基于酶生物传感器检测植物多酚的研究
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摘要
植物多酚作为一类储量丰富的可再生绿色资源,不仅在农业、生态环境、食品、医药化工等领域有重要意义,而且与人们的日常生活密切相关,随着多酚在不同领域的应用研究取得越来越多的成果,其必将成为人类利用的重要资源之一。因此,开发简单、快速、有效的检测植物多酚的手段和方法将越来越受到人们的关注。而酶生物传感器能够将酶的专一性、灵敏性和电学的简便、迅速巧妙地结合起来,可以在一个复杂的体系中,不受其他物质的干扰,快速准确的测出某些物质的含量。因此,开发酶生物传感器在植物多酚的检测方面将具有十分重要的意义。在传感器的构建过程中,固定材料的选择及固定方法的有效应用决定着酶生物传感器的稳定性、选择性和灵敏性等主要性能。本论文探讨设计了三种新型的酶生物传感器,并对其在植物多酚检测中的应用进行了研究。研究内容主要分为以下三部分:
(1)以Zn(Ac)_2和N,N-二甲基甲酰胺为原料,采用水热合成法在160°C下合成出了纳米ZnO,并用X-射线衍射、透射电镜表征了所制备纳米ZnO的结构,同时以制备的纳米ZnO固定酪氨酸酶制备了多酚生物传感器,结果表明高等电点的纳米ZnO在很大程度上促进了酪氨酸酶与电极之间的直接电子传递,能够有效地用于多酚的测定。
(2)通过将酪氨酸酶固定在类水滑石修饰的玻碳电极表面,构建了一种新型的酪氨酸酶生物传感器,用于对植物多酚的检测。利用静电吸附作用,类水滑石将表面带有负电荷的酪氨酸酶牢固的修饰在玻碳电极表面,并且使其保持了原有的生物活性。通过循环伏安法、电化学交流阻抗法对修饰电极进行了表征。同时,对检测条件如底液的pH值、温度、酪氨酸酶固定量进行了研究和优化。在最优条件下,对邻苯二酚、咖啡酸、槲皮素在浓度为3–300,0.888–444,0.066–396μmol/L范围内进行了检测,检测限分别为0.1,0.05和0.003μmol/L (S/N=3)。此外,论文对酶电极的重复性和稳定性进行了研究。通过对实际样品中植物多酚含量的测定,对所构建的Tyr/HTLc/GCE生物传感器的实际应用也进行了研究。
(3)通过将血红蛋白固定在Cu_2S纳米棒和Nafion纳米复合材料修饰的玻碳电极表面,构建了一种新型的血红蛋白生物传感器,在H_2O_2存在的条件下,用于对植物多酚的检测。Cu_2S纳米棒/Nafion无机-有机混合材料不仅为血红蛋白提供了生物适宜的微环境,而且提高了传感器检测多酚的灵敏度。通过线性扫描伏安法和电化学交流阻抗法对修饰电极进行了表征。同时,对测量条件如底液的pH值、H_2O_2浓度、应用电位进行了研究和优化。在最优条件下,对邻苯二酚、对苯二酚、间苯二酚在浓度为7.0–110,0.6–10和8–100μmol/L范围内进行了检测,检测限分别为0.5,0.03,0.6μmol/L (S/N=3)。该生物传感器对多酚检测的响应时间仅为8 s,且具有良好的稳定性和重现性。同时通过对实际样品中植物多酚含量的测定,对所构建的生物传感器的实际应用进行了研究。
Being one kind of rich reserves and renewable green resources, plant polyphenols not only play an important role in agriculture, environment, food, medicine and chemical industry, but also are closely related to people’daily life, and will certainly to become of capital stocks for humanity use. Therefore, the development of simple, fast and effective methods for plant polyphenols detection has gained more and more people's attention. Combining the specifity, sensitive of enzymes and the simply, rapid of electrochemistry, enzyme-based biosensors can determine the content of certain substances in a complex system quickly and accurately, and not affected by other materials. Therefore, the development of enzyme biosensors has the very vital significance in plant polyphenols detection. However, the methods and materials used to immobilize enzymes are one of the crucial factors for improving the stability, selectivity and sensitivity of biosensor in the preparation. Three new-typed enzyme-based biosensors were prepared, and their application in detection of plant polyphenols was studied in this paper. The dissertation is divided into three main parts:
(1) Using Zn(Ac)_2 and N,N-Dimethylformamide as raw materials, nanometersized ZnO was prepared at 160°C by hydrothermal reaction method. The X-ray diffraction (XRD) and Transmission electron microscopy (TEM) were used to analyse the structure of nanometersized ZnO. At the same time, the application of nanometersized ZnO using in modifying electrodes to detect the polyphenol was preliminarily studied, the results showed that the high-isoelectric point nano-ZnO promoted the direct electron transfer between the tyrosinase and the electrode to a large extent, could be effectively used for the determination of polyphenol.
