收光谱法、荧光光谱法以及电化学方法研究了莠去津与鲱鱼精DNA的相互作用,探讨了莠去津对DNA的损伤及其毒性作用机制。结果表明,莠去津与鲱鱼精DNA作用后,莠去津的紫外光谱呈现减色效应,并有轻微红移现象,而其荧光光谱强度明显增强;循环伏安法显示莠去津与DNA作用能引起莠去津还原电位正移,峰电流减小。以上实验结果表明,莠去津平面分子能够嵌插到DNA双螺旋链中,形成较稳定的加合物。
(3)将4.0代树状高分子(G4 PAMAM)与壳聚糖按一定比例混合后,利用壳聚糖的成膜特性,将G4 PAMAM固定在玻碳电极上,然后利用EDC的偶联活化作用将氨基基团与ssDNA 5′端的磷酸基团共价结合,选用[Co(phen)3]3+做指示剂,制备了一种新型的DNA电化学生物传感器。通过循环伏安法、示差脉冲伏安法和交流阻抗法对DNA的固定杂交情况进行了表征,并对溶液中的莠去津进行了检测。结果表明,此方法能增加修饰层上DNA探针的固定量,并使固定的ssDNA保持伸展状态,有利于杂交的进行,同时发现该传感器对莠去津具有较灵敏的响应。
DNA electrochemical biosensor is noted widely in recent years for rapid development. It has been used in many fields such as food industry, disease diagnosis, pharmaceutical analysis and environmental monitoring due to the high sensitivity, better specificity, rapid response, easy handling, no pollution and low cost. DNA is the basic genetic material of organism, the interaction of some chemicals and DNA make the formation of DNA adducts, which can evaluate and predict potential carcinogenicity of pollutants because of reflecting the interaction condition between chemical pollutants and DNA. So DNA electrochemical biosensor holds some promise of a novel detection technique, and it has a broad prospect of application in the pollutant monitoring field.
However, the method and material used to immobilize biomolecules is one of the crucial factors for improving the stability, selectivity and sensitivity of biosensor in the preparation, and the selection of appropriate indicator is also the key technology. Two new-typed DNA electrochemical biosensor was prepared, and the interaction between atrazine and Herring Sperm DNA was studied in this paper. The dissertation is divided into three main parts:
(1) A novel DNA electrochemical biosensor was prepared by immobilizing the first generation polyamidoamine (G1 PAMAM) dendrimer on the glassy carbon electrode (GCE) surface by coupled activation agent, which was treated via electrochemical oxidation, then ssDNA was also immobilized through covalently action. Using methylene blue as the indicator, the DNA electrochemical biosensor was characterized by cyclic voltammetry and differential pulse voltammetry. The results indicated that the complementary ssDNA segment in the solution could be recognized and detected through mensurating the oxidation-deoxidation peaks current of embed methylene blue in dsDNA.
(2) In order to explore the DNA damage effects induced by atrazine, ultraviolet absorption spectrometry, fluorescence spectrometry and cyclic voltammetry were used to study the the interaction between atrazine and Herring Sperm DNA. The results showed that a hypochromic effect and a slight red shift on the UV spectrogram of atrazine were observed after the interaction with Herring Sperm DNA, while the fluorescence increased obviously. Cyclic voltammetry indicated that the reductive potential of atrazine shifted positively with the peak current decreasing after the interaction with DNA. The above results proved that atrazine could be inserted into the double-helix of DNA and formed a stable DNA adduct.
(3) The mixture of the fourth generation polyamidoamine (G4 PAMAM) dendrimer and chitosan with certain proportion was immobilized on the glassy carbon electrode (GCE) surface by the film forming properties of chitosan, then the amino group and the 5′-phosphate group of ssDNA had a covalently action by coupled activation agent of EDC. Using [Co(phen)3]3+ as the indicator, a new DNA electrochemical biosensor was prepared. The immobilization and hybridization of ssDNA was characterized by cyclic voltammetry, differential pulse voltammetry and electrochemical impedance spectroscopy. Experiment results showed that the immobilized amount of DNA probe on modified layer increased, and the immobilized ssDNA kept a extension state, which was beneficial to the hybridization of DNA. Furthermore, this biosensor had a sensitive response for atrazine.
引文
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