碳纳米管修饰电极在毛细管电泳中的应用
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摘要
由于CNTs具有良好的导电性、催化活性和较大的比表面积,用碳纳米管对电极进行修饰可以降低电极反应的过电势,增加峰电流,改善分析性能,提高方法选择性和灵敏度,因此,近年来碳纳米管作为修饰电极材料也已广泛应用于食品化学、生命电分析化学、药物化学、环境监测及其它领域。毛细管电泳-电化学检测法(CE-EC)由于具有分离效率高、快速、简便、检测限低等优点,适合低浓度复杂样品的检测,因而得到了广泛的应用。将化学修饰电极微型化,并应用于毛细管电泳检测生命物质中,将会扩大检测的范围,提高检测的灵敏度和选择性。
本论文以电化学分析系统为技术依托,研制了碳纳米管修饰葡萄糖生物传感器,在此基础上将碳纳米管修饰葡萄糖生物传感器微型化并与毛细管电泳联用,实现了对血清中葡萄糖的快速灵敏检测。探索了毛细管电泳-电化学检测过氧化氢的实验条件,为下一步细胞内过氧化氢的检测奠定基础。论文共分四章。
第一章,综述了碳纳米管修饰电极在食品化学、生命化学、药物化学、环境检测等领域中的应用研究进展。主要介绍了碳纳米管在化学修饰电极和电化学生物传感器方面的应用情况,以及碳纳米管修饰电极与毛细管电泳联用的研究进展。碳纳米管作为一种新型的电极修饰剂,由于本身所拥有的独特性质使其具有广阔的发展前景。随着碳纳米管功能化的发展,其各种生物兼容性的多元功能化更加赋予了碳纳米管许多潜在的优势,在未来的生物、医药检测中将发挥重要的作用。
第二章,通过电化学沉积将壳聚糖、葡萄糖氧化酶和碳纳米管固定到镀铂金电极上,制备了一种新型葡萄糖生物传感器。以计时安培检测法,探讨了影响该生物传感器灵敏度的各因素,利用该生物传感器定量测定了人血清中的葡萄糖,测得的结果与对照值基本相符。该研究为测定人血清中的葡萄糖提供了新方法。研究表明:镀铂金电极上的铂颗粒多孔的结构,增加了酶在传感器上负载量,提高了电流响应的灵敏度;碳纳米管具有较大的比表面积和较快的电子传递速率,能保证葡萄糖的催化产物(H2O2)在膜内快速扩散和反应,而不聚集在电极表面,从而提高了电极的响应电流,缩短了响应时间,葡萄糖氧化酶也可以固定在碳纳米管的表面和内部并保持其活性,从而增加酶在电极表面的吸附量;传感器表面的Nafion膜有效阻止了UA和AA向电极表面的扩散;壳聚糖也可能在一定程度上抑制了这些物质的电化学反应,从而减小了这些电活性物质对葡萄糖测定的干扰。该传感器线性范围宽,灵敏度高,响应速度快,尿酸、抗坏血酸等血液中可能存在的电活性物质对葡萄糖的测定无干扰。
第三章,在第二章报道的葡萄糖生物传感器制作方法基础上,研制了一种微型化的葡萄糖生物传感器。将葡萄糖生物传感器与毛细管电泳联用,探索了检测电位、分离电压、缓冲溶液pH值及浓度等条件的影响,该微型生物传感器表现出良好的灵敏度和重现性。膜中Nafion和壳聚糖的存在有效抑制了抗坏血酸和尿酸的干扰,减少了葡萄糖氧化酶的流失和高压对酶活性的影响,增大了传感器的重现性和寿命。成功检测了健康人和糖尿病患者血清中的葡萄糖。与葡萄糖检测中常用的铜电极相比,该传感器灵敏度高,抗干扰能力强,有望用做芯片毛细管电泳的工作电极,能显著提高测定的速率和效率。
第四章,以金微盘电极和离子液体修饰单壁碳纳米管糊微盘电极作为毛细管电泳电化学工作电极,试验了两种电极对过氧化氢的响应情况。以金微盘电极与毛细管电泳联用,采用不易受溶液中氧干扰的正电位做为检测电势,实现了过氧化氢的定量检测。探讨了分离电压、缓冲溶液pH和工作电位等条件对H2O2检测的影响。该方法有望用于单细胞中H2O2的测定。
Carbon nanotubes (CNTs) have led to many new technical developments and applications such as being used for the modified electrodes due to their high chemical stability, high surface area, unique electronic properties, and relatively high mechanical strength. Such properties of carbon nanotubes make them also extremely attractive for the task of electrochemical detection. CNTs-modified electrodes have been shown useful for improving the electrochemical behavior of important analytes. Higher sensitivity and stability have thus been demonstrated in voltammetric analysis. Capillary electrophoresis (CE) with electrochemical detection (ECD) has already been applied to analyse many substances owing to its inherent high sensitivity, simplicity of instrumention needed, short analysis time, efficient separation capabilities, and minute consumption of sample. When CNTs-modified microelectrodes are used as detector of CE, the kinds of substance determined will increase greatly and the sensitivity and selectivity will enhance laargely.
