新型纳米材料在环境与生物传感研究中的应用
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摘要
20世纪80年代初发展起来的纳米材料,由于具有奇异的物理、化学特性和广阔的应用前景而享有“跨世纪的新材料”之称。纵观20多年来纳米材料的发展,可将其分为三类：纳米微粒、纳米固体和纳米组装体系。纳米微粒是处于1～100 nm之间的各种粒子的总称；纳米固体是由纳米微粒聚集而成的凝聚体；纳米组装体系则是由人工组装合成的,具有纳米结构的材料的总称,也叫纳米尺度的图案材料。纳米材料独特的结构，使其具有四大效应：表面效应、小尺寸效应、量子尺寸效应和宏观量子隧道效应。纳米颗粒具有独特的化学和物理性质,可应用于电学、光学、催化、磁性材料、生物技术、药物输送等领域。用纳米材料和纳米技术修饰的电极以及纳米材料荧光量子点与分子印迹技术相结合的应用都给分析检测领域带来了更多高效率的检测手段。
近年来,化学修饰电极已经受到人们的广泛关注,化学修饰电极最突出的特点就是可以有目的性地对化学电极进行修饰,从而达到人们的需要。化学修饰电极在定量检测分析中的应用,是把分离、富集和测定三者融合的体系,在提高选择性和灵敏度方面有着自身特有的优越性,因而拓展了电分析化学检测物质的范围。由于其具有良好的选择性、高的灵敏度、分析速度快、测量成本低的特点,并且能够在复杂的体系中实现在线连续监测，已经在环境监测、生物、医学、医药、食品、及军事等领域体现出广阔的应用前景,引起了世界各国的关注。
本论文致力于化学修饰电极新的修饰方法的研究,通过探讨待测电活性分子与电极表面间催化过程,制备出选择性好,检测限低,稳定性好和灵敏度低的化学与生物传感器;同时,论文中将修饰电极用于蔬果中农药以及神经递质的研究和测定,取得了较好的测定结果。文中通过碳纳米管、氧化还原酶、PAMAM制备的金属纳米粒子、二氧化硅分子印迹材料结合量子点等多种材料作为反应平台,以SEM, TEM,等表征手段研究了传感器的表面形貌；化学修饰电极方面,通过在电极表面的功能化设计,以电流-时间曲线、交流阻抗、循环伏安等方法探讨电活性物质在修饰电极上的电化学性质及催化响应机理,从而构筑用于环境和食品安全检测的有效的新型化学修饰电极。本论文同时还探讨了纳米材料荧光量子点与硅球制备的分子印迹材料对于甲基对硫磷农药的检出。本论文将努力实现纳米技术、分子印迹技术、量子点技术、和环境科学、食品安全,神经递质检测这几方面有机的结合。具体内容如下：
1绪论(第一章)
本部分内容主要包括系统地介绍纳米材料的性质、纳米材料的制备以及纳米材料的应用领域,化学修饰电极的概述,纳米材料与化学修饰电极相结合构筑电极,及纳米材料修饰电极的应用。纳米材料与分子印迹技术,包括纳米分子印迹材料的制备,以及分子印迹技术与量子点结合的应用以及量子点的制备和量子点的应用研究。最后阐述了本论文的目的和意义,指出论文的创新之处及主要研究内容。
2基于碳纳米管结合聚酰胺-胺型树状高分子PAMAM合纳米金电极固定乙酰胆碱酯酶修饰电极的构筑及其对农药卡巴呋喃的定量检测(第二章)
本章中我们主要的工作是基于层层自组装技术将电极表面修饰乙酰胆碱酯酶/PAMAM-Au/CNTs多层膜,并通过差分脉冲伏安法和循环伏安法来检测农药样品。这里用到的碳纳米管提供了一个在光电极和酶之间的界面，因为纳米管拥有高的电导率并且能够使酶的氧化还原中心集中。此外，树形分子PAMAM用于提供合适的微环境来保留修饰电极中乙酰胆碱酯酶的活性。在膜中的纳米金类似于导体的作用,便于电子传递的发生。从电化学性质研究中,可以看出实验对于农药卡巴呋喃有较好的相应,线性范围4.8×10-9M到0.9×10-7M；检测限4.0×10-9M(S/N=3)。,该传感器具有快捷、简便、灵敏等优点,为农药残留快速分析和监测提供了一种新的方法。
3聚酰胺-胺型树状高分子PAMAM合铂纳米粒子修饰电极固定谷氨酸氧化酶电极的构筑以及在谷氨酸在体检测中的应用(第三章)
本文通过构筑聚酰胺-胺型树状高分子PAMAM合铂纳米粒子修饰电极固定谷氨酸氧化酶电极,结合微渗析技术实现了谷氨酸的在体检测。从实验结果可以看出,此修饰电极对于谷氨酸与酶作用以后产生的过氧化氢的检测无需使用辣根过氧化酶,就可以实现,使体系更加简单,增加了测定的准确度。结合微渗析技术,实验中对线性扫描伏安法和电流时间曲线进行了研究,实验结果标明此电极对于过氧化氢的电化学催化功能良好,在-0.2V就有很好的响应,线性范围从1.0μM到50.0μM,检测限0.5μM(S/N=3)。该实验为神经递质在生物体中的在线检测提供了有力的检测手段。
4基于荧光量子点的纳米二氧化硅分子印迹材料的研制及其对农药甲基对硫磷的特异性检测(第四章)
本章通过表面分子印迹技术与荧光量子点的有机结合,农药对硫磷分子为模板分子,二氧化硅包裹的量子点材料表面印迹甲基对硫磷分子。实现了对甲基对硫磷的选择性检测,文中对几种主要的干扰物进行了检测,结果体现了材料较好的选择性。同时,论文对该分子印迹材料的吸附动力学与印迹因子等进行了研究,甲基对硫磷的线性范围是0.013mg Kg-1到2.63mg Kg-1,检测限4.0×10-3mgKg-1(SN)同时研究还发现,对一系列与对硫磷结构和性质相似的农药,该分子印迹传感器对甲基对硫磷的检测具有较高的选择性。由此可见本工作较好地将分子印迹技术特异性强、稳定性好和量子点高的灵敏度相结合。该印迹传感器用于蔬菜中甲基对硫磷农药的检测,取得良好的结果,有望进行痕量农药分析。
The nanomaterials have attracted more interest in the study of researchers from the early 1980s, because of the special physical, chemical properties and broad application. Nano materials can be divided into three categories:the nanoparticles. nano solid and nano assembly system. Nanomaterial is the particle with the scale in 1～100 nm. The unique structure makes it has four effects:surface effect, small size effect, quantum size effect and macroscopic quantum tunnel effect. Nanoparticles with unique chemical and physical properties can be applied in the field of electrical, optical, catalytical, magnetic materials, biological technology, drug delivery, etc. The Use of nanomaterials and nanotechnology modified electrodes and nano materials luminescent quantum dots and combination of molecular imprinting technique to analyze the testing substance made the measurement rapid and convenient.
