新型电化学传感器对于生理活性物质的分析研究
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摘要
生理活性物质与人类的健康息息相关,例如活性氧,它涉及到衰老、癌症、以及神经系统相关疾病的病理过程,因此,寻找可靠且灵敏的分析方法来定性评估与定量检测生理活性物质非常重要。由于具有灵敏度高、响应快、操作简便、样品用量少、可微型化等特点,生物传感器在临床医学、食品工业、发酵工业、环境监测等领域已得到广泛应用。蓬勃发展的纳米技术,特别是各种具有特殊性质的纳米材料的出现及应用,又为生物传感器的发展打开了一片新天地。本论文结合了分析化学、电化学、材料科学等领域的研究,致力于利用新型的纳米材料来构建电化学生物传感器,将其应用于生理活性物质的电化学分析中,主要分为以下六章：
第一章,首先对电化学生物传感器和纳米材料的基本原理、研究现状、应用领域和发展前景进行了综述,阐述了生理活性物质,尤其是活性氧的研究意义,然后在此基础上提出本论文的研究设想,即利用新型的纳米材料来构建电化学生物传感器,将其应用于生理活性物质的电化学分析中。
第二章,基于二氧化钛纳米粒子光催化体系的快速准确评估抗氧化药物的电化学传感器研究：为检测活性氧中毒性最强的·OH自由基,以及筛选能够有效清除-OH自由基的抗氧化药物,我们以二氧化钛纳米粒子的光催化反应作为实验中·OH自由基的发生体系,以4-羟基苯甲酸(4-hydroxybenzoic acid,4-HBA)与·OH自由基的羟基化反应作为研究模型,利用羟基化后的专一产物3,4-二羟基苯甲酸(3,4-dihydroxybenzoic acid,3,4-DHBA)的电化学响应来间接表征体系中·OH自由基的生成量。向反应体系中添加抗氧化药物后,再利用3,4-DHBA的电化学信号的抑制程度成功实现了抗氧化药物性能的精确评估。同时,我们也利用传统的荧光检测法验证了该电化学方法的准确性。荧光法的反应机理是对苯二酸(terephthalic acid, TA)捕获.OH生成具有荧光效应的2-羟基对苯二酸(2-hydroxyterephthalic acid,2-TA),利用添加抗氧化药物后所导致2-TA的荧光信号的抑制程度来反映抗氧化药物的抗氧化能力。两种方法结果的一致性表明了电化学方法的准确性与可靠性,最后,利用该电化学传感器成功评估了从6种天然抗氧化植物萃取出的生理活性物质的抗氧化性能,为将来进一步的实际应用奠定了良好的基础。
第三章,基于大孔有序的泡沫氧化硅材料的过氧化氢电化学生物传感器研究：基于大孔材料优异的传质性能、良好的蛋白分子吸附固定特性等,我们以大孔材料(MOSF)作为生物载体,通过静电作用力吸附细胞色素c,并利用细胞色素c对过氧化氢的电催化作用,构建了一个基于细胞色素c直接电子传递的过氧化氢生物传感器。该传感器针对过氧化氢检测的线性范围以及检测限方面均取得了令人满意的结果,为进一步的实际应用提供了一个良好的平台。随后,为进一步研究材料表面环境对所吸附的蛋白的影响,我们利用后修饰法在MOSF表面分别修饰了-CN,-SH,-NH2官能团,并运用同样的组装方法,将细胞色素c固定在材料的孔径中,研究了该氧化还原蛋白的直接电化学行为,并与未修饰官能团的MOSF材料做了比较。
第四章,基于介孔碳纳米球层层组装技术的过氧化氢电化学生物传感器研究：通过层层组装的方法,利用介孔碳小球与阳离子聚电解质构建了一种基于Cytc的H202生物传感器。由于静电吸引作用,修饰于ITO电极上的介孔碳纳米球对于Cytc显示了很好的吸附特性。峰形良好的氧化还原峰证实了在没有任何外加电子媒介体的存在下,吸附于介孔碳纳米球中的Cytc与电极成功发生了直接电子转移。该传感器对H202具有良好的电催化作用,并且可以通过控制修饰电极的组装层数来调变传感性能,从而达到合理设计传感器的目的。
第五章,基于介孔碳材料的烟酰胺腺嘌呤二核苷酸(NADH)的电催化氧化研究：NADH在固体电极上通常具有较高的氧化过电位,极易引起电极表面的污染与钝化。本章利用碳介孔材料(CMM)修饰的玻碳电极来研究NADH的电化学氧化。CMM所具有的大比表面积、高电子传递能力以及丰富的边片状缺陷位和表面含氧官能团,对NADH的氧化表现出了较高的电催化性能,将NADH的氧化过电位降低了595mV(与未修饰的裸玻碳电极相比),成功实现了NADH在低电位条件下(0.1Vvs. SCE,pH7.2)稳定灵敏的响应,检测下限为1.0μM,线性响应范围为10～600μM。这为基于以NADH为辅酶的脱氢酶的电流型生物传感器的发展提供了新的研究途径。
第六章,二维平面上单分散金原子阵列修饰的石墨烯材料的制备及其电化学性质的初步研究：石墨烯是继富勒烯和碳纳米管之后科学界的又一重大发现,石墨烯的发现,充实了碳材料家族,形成了从零维的富勒烯、一维的碳纳米管、二维的石墨烯到三维的金刚石和石墨的完整体系。目前已有一些关于石墨烯以及纳米粒子/石墨烯复合材料(NPs/graphene)的合成报道与应用研究。在这部分工作中,我们发展了一种基于石墨烯的二维平面,快速可控的溅射方法来形成金团簇纳米阵列。通过调整溅射电流的大小、角度和时间,得到了一系列不同尺寸的金团簇纳米颗粒阵列,最小可达1.05nm且无团聚现象发生,形成的纳米颗粒形貌均一,分散性良好,并且这种复合膜表现出良好的热力学稳定性、优良的导电性以及极高的金负载量,这些性能为生物传感器的研制和电化学研究提供了一个广阔的研究平台。
第七章,对全文进行了总结,总结了研究中存在的一些不足,并提出了后续工作的目标和研究思路。