(2) A tyrosinase (Tyr) biosensor has been constructed by immobilizing tyrosinase on the surface of Mg-Al-CO3 hydrotalcite-like compound film (HTLc) modified glassy carbon electrode (GCE) for the determination of polyphenols. The negatively charged tyrosinase was adsorbed firmly on the surface of positively charged HTLc/GCE by the electrostatic interactions and retained its activity to a great degree. The modified electrode was characterized by cyclic voltammetry and AC impedance spectra. Polyphenols were determined by the direct reduction of biocatalytically generated quinone species. The different parameters, including pH, temperature and enzyme loading were investigated and optimized. Under the optimum conditions, Tyr/HTLc electrode gave linear response ranges of 3–300, 0.888–444 and 0.066–396μmol/L with the detection limits (S/N=3) of 0.1, 0.05 and 0.003μmol/L for catechol, caffeic acid and quercetin. In addition, the repeatability and stability of the enzyme electrode were estimated. Total polyphenol contents of real samples were also determined to study the potential applicability of the Tyr/HTLc/GCE biosensor.
(3) A hemoglobin (Hb) biosensor was fabricated based on Hb immobilized onto Cu_2S nanorods/Nafion nanocomposite film for the detection of polyphenols in the presence of H_2O_2. The nanostructured inorganic–organic hybrid material formed by Cu_2S nanorods and Nafion provided a biocompatible microenvironment for Hb and increased the sensitivity for polyphenols detection. The modified electrodes were characterized by electrochemical impedance spectroscopy and linear sweep voltammetry. Parameters such as pH, H_2O_2 concentration and applied potential were optimized. Under optimum conditions, the biosensor gave linear response ranges of 7.0–110, 0.6–10 and 8–100μmol/L with the detection limits (S/N=3) of 0.5, 0.03 and 0.6μmol/L for catechol, hydroquinone and resorcin, respectively. The developed biosensor exhibited a short response time within only 8 s with good stability and reproducibility. Such new Hb biosensor showed great promise for rapid, simple analysis of polyphenols contents in real samples.
(1)以Zn(Ac)_2和N,N-二甲基甲酰胺为原料,采用水热合成法在160°C下合成出了纳米ZnO,并用X-射线衍射、透射电镜表征了所制备纳米ZnO的结构,同时以制备的纳米ZnO固定酪氨酸酶制备了多酚生物传感器,结果表明高等电点的纳米ZnO在很大程度上促进了酪氨酸酶与电极之间的直接电子传递,能够有效地用于多酚的测定。
(2)通过将酪氨酸酶固定在类水滑石修饰的玻碳电极表面,构建了一种新型的酪氨酸酶生物传感器,用于对植物多酚的检测。利用静电吸附作用,类水滑石将表面带有负电荷的酪氨酸酶牢固的修饰在玻碳电极表面,并且使其保持了原有的生物活性。通过循环伏安法、电化学交流阻抗法对修饰电极进行了表征。同时,对检测条件如底液的pH值、温度、酪氨酸酶固定量进行了研究和优化。在最优条件下,对邻苯二酚、咖啡酸、槲皮素在浓度为3–300,0.888–444,0.066–396μmol/L范围内进行了检测,检测限分别为0.1,0.05和0.003μmol/L (S/N=3)。此外,论文对酶电极的重复性和稳定性进行了研究。通过对实际样品中植物多酚含量的测定,对所构建的Tyr/HTLc/GCE生物传感器的实际应用也进行了研究。
(3)通过将血红蛋白固定在Cu_2S纳米棒和Nafion纳米复合材料修饰的玻碳电极表面,构建了一种新型的血红蛋白生物传感器,在H_2O_2存在的条件下,用于对植物多酚的检测。Cu_2S纳米棒/Nafion无机-有机混合材料不仅为血红蛋白提供了生物适宜的微环境,而且提高了传感器检测多酚的灵敏度。通过线性扫描伏安法和电化学交流阻抗法对修饰电极进行了表征。同时,对测量条件如底液的pH值、H_2O_2浓度、应用电位进行了研究和优化。在最优条件下,对邻苯二酚、对苯二酚、间苯二酚在浓度为7.0–110,0.6–10和8–100μmol/L范围内进行了检测,检测限分别为0.5,0.03,0.6μmol/L (S/N=3)。该生物传感器对多酚检测的响应时间仅为8 s,且具有良好的稳定性和重现性。同时通过对实际样品中植物多酚含量的测定,对所构建的生物传感器的实际应用进行了研究。