In this thesis, we develop a method to make a novel glucose biosensor, the concentration of glucose in human serum successfully. A simple and convenient method of H2O2 determination was developed by CE- ECD.
In the first chapter, The carbon nanotubes modified electrode and its applications in the food chemistry, biologic chemistry, medicine chemistry, environmental chemistry test and capillary electrophoresis are reviewed. We mainly summarized the analytical applications and progresses of carbon nanotubes in chemical modified electrodes and electrochemical biosensors.
In the second chapter, a new amperometric biosensor for the quantitative measurement of glucose was reported. The biosensor was based on the immobilization of single walled carbon nanotubes(SWNTs)–glucose oxidase–chitosan biocomposite at platinized Au electrode. The effect of Pt deposit time, the content of SWNTs and GOx, pH of the buffer and the operating potential was studied. The production mechanism was discussed. The experimental result illustrated that the biosensor has the properties of good reproducibility and stability, high sensitivity and rapid response to the determination of glucose. The experiment also showed that there was no interference from the electro-active interfering species such as ascorbic acid and uric acid when they are at endogenous level in serum. The biosensor was successfully used to detect the glucose in serum rapidly and accurately. The existence of SWNTs increased the number of glucose oxidase and the speed of electron delivery in biomembrane.
In the third chapter, we made a kind of Au microelectrode and developed a novel method for the determination of glucose in human serum based on capillary electrophoresis coupled with novel glucose biosensor which was homemade and based on the immobilization of SWNTs–glucose oxidase–chitosan biocomposite at platinized Au electrode by one-step electrodeposition according to the method reported in the second chapter. Factors influencing the performance, including separation voltage, detection potential, pH value and the concentration of the buffer were studied. The biosensor exhibited good stability and durability in the analytical procedures. Finally, glucose in human serum from two healthy individuals and two diabetics was successfully determined. It proved that the method can be applied to the determination of glucose in human blood using CE-ECD. The reason of its good reproducibility and durability is probably that the immobilized enzyme was firmly retained on the biosensor surface as a result of the presense of chitosan and Nafion.
In the fourth chapter, a method using capillary electrophoresis with amperometric detection was developed for the determination of hydrogen peroxide. Gold microelectrode and ionic liquid modified-SWNTs paste microelectrode made by ourselves were used as the detector of capillary zone electrophoresis with electrochemical end-column amperometric detection to determine hydrogen peroxide. Gold microelectrode showed higher sensitivity to H2O2 was used as the detector of capillary electrophoresis. Factors influencing the performance, including separation voltage, detection potential and pH value of the buffer were studied. Because of the higher sensitivity of this method, it was hopeful to determinate the concentration of H2O2 in single cell.
本论文以电化学分析系统为技术依托,研制了碳纳米管修饰葡萄糖生物传感器,在此基础上将碳纳米管修饰葡萄糖生物传感器微型化并与毛细管电泳联用,实现了对血清中葡萄糖的快速灵敏检测。探索了毛细管电泳-电化学检测过氧化氢的实验条件,为下一步细胞内过氧化氢的检测奠定基础。论文共分四章。
第一章,综述了碳纳米管修饰电极在食品化学、生命化学、药物化学、环境检测等领域中的应用研究进展。主要介绍了碳纳米管在化学修饰电极和电化学生物传感器方面的应用情况,以及碳纳米管修饰电极与毛细管电泳联用的研究进展。碳纳米管作为一种新型的电极修饰剂,由于本身所拥有的独特性质使其具有广阔的发展前景。随着碳纳米管功能化的发展,其各种生物兼容性的多元功能化更加赋予了碳纳米管许多潜在的优势,在未来的生物、医药检测中将发挥重要的作用。
第二章,通过电化学沉积将壳聚糖、葡萄糖氧化酶和碳纳米管固定到镀铂金电极上,制备了一种新型葡萄糖生物传感器。以计时安培检测法,探讨了影响该生物传感器灵敏度的各因素,利用该生物传感器定量测定了人血清中的葡萄糖,测得的结果与对照值基本相符。该研究为测定人血清中的葡萄糖提供了新方法。研究表明:镀铂金电极上的铂颗粒多孔的结构,增加了酶在传感器上负载量,提高了电流响应的灵敏度;碳纳米管具有较大的比表面积和较快的电子传递速率,能保证葡萄糖的催化产物(H2O2)在膜内快速扩散和反应,而不聚集在电极表面,从而提高了电极的响应电流,缩短了响应时间,葡萄糖氧化酶也可以固定在碳纳米管的表面和内部并保持其活性,从而增加酶在电极表面的吸附量;传感器表面的Nafion膜有效阻止了UA和AA向电极表面的扩散;壳聚糖也可能在一定程度上抑制了这些物质的电化学反应,从而减小了这些电活性物质对葡萄糖测定的干扰。该传感器线性范围宽,灵敏度高,响应速度快,尿酸、抗坏血酸等血液中可能存在的电活性物质对葡萄糖的测定无干扰。
第三章,在第二章报道的葡萄糖生物传感器制作方法基础上,研制了一种微型化的葡萄糖生物传感器。将葡萄糖生物传感器与毛细管电泳联用,探索了检测电位、分离电压、缓冲溶液pH值及浓度等条件的影响,该微型生物传感器表现出良好的灵敏度和重现性。膜中Nafion和壳聚糖的存在有效抑制了抗坏血酸和尿酸的干扰,减少了葡萄糖氧化酶的流失和高压对酶活性的影响,增大了传感器的重现性和寿命。成功检测了健康人和糖尿病患者血清中的葡萄糖。与葡萄糖检测中常用的铜电极相比,该传感器灵敏度高,抗干扰能力强,有望用做芯片毛细管电泳的工作电极,能显著提高测定的速率和效率。
第四章,以金微盘电极和离子液体修饰单壁碳纳米管糊微盘电极作为毛细管电泳电化学工作电极,试验了两种电极对过氧化氢的响应情况。以金微盘电极与毛细管电泳联用,采用不易受溶液中氧干扰的正电位做为检测电势,实现了过氧化氢的定量检测。探讨了分离电压、缓冲溶液pH和工作电位等条件对H2O2检测的影响。该方法有望用于单细胞中H2O2的测定。
