【作者】 章仁毅；

【导师】 何品刚； 方禹之；

【作者基本信息】 华东师范大学 ， 分析化学， 2010， 硕士

【摘要】 近年来为了了解蛋白质的结构,蛋白质发生电子传递的机理,以及在电极表面模拟生物体内的电子传递过程,基于氧化还原蛋白直接电化学的生物传感器受到了广泛的关注和研究。但生物活性物质的电活性中心多被包埋在多肽链中,同时它也容易在电极表面强烈吸附造成电极的钝化,因此在裸电极表面难以实现其直接电子转移。碳纳米管以其特有的力学、电学和化学性质以及独特的管状分子结构和应用价值,已成为研究热点。碳纳米管由于易于表面功能化、大的比表面积、促进电子传递反应效应,能提高生物电化学传感器电极的灵敏度；其良好生物相容性,有利于保持固定化的生物体的活性。在引进其他纳米材料如金属纳米颗粒、量子点等后亦能进一步促进生物活性物质和传感器电极的电子传递速率,更持久地保持生物体的活性,为开发更高效,简便,耐用的生物电化学传感器提供新的思路。本文利用定向碳纳米管阵列优良的物理、化学、电催化性能以及它们良好的生物兼容性,结合纳米颗粒的量子效应和大的比表面积效应,制备了2种不同类型的定向碳纳米管阵列修饰电极。采用化学气相沉积法在石英基底上成功制备了大面积的定向碳纳米管阵列,并将其制成定向碳纳米管阵列电极,将血红蛋白、葡萄糖氧化酶采用多种方法固定到阵列电极界面上,制备的生物传感器具有较高的灵敏度、较低的检测下限以及快的响应速度等优势。第一章绪论首先系统介绍了碳纳米管的发现及应用研究,包括碳纳米管的结构和分类、性质、制备方法、分离和纯化以及应用现状。着重介绍定向碳纳米管阵列在生物和化学传感器中的研究进展。第二章常压下单控温化学气相沉积法制备定向碳纳米管阵列本实验以石英为基底,以金属有机化合物酞菁铁为原料,利用其高温裂解可同时产生碳源和催化剂,采用常压下单控温方式在最高温890℃时制备了大面积的定向碳纳米管阵列,实现了在常压条件下,仅控制单一温度就可以进行化学气相沉积制备定向碳纳米管阵列,有效地降低了实验成本,简化了实验器材。通过对工艺参数的改进和优化,利用化学气相沉积法成功实现了定向碳纳米管阵列的批量制备,获得了大面积的、高度定向排列的碳纳米管,实验制备的定向碳纳米管阵列管长均匀约10μm,管径约50-70 nm。实验探讨了电阻炉最高炉温、升华区位置和酞菁铁用量、载气流速和比例等制备参数以及不同基底上定向碳纳米管阵列生长的情况,对碳纳米管的制备和实际应用研究具有重要意义。第三章葡萄糖氧化酶/CdS纳米颗粒／壳聚糖/Pt纳米颗粒修饰定向碳纳米管阵列电极的直接电化学研究本章采用化学气相沉积法,以酞菁铁为原料,在石英基底上制备了大面积的定向碳纳米管阵列。将制得的定向碳纳米管阵列表面喷金处理后,用40%的HF溶液使得定向碳纳米管阵列和石英片基片相互分离,制成ACNTs工作电极。场发射扫描电镜图表明,制备的定向碳纳米管阵列生长密度高、排列紧密、长度接近、直径均匀、定向性好。使用了铂和CdS两种类型的纳米颗粒及葡萄糖氧化酶(GOD)对定向碳纳米管阵列电极进行修饰并以此电极作为工作电极：首先在定向碳纳米管阵列上电沉积的铂纳米颗粒(Ptnano)；当溶液pH值大于壳聚糖的pKa时,葡萄糖氧化酶(GOD)、CdS纳米颗粒和壳聚糖(CS)的混合溶液电沉积到ACNTs-Ptnano电极表面。实验表明,铂纳米粒子能有效地增加电极表面积、提高电极的电子传递速率；CdS纳米粒子能有效地促进葡萄糖氧化酶与定向碳纳米管阵列电极之间的直接电子传递,实现了葡萄糖氧化酶的活性中心FAD/FADH2的直接电化学。这种基于CS-GOD-CdS/ACNTs-Ptnano电极的葡萄糖生物传感器显示了良好的传感性能,其检测线性范围为0.4 mmol/L-21.2 mmol/L,最低检测限为46.8μmol/L (S/N=3)，表观米氏常数Kmapp为11.86mmol/L。电极保存2个星期后,该传感器仍保持了良好的选择性、重复性和稳定的电化学信号。第四章血红蛋白在重氮化修饰定向碳纳米管阵列电极上的直接电化学及电催化性质研究本章通过在溶液中电化学还原4-羧基苯基重氮盐,对定向碳纳米管阵列电极进行功能化修饰,得到基于表面羧基化的定向碳纳米管阵列电极,并且成功地将血红蛋白(Hb)分子固定在定向碳纳米管阵列电极表面。制备的定向碳纳米管阵列电极具有良好的生物兼容性和导电性,电极表面的羧基提供了更多的蛋白质结合位点,使得血红蛋白分子在此功能界面上可实现直接电子转移。采用循环伏安法和计时电流法对Hb分子的直接电化学和生物电催化活性进行监测。结果表明,在0.1mol/L pH=7.0的PBS溶液中,Hb的式量电位为-0.312 V (vs.Ag/AgCl),异相电子传递速率常数为0.95±0.05 cm/s。该电极对H202有很好的电催化还原作用,其表观米氏常数Kmapp为0.15 mmol/L。更多还原

【Abstract】 In recent years, in order to understand the structure and the mechanism of electron transfer, as well as on the electrode surface simulations of biological electron transfer processes of proteins in vivo, the direct electrochemistry of redox protein based bio-sensors has been widespread concern and research. However, the electrical bio-active substances are often embedded in the active center of multi-polypeptide chain, while it is also easily and strongly adsorptable on the electrode surface to cause electrode passivation, and therefore the bare electrode surface is difficult to achieve the direct electron transfer.Carbon nanotubes for its unique mechanical, electrical and chemical properties, as well as a unique molecular structure of tubular and application value have become a research hotspot. As the easy-to surface functionalization of carbon nanotubes, a large surface area to promote the electron transfer reaction