Physiologically active substances, such as reactive oxygen species (ROS), are related to human health. The ROS plays an important role in many pathological processes, such as aging, cancer and neurological diseases. Therefore, it is necessary to establish new reliable and sensitive method to make a qualitative and quantitative assessment of physiological active substances. Due to the high sensitivity, fast response, simple operation, and small consumption of sample, biosensors have been widely used in clinical medicine, food industry, fermentation industry, environmental monitoring and other fields. The vigorous development of nanotechnology, in particular, the appearance of a variety of nanomaterials with special properties, has opened a new world for bio-sensors. Combining the analytical chemistry, electrochemistry, material science and other areas of research, this thesis committed to use new nanomaterials for construction of biosensors which are applied to the electroanalysis of physiologically active substances. This dissertation includes six chapters as follows:
In Chapter One, we introduced the research and development of nanomaterials and biosensors, respectively. Then we proposed a scheme of nanomaterials-based electrochemical sensors for electroanalysis of physiologically active substances. Finally we outlined the experimental ideas and the research purposes of this thesis.
In Chapter Two, we studied a simple and sensitive electrochemical method for the determination of antioxidant capacity.4-hydroxybenzoic acid (4-HBA) was used as a trapping agent for TiO2photogenerated-OH radicals, leading to3,4-dihydroxybenzoic acid (3,4-DHBA). Square-wave voltammetry was used to quantify the amount of3,4-DHBA formed from the reaction between4-HBA and·OH. Addition of antioxidants induced the competition between4-HBA and antioxidants toward-OH elimination, resulting in a decrease of the measured current. The IC50for different standard antioxidants was calculated and expressed in Trolox equivalent antioxidant capacity (TEAC). The validation of the proposed electrochemical method was also performed using an alternative detection method based on fluorimetry. In this method, the·OH radicals generated through the photocatalytic oxidation of water ware trapped by terephthalic acid (TA), producing fluorescent2-hydroxy-terephthalic acid (2-HTA). And similar trends were observed when comparing the antioxidant capacity calculated using the two methods. The applicability of this method has been shown by analyzing the antioxidant capacity of six plant extracts. Therefore, it can be concluded that the proposed electrochemical method is a good alternative strategy for antioxidant assessment due to its fast response, accuracy and inexpensive apparatus.
