【作者】 钟霞；

【导师】 袁若；

【作者基本信息】 西南大学 ， 分析化学， 2013， 博士

【摘要】 葡萄糖含量的测定在很多领域都有着十分重要的意义。现代的电化学葡萄糖生物传感技术将纳米材料与电化学分析检测技术有机的结合在一起,由此产生了一系列性能优良的电化学葡萄糖生物传感器。本文着重于设计和合成新型的纳米复合材料,并结合电化学或电致化学发光检测技术构建了几种新型的电化学葡萄糖传感器。本论文由六个部分组成。第一章绪论在这一章里对生物传感器的基本原理及分类、电化学生物传感器、纳米材料的定义和特性进行了介绍；对几种常见的纳米材料及其在生物传感器中的应用、电化学葡萄糖生物传感器的发展、纳米材料在葡萄糖生物传感器的应用与发展、电致化学发光葡萄糖生物传感器作了简要的概述。第二章纳米材料的引入为葡萄糖生物传感器的直接电化学带来了新契机,由各种纳米材料构筑的直接电子转移的葡萄糖生物传感器已经成为如今研究的热点。很多的纳米材料都存在着容易从电极表面渗漏的问题,这使得测定时的电化学信号很不稳定,传感器的性能因此降低。由纳米材料构建的性能优良的葡萄糖生物传感器不仅应该克服纳米材料渗漏的问题,而且该纳米材料还能够在酶的氧化还原活性中心和电极表面之间进行有效的电子传递,使得响应时间缩短、灵敏度提高。因此,在本章中,首先基于电活性物质普鲁士蓝、石墨烯以及生物相容性好的壳聚糖合成了壳聚糖/普鲁士蓝/石墨烯的纳米复合物(CS-PB-GR),将壳聚糖用于共建壳聚糖/普鲁士蓝/石墨烯的纳米复合物不仅增强了该复合材料的生物相容性,而且有效的解决了聚普鲁士蓝纳米粒子的渗漏问题,可以有效的提高该复合纳米材料的性能。将该纳米复合材料结合纳米金和半刀豆球蛋白A(ConA)在玻碳电极上构建了：葡萄糖氧化酶/Con A/葡萄糖氧化酶／纳米金/CS-PB-GR/葡萄糖酶传感器。该传感器还具有如下优势：纳米金和Con A的引入,可以有效的提高葡萄糖氧化酶的固载量；CS-PB-GR纳米复合材料中的PB纳米粒子和电极表面的GOD可以形成一种类双酶的体系,起到信号放大的作用；石墨烯和PB纳米粒子能够的提高电子在酶的活性中心(FAD)和电极表面之间的迁移速率。用该方法制得的葡萄糖传感器具有响应快、灵敏度高、选择性好等优点。第三章随着对碳材料性质研究的进一步深入,富勒烯作为一种生物传感材料也开始被应用于葡萄糖生物传感器研究领域。C60分子具有一个大的共轭离域π键,亲电子能力很强,可作为电子受体。这使得它具有了许多特殊的物理和化学性质,并且呈现出令人期待的应用前景。C60易溶于甲苯、苯、烷烃和二硫化碳等非极性有机溶剂,但它不溶于水,而且导电性能不高,因此使得C60在生物传感器中的应用受到了一定的局限。为了改善C60导电性能不高的这一不足,我们设计、合成了铂包裹的C60纳米线。将合成的Pt@C60内米线和葡萄糖氧化酶滴涂在电极表面,用壳聚糖固定成膜,制备了葡萄糖酶传感器。研究表明,由于引入了导电性能好、催化性能高、生物兼容性好的铂纳米材料,使得基于Pt@C60纳米线构建的酶生物传感器对葡萄糖具有很好的催化性能。该传感器也具有制作简单、响应时间短、选择性好、稳定性好等优点。第四章表面活性剂作为一种含有极性和非极性官能团的两性分子,它能强烈吸附在固-液界面上,将表面活性剂溶液滴涂到电极表面会形成有序的多重双层生物模拟膜,能加快电子在酶和电极之间的交换速率。为了改进C60的导电性能同时提高基于C60纳米粒子的成膜能力,我们以阳离子表面活性剂四辛基溴化铵(TOAB)作为稳定剂和相迁移试剂,合成了Au@C60纳米粒子,并在玻碳电极上制备了：葡萄糖氧化酶／Au@C60葡萄糖酶生物传感器。纳米金的引入使得C60的亲电子能力得到进一步提高,制得的Au@C60可以在电极表面直接成膜而且还能够有效的固载葡萄糖氧化酶；同时Au@C60表面带正电荷的TOAB,也可以增加酶的固载量；而且合成的Au@C60纳米粒子能够实现电子在葡萄糖氧化酶的活性中心和电极表面之间的直接电子转移。该传感器制作简单、响应时间短、选择性好、稳定性好。第五章为了扩大C60纳米粒子在生物传感器中的进一步应用,我们设计合成了水溶性的C60衍生物,利用该衍生物,在水溶液中和四氯钯酸钾合成了一种新型的钯纳米粒子(Pd@Cys-C60)。将该粒子滴涂在玻碳电极表面构建了无酶的葡萄糖生物传感器,无酶的葡萄糖传感器也是当今研究的热点领域。在Pd纳米粒子和C60的协同作用下,Pd@Cys-C60纳米粒子能有效的催化葡萄糖。该无酶的葡萄糖生物传感器构造简单、不需要在特殊条件下保存、不受酶易变性失活的影响、使用寿命长,而且该非酶传感器的稳定性和重现性比普通的酶生物传感器要好。第六章电致化学发光是把电化学和化学发光相结合发展起来的一门新的检测技术,它不仅具有电化学分析的一些优点,而且还具有化学发光分析的诸多特点。鲁米诺是电致化学发光中最常用的发光试剂,当溶液中有过氧化氢存在时,产生的发光信号的强度与过氧化氢的浓度成正比。由于葡萄糖氧化酶在氧化葡萄糖时可以产生过氧化氢,因此可以通过间接测定过氧化氢的方法来检测葡萄糖,从而制备高灵敏的电致化学发光葡萄糖生物传感器。研究表明,纳米金可以增强鲁米诺-过氧化氢体系的电致化学发光强度。因此,在本章中用葡萄糖氧化酶交联戊二醛的方法合成了Ausbeii@GOD纳米粒子,并结合电致化学发光检测技术,在玻碳电极上制备了：葡萄糖氧化酶/Aushell@GOD/壳聚糖/酶传感器,并将该传感器应用于葡萄糖的电致化学发光检测。研究表明,合成的Aushell@GOD纳米粒子,可以有效的催化Luminol-H2O2体系的电致化学发光,该电致化学发光葡萄糖酶生物传感器制备简单、响应快速、线性范围宽、灵敏高,选择性好。更多还原

【Abstract】 The determination of glucose has vital significance in many areas. Modern electrochemical glucose biosensor perfectly combines the nano-materials with electrochemical detection technologies, which produced a series of electrochemical glucose biosensor with excellent performance. This article focuses on the study and synthesis of novel nanocomposite and constructs several distinctive glucose sensors employing electrochemical as well as electro-chemiluminescence detection techniques. This paper consists of six parts.Chapter one:In this chapter, the basic principles and classification of biosensors, electrochemical biosensor as well as the definition and characteristics of nanomaterials are introduced. In addition, several aspects are briefly summarized including applications of common nanomaterials in biosensors, development of