effect, can improve the sensitivity of bio-electrochemical sensor electrode; its good biocompatibility is conducive to maintaining the activity of immobilized organisms. In the introduction of other nanomaterials such as metal nano-particles, quantum dots, etc. After the bio-active substances can also further promote and sensor electrode electron transfer rate, more durable to maintain the activity of organisms, in order to develop more efficient, simple, durable, bio-electrochemical sensors provide a new way of thinking.In this paper, aligned carbon nanotube array of excellent physical, chemical, electro-catalytic properties and their good biological compatibility, combined with nano-particles which have quantum effects and large surface area effect, prepared two kinds of different types of modificated aligned carbon nanotube arrays electrode. Using chemical vapor deposition on quartz substrates were successfully prepared large area aligned carbon nanotube arrays, and it’s made of aligned carbon nanotube array electrode. We used a variety of ways to fix the hemoglobin, glucose oxidase onto the array electrode interface and prepared bio-sensors which have higher sensitivity, lower detection limit and fast response speed advantages.Chapter One:PrefaceFirst, a systematic introduction to the discovery and application of carbon nanotubes, including carbon nanotubes of the structure and classification, properties, preparation methods, separation and purification as well as the application status. Aligned carbon nanotube arrays in biological and chemical sensors are mainly focuses on and reviewed.Chapter Two:Single temperature controlled aligned carbon nanotube arrays synthesizing by chemical vapor deposition method under atmospheric pressureIn this experiment, we used quartz as substrate, the metal phthalocyanine compounds of iron as raw material which can also produce carbon source and catalyst by its high-temperature pyrolysis, to prepare a large area carbon nanotube arrays with only a single temperature of the maximum temperature 890℃controlled under atmospheric pressure that will effectively reduce the test cost, simplify the experimental equipment. Through the improvement and optimization of process parameters, using chemical vapor deposition was achieved a large area, highly aligned carbon nanotube arrays.The even length of carbon nanotubes is about 10μm while its diameter about 50 - 70 nm. Experimental Investigation of the maximum furnace temperature, sublimation zone and the amount of iron phthalocyanine, carrier gas flow rate and the ratio of the preparation parameters and the substrate to the growth of aligned carbon nanotube arrays on the situation, the preparation and practical application of carbon nanotube research has significance.Chapter Three: Glucose oxidase/CdS nanoparticles/chitosan/Pt nanoparticles modified aligned carbon nanotube array electrode and its direct electrochemistryThis chapter we used the chemical vapor