In Chapter Three, an amperometric biosensor for hydrogen peroxide (H2O2) has been constructed by immobilizing cytochrome c (Cyt c) on a macroporous material modified indium/tin oxide (ITO) electrode. Cyclic voltammetry showed that the direct and quasi-reversible electron transfer of Cyt c was achieved without the aid of an electron mediator. A surface-controlled electrode process was observed with the apparent heterogeneous electron-transfer rate constant (ks) of29.2s-1. The prepared biosensor exhibited an excellent electrocatalytic response to the reduction of H2O2. The amperometric responses increased steadily with the concentration of H2O2in the range from5μM to2mM, with a detection limit of0.5μM at pH7.4. This investigation not only provided a method for the direct electron transfer of Cyt c on macroporous materials, but also established a feasible approach for durable and reliable detection of H2O2. Furthermore, in view of the adjustable surface of MOSF, we also investigated the electrochemical behavior of Cyt c immobilized on MOSF substrates which were modified with different organic functionalities, including-CN,-SH, and-NH2.
In Chapter Four, a Cytochrome c-based biosensor for hydrogen peroxide (H2O2) has been constructed via layer-by-layer (LBL) assembly of mesoporous carbon nanospheres and polydiallyldimethylammonium (PDDA). Cyclic voltammetry showed that the direct and quasi-reversible electron transfer of Cyt c was achieved without the aid of an electron mediator. The prepared biosensor exhibited good electrocatalytic responses to the reduction of H2O2. In addition, assembling carbon nanospheres on the electrode by means of LBL technology could control the amount of Cyt c immobilized on the electrode, and thus control its biosensor properties by regulating the thickness of the mesoporous carbon nanospheres-assembled layers.this makes the rational design of enzyme electrodes possible.
In Chapter Five, the electrochemical oxidation of β-nicotinamine adenine dinucleotide (NADH) was investigated at a glassy carbon electrode modified with carbon mesoporous materials (CMM). Due to the large surface area and electro-catalytic properties of CMM, the overpotential of the electrodes toward the oxidation of NADH is decreased by595mV in aqueous solution at neutral pH. The anodic peak currents increase steadily with the concentration of NADH in the range from2μM to1.1mM, the detection limit being1.0μM at pH7.2and a potential of +0.3V vs. SCE. The results enable NADH to be sensed at a low potential and are promising with respect to the design of dehydrogenasebased amperometric biosensors.
In Chapter Six, we developed a rapid, solventless sputtering method to generate the2D bulk gold NPs/graphene hybrid superstructures in10seconds. The size and morphology of gold NPs are fine controlled in a wide range down to1.05nm by adjusting the sputtering current, sputtering angle, and sputtering time. The hybrids achieve ultra-high gold carriage and high thermal stability. We also investigate the properties of the hybrids via electrochemical methods. This composite structure enables a new class of electrode materials and offers an excellent platform for electrochemical applications.
In Chapter Seven, we summarized and proposed the objects and schemes for further research.