electro-chemical glucose biosensors, the application of nanomaterials in development of the glucose biosensor and ECL glucose biosensors.Chapter two:Introduction of nanomaterials brings new opportunities for the direct electrochemistry of glucose biosensor. Glucose biosensors based on various nanomaterials to realize the direct electron transfer of glucose oxidase have become hot research now. However, most of the nanomaterials are easy to leak from the electrode surface, which makes the measured electrochemical signal unstable, thus reducing the performance of the sensor. Glucose biosensor constructed by nanomaterials with good performance can not only overcome the problem of leakage of nanomaterials, but also achieve direct electron transfer between the redox-active center of the enzyme and the electrode surface, thus improved the response time and the sensitivity. Therefore, in this chapter, firstly, the Chitosan/Prussian blue/graphene nanocomposite (CS-PB-GR) was synthesized based on the electroactive species including Prussian blue, graphene and chitosan. The application of chitosan for building chitosan/Prussian blue/graphene nanocomposites not only enhanced the biocompatibility of the complex but also effectively avoided the leakage of poly Prussian blue nanoparticles, which strongly improved the performance of composite nanomaterials. The CS-PB-GR nanocomposites, nano-Au and half sword bean globulin a (Con A) were applied to construct the GOD/Con A/GOD/nano-Au/CS-PB-GR/glassy carbon electrode. The advantages of this method were summarized as follows: The introduction of nano-Au and Con A effectively improved the amount of glucose oxidation. A pseudobienzymatic system was formed with PB nanoparticles and GOD to enhance the esponse signal. The electron transfer rate between the activity center (FAD) of enzyme and electrode surface was significantly accelerated by the application of grapheme and PB nanoparticles. The biosensor exhibits good electrocatalytic behavior towards detection of glucose with fast response, high sensitivity and selectivity and realized the direct electron transfer.Chapter three:With further research on the carbon materials, fullerenes, as a biomaterial, began to be applied in glucose biosensor. C60molecule has a large conjugated delocalizedπbond and behaves strong electron affinity, which can be used as the electron acceptor. The characters mentioned above endow C60special physical and chemical properties, which prompt such material present potential application prospects. C60is soluble in non-polar organic solvent such as toluene, benzene, paraffin, and carbon disulfide. However, C60has poor water soluble and conductivity, which limited its application in biosensor. In order to overcome this shortcoming of C60, we synthesized platinum wrapped C60nanowires. The synthesized Pt@C60nanowires, glucose oxidase and CS were dropped onto the electrode surface to prepare a glucose sensor. Results showed that the enzyme biosensor based on Pt@C60nanowires behaved good catalytic performance towards glucose, due to the introduction of platinum nanomaterials with good electrical conductivity, high catalytic performance and good biocompatibility. Besides, the sensor exhibited short