Being one kind of rich reserves and renewable green resources, plant polyphenols not only play an important role in agriculture, environment, food, medicine and chemical industry, but also are closely related to people’daily life, and will certainly to become of capital stocks for humanity use. Therefore, the development of simple, fast and effective methods for plant polyphenols detection has gained more and more people's attention. Combining the specifity, sensitive of enzymes and the simply, rapid of electrochemistry, enzyme-based biosensors can determine the content of certain substances in a complex system quickly and accurately, and not affected by other materials. Therefore, the development of enzyme biosensors has the very vital significance in plant polyphenols detection. However, the methods and materials used to immobilize enzymes are one of the crucial factors for improving the stability, selectivity and sensitivity of biosensor in the preparation. Three new-typed enzyme-based biosensors were prepared, and their application in detection of plant polyphenols was studied in this paper. The dissertation is divided into three main parts:
(1) Using Zn(Ac)_2 and N,N-Dimethylformamide as raw materials, nanometersized ZnO was prepared at 160°C by hydrothermal reaction method. The X-ray diffraction (XRD) and Transmission electron microscopy (TEM) were used to analyse the structure of nanometersized ZnO. At the same time, the application of nanometersized ZnO using in modifying electrodes to detect the polyphenol was preliminarily studied, the results showed that the high-isoelectric point nano-ZnO promoted the direct electron transfer between the tyrosinase and the electrode to a large extent, could be effectively used for the determination of polyphenol.
(2) A tyrosinase (Tyr) biosensor has been constructed by immobilizing tyrosinase on the surface of Mg-Al-CO3 hydrotalcite-like compound film (HTLc) modified glassy carbon electrode (GCE) for the determination of polyphenols. The negatively charged tyrosinase was adsorbed firmly on the surface of positively charged HTLc/GCE by the electrostatic interactions and retained its activity to a great degree. The modified electrode was characterized by cyclic voltammetry and AC impedance spectra. Polyphenols were determined by the direct reduction of biocatalytically generated quinone species. The different parameters, including pH, temperature and enzyme loading were investigated and optimized. Under the optimum conditions, Tyr/HTLc electrode gave linear response ranges of 3–300, 0.888–444 and 0.066–396μmol/L with the detection limits (S/N=3) of 0.1, 0.05 and 0.003μmol/L for catechol, caffeic acid and quercetin. In addition, the repeatability and stability of the enzyme electrode were estimated. Total polyphenol contents of real samples were also determined to study the potential applicability of the Tyr/HTLc/GCE biosensor.
(3) A hemoglobin (Hb) biosensor was fabricated based on Hb immobilized onto Cu_2S nanorods/Nafion nanocomposite film for the detection of polyphenols in the presence of H_2O_2. The nanostructured inorganic–organic hybrid material formed by Cu_2S nanorods and Nafion provided a biocompatible microenvironment for Hb and increased the sensitivity for polyphenols detection. The modified electrodes were characterized by electrochemical impedance spectroscopy and linear sweep voltammetry. Parameters such as pH, H_2O_2 concentration and applied potential were optimized. Under optimum conditions, the biosensor gave linear response ranges of 7.0–110, 0.6–10 and 8–100μmol/L with the detection limits (S/N=3) of 0.5, 0.03 and 0.6μmol/L for catechol, hydroquinone and resorcin, respectively. The developed biosensor exhibited a short response time within only 8 s with good stability and reproducibility. Such new Hb biosensor showed great promise for rapid, simple analysis of polyphenols contents in real samples.
引文
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