第一章,首先对电化学生物传感器和纳米材料的基本原理、研究现状、应用领域和发展前景进行了综述,阐述了生理活性物质,尤其是活性氧的研究意义,然后在此基础上提出本论文的研究设想,即利用新型的纳米材料来构建电化学生物传感器,将其应用于生理活性物质的电化学分析中。
第二章,基于二氧化钛纳米粒子光催化体系的快速准确评估抗氧化药物的电化学传感器研究：为检测活性氧中毒性最强的·OH自由基,以及筛选能够有效清除-OH自由基的抗氧化药物,我们以二氧化钛纳米粒子的光催化反应作为实验中·OH自由基的发生体系,以4-羟基苯甲酸(4-hydroxybenzoic acid,4-HBA)与·OH自由基的羟基化反应作为研究模型,利用羟基化后的专一产物3,4-二羟基苯甲酸(3,4-dihydroxybenzoic acid,3,4-DHBA)的电化学响应来间接表征体系中·OH自由基的生成量。向反应体系中添加抗氧化药物后,再利用3,4-DHBA的电化学信号的抑制程度成功实现了抗氧化药物性能的精确评估。同时,我们也利用传统的荧光检测法验证了该电化学方法的准确性。荧光法的反应机理是对苯二酸(terephthalic acid, TA)捕获.OH生成具有荧光效应的2-羟基对苯二酸(2-hydroxyterephthalic acid,2-TA),利用添加抗氧化药物后所导致2-TA的荧光信号的抑制程度来反映抗氧化药物的抗氧化能力。两种方法结果的一致性表明了电化学方法的准确性与可靠性,最后,利用该电化学传感器成功评估了从6种天然抗氧化植物萃取出的生理活性物质的抗氧化性能,为将来进一步的实际应用奠定了良好的基础。
第三章,基于大孔有序的泡沫氧化硅材料的过氧化氢电化学生物传感器研究：基于大孔材料优异的传质性能、良好的蛋白分子吸附固定特性等,我们以大孔材料(MOSF)作为生物载体,通过静电作用力吸附细胞色素c,并利用细胞色素c对过氧化氢的电催化作用,构建了一个基于细胞色素c直接电子传递的过氧化氢生物传感器。该传感器针对过氧化氢检测的线性范围以及检测限方面均取得了令人满意的结果,为进一步的实际应用提供了一个良好的平台。随后,为进一步研究材料表面环境对所吸附的蛋白的影响,我们利用后修饰法在MOSF表面分别修饰了-CN,-SH,-NH2官能团,并运用同样的组装方法,将细胞色素c固定在材料的孔径中,研究了该氧化还原蛋白的直接电化学行为,并与未修饰官能团的MOSF材料做了比较。
第四章,基于介孔碳纳米球层层组装技术的过氧化氢电化学生物传感器研究：通过层层组装的方法,利用介孔碳小球与阳离子聚电解质构建了一种基于Cytc的H202生物传感器。由于静电吸引作用,修饰于ITO电极上的介孔碳纳米球对于Cytc显示了很好的吸附特性。峰形良好的氧化还原峰证实了在没有任何外加电子媒介体的存在下,吸附于介孔碳纳米球中的Cytc与电极成功发生了直接电子转移。该传感器对H202具有良好的电催化作用,并且可以通过控制修饰电极的组装层数来调变传感性能,从而达到合理设计传感器的目的。
第五章,基于介孔碳材料的烟酰胺腺嘌呤二核苷酸(NADH)的电催化氧化研究：NADH在固体电极上通常具有较高的氧化过电位,极易引起电极表面的污染与钝化。本章利用碳介孔材料(CMM)修饰的玻碳电极来研究NADH的电化学氧化。CMM所具有的大比表面积、高电子传递能力以及丰富的边片状缺陷位和表面含氧官能团,对NADH的氧化表现出了较高的电催化性能,将NADH的氧化过电位降低了595mV(与未修饰的裸玻碳电极相比),成功实现了NADH在低电位条件下(0.1Vvs. SCE,pH7.2)稳定灵敏的响应,检测下限为1.0μM,线性响应范围为10～600μM。这为基于以NADH为辅酶的脱氢酶的电流型生物传感器的发展提供了新的研究途径。
第六章,二维平面上单分散金原子阵列修饰的石墨烯材料的制备及其电化学性质的初步研究：石墨烯是继富勒烯和碳纳米管之后科学界的又一重大发现,石墨烯的发现,充实了碳材料家族,形成了从零维的富勒烯、一维的碳纳米管、二维的石墨烯到三维的金刚石和石墨的完整体系。目前已有一些关于石墨烯以及纳米粒子/石墨烯复合材料(NPs/graphene)的合成报道与应用研究。在这部分工作中,我们发展了一种基于石墨烯的二维平面,快速可控的溅射方法来形成金团簇纳米阵列。通过调整溅射电流的大小、角度和时间,得到了一系列不同尺寸的金团簇纳米颗粒阵列,最小可达1.05nm且无团聚现象发生,形成的纳米颗粒形貌均一,分散性良好,并且这种复合膜表现出良好的热力学稳定性、优良的导电性以及极高的金负载量,这些性能为生物传感器的研制和电化学研究提供了一个广阔的研究平台。
第七章,对全文进行了总结,总结了研究中存在的一些不足,并提出了后续工作的目标和研究思路。
Physiologically active substances, such as reactive oxygen species (ROS), are related to human health. The ROS plays an important role in many pathological processes, such as aging, cancer and neurological diseases. Therefore, it is necessary to establish new reliable and sensitive method to make a qualitative and quantitative assessment of physiological active substances. Due to the high sensitivity, fast response, simple operation, and small consumption of sample, biosensors have been widely used in clinical medicine, food industry, fermentation industry, environmental monitoring and other fields. The vigorous development of nanotechnology, in particular, the appearance of a variety of nanomaterials with special properties, has opened a new world for bio-sensors. Combining the analytical chemistry, electrochemistry, material science and other areas of research, this thesis committed to use new nanomaterials for construction of biosensors which are applied to the electroanalysis of physiologically active substances. This dissertation includes six chapters as follows:
In Chapter One, we introduced the research and development of nanomaterials and biosensors, respectively. Then we proposed a scheme of nanomaterials-based electrochemical sensors for electroanalysis of physiologically active substances. Finally we outlined the experimental ideas and the research purposes of this thesis.