response time, good selectivity and good stability.Chapter four:As an amphiphilic molecule, the surface active agent containing polar and non-polar functional groups can strongly adsorb on the solid-liquid interface. The application of surfactant solution could form an orderly double biomimetic membrane, which can speed up the exchange rate of electrons between the enzyme and the electrode. To overcome the water-insoluble and poor conductivity of C60, Au@C60core-shell nanoparticles were synthesized with cationic surfactants tetraoctylammonium bromide (TOAB) as a stabilizing agent and phase transfer reagent. Furthermore, the glucose enzyme biosensor was fabricated based on glucose oxidase/Au@C60. The utility of nano-Au enhanced the electronic affinity of C60, which ensured the effective immobilization of glucose oxydase by Au@C60. Simultaneously, the positive charged TOAB also increased the enzyme loading amount. Moreover, the obtained Au@C60nanoparticles can promote the direct electron transfer between the active center of glucose oxydase and the electrode surface. The fabrication process of the sensor was simple, and the sensor exhibited short response time, good selectivity and excellent stability.Chapter five:In order to expand the application of C60nanoparticles in biosensors, a new type of Palladium Nanoparticles (Pd@Cys-C60) were synthesizes employing C60and potassium tetrachloropalladate. The nanoparticles were modified onto the glassy carbon electrode surface to construct non-enzymatic glucose sensor. Pd@Cys-C60nanoparticles behaved excellent catalysis to glucose due to the synergetic effect of Pd nanoparticles and C60. The non-enzymatic glucose sensor can be saved under common conditions and are free of influence by the deactivation of the enzyme variability. Moreover the stability and reproducibility of this non-enzyme sensor are better than the ordinary enzyme biosensor.Chapter six:ECL is a new testing technology combining electro-chemistry and chemiluminescence, which not only obtains the advantages of electrochemical analysis, but also exhibits many characteristics of chemiluminescence analysis. Luminol is most commonly used reagent in the luminescent ECL. The ECL intensity of luminol-H2O2is directly proportional to the quantity of H2O2. GOD can produce H2O2during their substrate-specific enzymatic reaction and the intensity of the ECL signal is directly proportional to the concentration of H2O2which was generated by enzymatical catalysis. Therefore, a sensitive ECL glucose biosensor could be designed for measure of glucose by detecting H2O2indirectly. Gold nanoparticles can enhance the ECL intensity of the luminol-H2O2system. Thus, in this chapter, hollow Aushell@GOD nanoparticles were synthesized using glutaraldehyde cross-link with glucose oxidase. Then, the GOD/Aushell@GOD/chitosan/GCE was prepared to detect glucose through ECL technology. Results showed that, the obtained Aushell@GOD nanoparticles exhibited excellent catalytic effect towards the electro-chemiluminescence of luminol-H2O2system. The preparation of this glucose enzyme biosensor is simple, and the electro-chemiluminescence biosensor showed fast response, wide linear range, high sensitivity and good selectivity.更多还原