In recent years, chemical modified electrode has become hotpot in the research. The most obvious advantage of modified electrode is that they can be modified with functional group as people needed. The application of the quantitative measurement is the combination system of separation, gathering and measurement to extend the range of electrochemical analysis. Because of its excellent selectivity, high sensitivity, rapid response, low cost and continuous on-line measurement in complex system. It has been widely used in the environment monitoring, biology, medical science, medicine, food and military and promising for these fields.
This paper are focusing on the research of modified method for electrode and probe the catalytic process of electro-active molecule and surface of the electrode to prepare the good selective, high sensitive biosensors. Meanwhile, we made use of the modified electrode to study the pesticide in vegetables and fruits and neurotransmitters in brains. The results of the tests are proved to be good. In this paper, we used the materials such as carbon nanotubes, redox enzyme, PAMAM preparared metal nanoparticles and silica molecularly imprinted materials with quantum dots as reaction platform. We characterized the surface topography by SEM. TEM to study the sensors. In the aspect of chemical modified electrodes. we used the methods of current-time curves, ac impedance, circular voltammetric to investigate electrochemical properties and catalytic response mechanism of the active substances on modified electrodes on the desighed functionally electrodes and thus build the effective new chemical modified electrodes for environmental and food safety detection. This paper also discussed the luminescent quantum dots and silicon ball of molecularly imprinted materials for detection of parathion-methyl. This thesis will strive to combination of nanotechnology, molecular imprinting technique, quantum dot technology, and environmental science, food safety, neurotransmitters detection The primary research work is as follows:
Chapter 1 Overview
In this part, a detailed outlines and reviews mainly on the characteristics, preparation and application of nano materials. It also contains the summary of the chemical modified electrode and the combination of nano materials and electrode.. After a review of CMEs. the development and application of nanotechnology and molecularly imprinted technology into biosensors and application of quantum dots with MIP technology were highlighted. Finally, we emphatically pointed out the purpose and significance of the dissertation, its innovation spot and content as well.