In Chapter Two, we studied a simple and sensitive electrochemical method for the determination of antioxidant capacity.4-hydroxybenzoic acid (4-HBA) was used as a trapping agent for TiO2photogenerated-OH radicals, leading to3,4-dihydroxybenzoic acid (3,4-DHBA). Square-wave voltammetry was used to quantify the amount of3,4-DHBA formed from the reaction between4-HBA and·OH. Addition of antioxidants induced the competition between4-HBA and antioxidants toward-OH elimination, resulting in a decrease of the measured current. The IC50for different standard antioxidants was calculated and expressed in Trolox equivalent antioxidant capacity (TEAC). The validation of the proposed electrochemical method was also performed using an alternative detection method based on fluorimetry. In this method, the·OH radicals generated through the photocatalytic oxidation of water ware trapped by terephthalic acid (TA), producing fluorescent2-hydroxy-terephthalic acid (2-HTA). And similar trends were observed when comparing the antioxidant capacity calculated using the two methods. The applicability of this method has been shown by analyzing the antioxidant capacity of six plant extracts. Therefore, it can be concluded that the proposed electrochemical method is a good alternative strategy for antioxidant assessment due to its fast response, accuracy and inexpensive apparatus.
In Chapter Three, an amperometric biosensor for hydrogen peroxide (H2O2) has been constructed by immobilizing cytochrome c (Cyt c) on a macroporous material modified indium/tin oxide (ITO) electrode. Cyclic voltammetry showed that the direct and quasi-reversible electron transfer of Cyt c was achieved without the aid of an electron mediator. A surface-controlled electrode process was observed with the apparent heterogeneous electron-transfer rate constant (ks) of29.2s-1. The prepared biosensor exhibited an excellent electrocatalytic response to the reduction of H2O2. The amperometric responses increased steadily with the concentration of H2O2in the range from5μM to2mM, with a detection limit of0.5μM at pH7.4. This investigation not only provided a method for the direct electron transfer of Cyt c on macroporous materials, but also established a feasible approach for durable and reliable detection of H2O2. Furthermore, in view of the adjustable surface of MOSF, we also investigated the electrochemical behavior of Cyt c immobilized on MOSF substrates which were modified with different organic functionalities, including-CN,-SH, and-NH2.
In Chapter Four, a Cytochrome c-based biosensor for hydrogen peroxide (H2O2) has been constructed via layer-by-layer (LBL) assembly of mesoporous carbon nanospheres and polydiallyldimethylammonium (PDDA). Cyclic voltammetry showed that the direct and quasi-reversible electron transfer of Cyt c was achieved without the aid of an electron mediator. The prepared biosensor exhibited good electrocatalytic responses to the reduction of H2O2. In addition, assembling carbon nanospheres on the electrode by means of LBL technology could control the amount of Cyt c immobilized on the electrode, and thus control its biosensor properties by regulating the thickness of the mesoporous carbon nanospheres-assembled layers.this makes the rational design of enzyme electrodes possible.
In Chapter Five, the electrochemical oxidation of β-nicotinamine adenine dinucleotide (NADH) was investigated at a glassy carbon electrode modified with carbon mesoporous materials (CMM). Due to the large surface area and electro-catalytic properties of CMM, the overpotential of the electrodes toward the oxidation of NADH is decreased by595mV in aqueous solution at neutral pH. The anodic peak currents increase steadily with the concentration of NADH in the range from2μM to1.1mM, the detection limit being1.0μM at pH7.2and a potential of +0.3V vs. SCE. The results enable NADH to be sensed at a low potential and are promising with respect to the design of dehydrogenasebased amperometric biosensors.
In Chapter Six, we developed a rapid, solventless sputtering method to generate the2D bulk gold NPs/graphene hybrid superstructures in10seconds. The size and morphology of gold NPs are fine controlled in a wide range down to1.05nm by adjusting the sputtering current, sputtering angle, and sputtering time. The hybrids achieve ultra-high gold carriage and high thermal stability. We also investigate the properties of the hybrids via electrochemical methods. This composite structure enables a new class of electrode materials and offers an excellent platform for electrochemical applications.
In Chapter Seven, we summarized and proposed the objects and schemes for further research.
引文
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