Carbon nanotubes (CNTs) have led to many new technical developments and applications such as being used for the modified electrodes due to their high chemical stability, high surface area, unique electronic properties, and relatively high mechanical strength. Such properties of carbon nanotubes make them also extremely attractive for the task of electrochemical detection. CNTs-modified electrodes have been shown useful for improving the electrochemical behavior of important analytes. Higher sensitivity and stability have thus been demonstrated in voltammetric analysis. Capillary electrophoresis (CE) with electrochemical detection (ECD) has already been applied to analyse many substances owing to its inherent high sensitivity, simplicity of instrumention needed, short analysis time, efficient separation capabilities, and minute consumption of sample. When CNTs-modified microelectrodes are used as detector of CE, the kinds of substance determined will increase greatly and the sensitivity and selectivity will enhance laargely.
In this thesis, we develop a method to make a novel glucose biosensor, the concentration of glucose in human serum successfully. A simple and convenient method of H2O2 determination was developed by CE- ECD.
In the first chapter, The carbon nanotubes modified electrode and its applications in the food chemistry, biologic chemistry, medicine chemistry, environmental chemistry test and capillary electrophoresis are reviewed. We mainly summarized the analytical applications and progresses of carbon nanotubes in chemical modified electrodes and electrochemical biosensors.
In the second chapter, a new amperometric biosensor for the quantitative measurement of glucose was reported. The biosensor was based on the immobilization of single walled carbon nanotubes(SWNTs)–glucose oxidase–chitosan biocomposite at platinized Au electrode. The effect of Pt deposit time, the content of SWNTs and GOx, pH of the buffer and the operating potential was studied. The production mechanism was discussed. The experimental result illustrated that the biosensor has the properties of good reproducibility and stability, high sensitivity and rapid response to the determination of glucose. The experiment also showed that there was no interference from the electro-active interfering species such as ascorbic acid and uric acid when they are at endogenous level in serum. The biosensor was successfully used to detect the glucose in serum rapidly and accurately. The existence of SWNTs increased the number of glucose oxidase and the speed of electron delivery in biomembrane.
In the third chapter, we made a kind of Au microelectrode and developed a novel method for the determination of glucose in human serum based on capillary electrophoresis coupled with novel glucose biosensor which was homemade and based on the immobilization of SWNTs–glucose oxidase–chitosan biocomposite at platinized Au electrode by one-step electrodeposition according to the method reported in the second chapter. Factors influencing the performance, including separation voltage, detection potential, pH value and the concentration of the buffer were studied. The biosensor exhibited good stability and durability in the analytical procedures. Finally, glucose in human serum from two healthy individuals and two diabetics was successfully determined. It proved that the method can be applied to the determination of glucose in human blood using CE-ECD. The reason of its good reproducibility and durability is probably that the immobilized enzyme was firmly retained on the biosensor surface as a result of the presense of chitosan and Nafion.
In the fourth chapter, a method using capillary electrophoresis with amperometric detection was developed for the determination of hydrogen peroxide. Gold microelectrode and ionic liquid modified-SWNTs paste microelectrode made by ourselves were used as the detector of capillary zone electrophoresis with electrochemical end-column amperometric detection to determine hydrogen peroxide. Gold microelectrode showed higher sensitivity to H2O2 was used as the detector of capillary electrophoresis. Factors influencing the performance, including separation voltage, detection potential and pH value of the buffer were studied. Because of the higher sensitivity of this method, it was hopeful to determinate the concentration of H2O2 in single cell.
引文
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