deposition method, iron phthalocyanine as raw materials in the quartz substrate to prepare a large area aligned carbon nanotube arrays.Spraying the surface of aligned carbon nanotube arrays by gold after treatment, and then with 40% HF solution makes the aligned carbon nanotube arrays and quartz film substrate separated and made the ACNTs to be working electrode. Field emission scanning electron micrographs showed that the growth of aligned carbon nanotube arrays prepared are high density, closely arranged, the length and diameter is close, orientation is good. We used two types of Pt and CdS nano-particles and glucose oxidase (GOD) to modify the aligned carbon nanotube arrays electrode as working electrode:first to electro-deposited platinum nano-particles (Ptnano) on the vertical carbon nanotube arrays; When the solution’s pH value is greater than the pKa of chitosan, the glucose oxidase (GOD), CdS nanoparticles and chitosan (CS) mixed solution will electro-deposite onto ACNTs-Ptnano electrode surface. Experiments show that Pt nanoparticles can effectively increase the electrode surface area, improve the electrode electron transfer rate; CdS nano-particles can be effective in promoting glucose oxidase and vertical carbon nanotube arrays of direct electron transfer between electrodes to achieve a glucose oxidase Direct Electrochemistry of active site FAD/FADH2. This electrode-based glucose biosensor CS-GOD-CdS/ACNTs-Ptnano showed good sensing properties, with the linear range of detection of 0.4 mM - 21.2 mM, the lowest detection limit of 46.8μmol/L (S/N = 3), the apparent Michaelis-Menten constant of 11.86 mmol/L. After Electrode Saved 2 weeks, the sensor is still maintaining a good selectivity, repeatability and stability of electrochemical signals.Chapter Four: The directly electrochemical and electrocatalytic properties of hemoglobin on the diazotization salt modified aligned carbon nanotube array electrodeThis chapter through the electrochemical reduction of 4 - carboxyl-phenyl diazonium salt in the solution, we functional modificated the aligned carbon nanotube array electrode, and successfully made hemoglobin (Hb) molecules fixed in aligned carbon nanotube arrays electrode surface. Prepared aligned carbon nanotube array electrode has a good biological compatibility and electrical conductivity; the electrode surface carboxyl group provides more protein binding sites, making this function interface in the hemoglobin molecule can achieve the direct electron transfer. Using cyclic voltammetry and chronoamperometry we can research the direct electrochemistry and bio-electrocatalytic activity monitoring of Hb molecules. The results show that in 0.1mol/L pH = 7.0 in PBS solution, Hb-type amount of the potential of -0.312 V (vs. Ag / AgCl), heterogeneous electron transfer rate constant of 0.95±0.05 cm/s. The electrode have a good electro-catalytic reduction reactions to H2O2, its apparent Michaelis-Menten constant Kmapp is 0.15 mmol/L.更多还原