【作者】 谢宗红；

【导师】 金利通；

【作者基本信息】 华东师范大学 ， 分析化学， 2007， 硕士

【摘要】 自1962年Clark和Lyons首先报道了基于生物传感技术的电化学葡萄糖生物传感器。发展至今，生物传感技术已成为一项前沿技术，它成为一个由生物、化学、医学、药学、电子技术等多种学科相互渗透的研究领域。生物传感器因其具有选择性高、分析速度快、操作简易等特点，而且可进行在线活体分析，在临床诊断、环境监测、食品工业等方面得到了高度重视和广泛应用。随着纳米技术的发展，将纳米技术引入生物传感器，不仅表现出纳米材料的优势，更为生物传感器的发展开辟了广阔的前景。本论文主要工作是新型纳米材料在酶生物传感器中的研究及其应用。纳米材料的引入，有效的提高了生物传感器的灵敏度和检测限，克服了传统生物传感器的不足，论文对可能的机理进行了讨论。另外，将纳米材料应用于生物传感器中，并与其他技术（微渗析、流动注射）联用，有效的实现了对葡萄糖，次黄嘌呤，有机磷农药的测定。本论文努力实现纳米技术、生物传感技术和生物科学技术的三者有机结合，具体内容如下：第一部分绪论首先介绍了生物传感器的原理及其发展和应用，其次介绍了纳米技术在生物传感器中的应用及新型纳米生物传感器与微渗析技术，流动注射技术联用在生物、化学和药理研究中的优点。纳米技术引入生物传感器后，不但体现了自身的优越性，还明显的提高了生物传感器的灵敏度，检测时间，并实现了实时检测。纳米生物传感器作为一个发展热点将被广泛用于化学、医学、生物和环境等领域的快速检测。最后简述了本论文的工作和意义。第二部分金属铁卟啉纳米粒子修饰的生物传感器及其用于鼠脑中葡萄糖测定的研究本章利用金属卟啉的化学稳定性，及其在电极上很好的可逆性和电催化活性，制备了金属铁卟啉纳米粒子并将其成功地应用于葡萄糖生物传感器的研制，得到了以金属铁卟啉纳米颗粒为电子媒介体的葡萄糖生物传感器。考察了该传感器在优化的实验条件下对葡萄糖的响应特性，实验结果表明该传感器在pH为6.9的磷酸缓冲溶液（PBS）条件下对葡萄糖的线性检测范围为1.0×10^-5～2.1×10^-3mol·L^-1，检测下限为5.0×10^-6mol·L^-1。研究表明，该传感器选择性好，灵敏度高，寿命长，且制作简单使用方便；可有效地排除了抗坏血酸、尿酸、L-半胱胺酸、谷氨酸的干扰；与微渗析技术联用，能成功地测定S．D．大鼠大脑中葡萄糖的含量，为生理以及临床医学的研究提供了新的检测手段。第三部分金属铁卟啉纳米粒子修饰的次黄嘌呤生物传感器研究嘌呤碱是一类重要的生物活性物质，次黄嘌呤（Hypoxanthine，HX）是一磷酸腺苷（AMP）的分解代谢产物。低氧时，AMP加速分解，HX堆积在组织和血液中，因此测定血液中的HX水平与脑缺氧、脑损伤程度的分析密切相关，对指导临床，判断疾病具有极为重要的意义。本章制备了新型金属铁卟啉的纳米粒子，将此纳米颗粒、黄嘌呤氧化酶依次修饰于光玻碳电极表面，得到了以金属铁卟啉纳米颗粒为电子媒介体黄嘌呤氧化酶生物传感器。考察了该传感器在优化的实验条件下对次黄嘌呤的响应特性，实验结果表明该传感器在pH为6.9的磷酸缓冲溶液（PBS 6.9）条件下对次黄嘌呤的线性检测范围为2.0×10^-6～3.4×10^-4mol·L^-1，检测下限为1.0×10^-6mol·L^-1，并能有效的消除了抗坏血酸（AA）、尿酸（UA）、L-半胱胺酸（L-cys）对电极的干扰。该传感器克服了传统传感器中介体易流失的缺点，延长了使用寿命，并提高了检测的灵敏度、稳定性和抗干扰性。与微渗析技术联用，能成功地测定S．D．大鼠大脑中次黄嘌呤的含量，为生理以及临床医学的研究提供了新的检测手段。第四部分金属锰卟啉纳米粒子修饰电极与流动注射法联用应用于抑制型有机磷农药生物传感器的研究有机磷农药是一种高效、广谱的农用杀虫剂，在农业生产中应用广泛。有机磷农药的过量或不当使用会引起的各种环境问题。因此，农药残留快速检测的研究对保证绿色农业的实现具有重要意义。本文制备了金属锰卟啉（MnTPP）纳米粒子，将此纳米粒子应用于乙酰胆碱酯酶（AChE）传感器中，制成新型MnTPP纳米粒子修饰AChE生物传感器。考察了该传感器在优化实验条件下对硫代乙酰胆碱（ATChI）的响应特性，并与流动注射法联用，成功应用于有机磷农药含量的测定。实验结果表明，该生物传感器在pH为7.3的磷酸缓冲溶液（PBS）条件下对有机磷农药敌百虫的线性检测范围为1.0×10^-9～1.0×10^-3mol·L^-1，检测下限为5.0×10¹⁰mol·L^-1。该传感器灵敏度高，稳定性好，可快速、灵敏、准确、简便的应用于有机磷农药的含量的检测。第五部分PVP-CdS量子点修饰的酶生物传感器及其应用于有机磷农药检测的研究量子点（quantum dots，QDs）是半径小于或接近于激光波尔半径的半导体纳米晶粒。作为准零维纳米材料有着特殊的光学和电学性质。本章制备了聚乙烯吡咯烷酮（PVP）修饰的硫化镉（CdS）量子点（PVP-CdS），将其修饰在玻碳电极上，制成安培型乙酰胆碱酯酶（AChE）生物传感器。并将此生物传感器成功地应用于有机磷农药敌百虫的检测。PVP-CdS量子点也具有较好的稳定性和生物兼容性，能最大限度保持酶的活性，实验结果表明，所得传感器灵敏度显著提高，成功地应用于农药敌百虫的测定。以硫代乙酰胆碱作底物，对农药敌百虫进行了测定，在25ppb～515ppb范围内校准曲线呈良好的线性关系，检出限为12ppb。更多还原

【Abstract】 Since the development of the first glucose biosensor in 1962 by Clark and Lyons, biosensors have developed to be a frontier and newly-interdisciplinary including biology, chemistry, medicine and electronics. Due to its simplicity, high sensitivity and potential ability for real-time and on-site analysis, biosensor has been widely applied in various fields including clinical diagnosis, environment monitoring, food control and industrial process, and so on.The development of nano-techonology provides a new opportunity to the development of biosensors. Nanobiosening technology is revealing its superiority not only have good effect on it, but also on biosensor. This dissertation focuses on 1) fabricating novel biosensors based on new nanomaterials and nanostructures, and integrated them with microdialysis, flow injection analysis, etc. 2) establishing the bases for application of them to detect glucose, hypoxanthine and organophosphate pesticides. We are adhering to an organic combination of nanotechnology, biosensing technology and clinical researches. The details are given as follows:Chapter One: PrefaceAt first, a critical review with regard to biosensors and the development of biosensors is given. Then the application of nanotechnology into biosensors attach with micro dialysis and flow inject analysis is highlighted. Nanotechnology is revolutionizing the development of biosensors. Sensitivity and other attributes of biosensors can be improved by using nanomaterials in their construction. Finally, the work and significance of this thesis was briefly introduced.Chapter Two: Biosensor based on Iron Porphyrin nanoparticles for amperometric detection of glucose Metalloporphyrin is a prototype of metal hexacyanoferrates with remarkable catalytic-, electro-, photo-, and biochemical properties. They are potentially used as electron-transfer mediators for the development of amperometric biosensors owing to their excellent electrochemical and electro-catalytic properties. In this paper, a type of iron （III）-porphyrin （FeTPP） nanoparticles was prepared and modified on the surface of glassy carbon electrode （GCE） as a novel redox electron mediator. Therefore a novel electrochemical sensor for glucose was developed. The effects of experimental conditions on biosensors’ response have been investigated. The linear range for the determination of glucose is 1.0×10^-5 ～ 2.1×10^-3 mol·L^-1 with a detection limit of 5.0×10^-6 mol·L^-1 （S/N = 3）. A possible mechanism for the operation of the glucose biosensor was also proposed. This biosensor exhibited high sensitivity, stability and anti-interference. Finally the level of glucose in S.D. rat brain was also detected in order to demonstrate the practical usage of this biosensor. The result was in satisfactory agreement with the references.Chapter Three: Amperometric biosensor for hypoxanthine based on immobilized xanthine oxidase on Iron Porphyrin nanoparticles modified glassy carbon electrodeThe determination of hypoxanthine （Hx） has considerable importance for the detection of human’s brain injure. The continuing development and application of analytical methods are proceeding at a rapid pace, and many methods have been proposed for the determination of trace amounts of Hx. In this paper, a type of iron （III）-porphyrin （FeTPP） nanoparticles was prepared and modified on the surface of glassy carbon electrode as a novel redox electron mediator. Therefore a novel electrochemical sensor for hypoxanthine was developed. The features of the enzymic biosensor were discussed in detail. Iron （III）-porphyrin （FeTPP） nanoparticles was dripped on the biosensor to exclude electro oxidizable interferants, such as ascorbic acid （AA） and uric acid （UA）. The resulting sensor is stable, highly sensitive and shows a fast response time. The response conditions were optimized. Coupled with micro dialysis, the Hx in rat brains were measured with satisfactory results.Chapter Four: Mn Porphyrin nanoparticles modified Acetylcholinesterase biosensor coupled with flow injection analyzer for detecion of PesticidePesticides represent a major hazard to public health throughout the world, it is necessary to monitor them in the environment and foods. Biosensor technology is well suited for field of monitoring OP compounds. In this paper Mn Porphyrin （MnTPP） nanoparticles （NPs） were prepared using mixing solvent techniques. MnTPP NPs has been used for the immobilization and stabilization of the acetylcholinesterase （AChE）. The use of MnTPP NPs is shown to provide significant enzyme stabilization, as well as the means for lowering the detection limit of the biosensor. The biosensor was optimized and the properties were described. Coupled with flow injection analyzer （FIA）, the biosensor was used for the detection of organophosphate pesticides （OPs） trichlorfon. The concentration of this enzymatic inhibitor had the linear range from 1.0×10^-9 mol·L^-1 to 1.0×10^-3 mol·L^-1and the detection limit was found to be 5.0×10^-10 mol·L^-1.’So, a novel amperometric OPs biosensor based on MnTPP NPs has been firstly developed and showed excellent detection limit, the good precision and the relative stability.Chapter Five: Development of Quantum Dots modified acetylcholinesterase biosensor for the detection of trichlorfonQuantum dots （QDs） are semiconductors particles that have all three dimensions confined to the nanometer length scales which close to their Bohr exciton radius. And QDs exhibit unique optical and electronic properties. Recently, people are interested in QDs for potential use in many fields, especially in biosensing and bioconjugates. In this paper, we conceive that QDs as the enzyme biosensor platform could be advantageous for the sensitive detection of OP compounds. PVP-CdS QDs has been used for the immobilization and stabilization of the acetylcholinesterase （AChE）. The electrocatalytic activity of PVP-CdS QDs leads to a greatly improved electrochemical detection and higher sensitivity and stability. The biosensor was used for the detection of organophosphate pesticides （OPs）, such as trichlorfon. The sensor performance, including pH, was optimized with respect to operating conditions. The biosensor had excellent operational lifetime stability with very low decrease in the activity of enzymes for more than 10 repeated measurements over a 2-week period.更多还原