Chapter 2 Layer-by-Layer self-assembled acetylcholinesterase/ PAMAM- Au on CNTs modified electrode for sensing pesticides electrode for sensing pesticides
In this paper. an acetylcholinesterase (AChE)/dendrimers polyamidoamine (PAMAM)-Au/Carbon nanotubes (CNTs) multilayer modified electrode based on LbL self-assembled technique was employed in the detection of carbofuran in samples. The configuration of the nanostructure on the electrode provided a favorable environment to the immobilization of AChE. The modified films also improved the electrocatalytic characteristics and electron transfer speed between the films and the surface of electrode. The PAMAM-Au nanoparticless were characterized by SEM and UV-VIS methods. A set of experimental conditions were also optimized for the detection of the pesticides. A linear response over cabofuran concentration in the range of was exhibited with a detection limit of. The biosensor showed high sensitivity, good stability and reproducibility with promising application.
Chapter 3 On-line microdialysis system with poly (amidoamine)-encapsulated Pt nanoparticles biosensor for glutamate sensing in vivo
In this work, an amine-terminated poly (amidoamine) dendrimer containing Pt nanoparticles (PAMAM/Pt)nanocomposite was synthesized and a novel amperometric H2O2 biosensor based on PAMAM/Pt and MWCNTs was developed. The resulting film of MWCNTs/PAMAM/Pt was characterized by transmission electron microscopy (TEM), linear sweep voltammetry (LSV) and amperometric i-t curve. It demonstrates excellent electrocatalytic responses toward the reduction of H2O2 at -200 mV (vs.SCE) without HRP participation. Immobilized with glutamate oxidase (GlutaOx), an effective glutamate biosensor, was fabricated, and the in vivo detection for glutamate was realized combining with the on-line microdialysis system. The glutamate biosensor showed good linear range from1.0μM to 50.0μM with the detection limit of 0.5μM (S/N=3). The basal level of glutamate in the striatum of rat was detected continuously with this on-line system and was calculated to be 5.80±0.12μM (n=3). This method was proved to be sensitive and selective and may be feasible in the further application of physiology and pathology.
Chapter 4 Determination of Parathion-methyl in Vegetables by Fluorescent-Labeled Molecular Imprinted Polymer
A novel sensor for the determination of parathion-methyl based on couple grafting of functional molecular imprinted polymers (MIPs) was fabricated which is developed by anchoring the MIP layer on surfaces of silica particles embedded CdSe quantum dots by surface imprinting technology. The synthesized molecular imprinted silica nanospheres (CdSe@SiO2@MIP) allows a high selectivity and sensitivity of parathion-methyl via Fluorescence intensity decreasing when the M1P material rebinding the parathion-methyl molecule. Compared with the MIP fabricated in traditional method, the template of parathion-methyl was easier to remove from the CdSe@SiO2@MIP imprinted material. Under the optimal conditions, the Fluorescence intensity of parathion-methyl at the imprinted sensor was detected by spectrofluorophotometer. The relative fluorescence intensity of CdSe@SiO2@MIP decreased linearly with the increasing concentration of parathion-methyl range from 0.013 mg Kg-1 to 2.63 mg Kg-1 with a detection limit (3σ2) of 0.004 mg Kg-1 (S/N=3), which is lower than the MIP in tradition. The imprinted film sensor was applied to detect parathion-methyl in vegetable samples without the interference of other organophosphate pesticides and showed a prosperous application in the field of food safety.
近年来,化学修饰电极已经受到人们的广泛关注,化学修饰电极最突出的特点就是可以有目的性地对化学电极进行修饰,从而达到人们的需要。化学修饰电极在定量检测分析中的应用,是把分离、富集和测定三者融合的体系,在提高选择性和灵敏度方面有着自身特有的优越性,因而拓展了电分析化学检测物质的范围。由于其具有良好的选择性、高的灵敏度、分析速度快、测量成本低的特点,并且能够在复杂的体系中实现在线连续监测，已经在环境监测、生物、医学、医药、食品、及军事等领域体现出广阔的应用前景,引起了世界各国的关注。
本论文致力于化学修饰电极新的修饰方法的研究,通过探讨待测电活性分子与电极表面间催化过程,制备出选择性好,检测限低,稳定性好和灵敏度低的化学与生物传感器;同时,论文中将修饰电极用于蔬果中农药以及神经递质的研究和测定,取得了较好的测定结果。文中通过碳纳米管、氧化还原酶、PAMAM制备的金属纳米粒子、二氧化硅分子印迹材料结合量子点等多种材料作为反应平台,以SEM, TEM,等表征手段研究了传感器的表面形貌；化学修饰电极方面,通过在电极表面的功能化设计,以电流-时间曲线、交流阻抗、循环伏安等方法探讨电活性物质在修饰电极上的电化学性质及催化响应机理,从而构筑用于环境和食品安全检测的有效的新型化学修饰电极。本论文同时还探讨了纳米材料荧光量子点与硅球制备的分子印迹材料对于甲基对硫磷农药的检出。本论文将努力实现纳米技术、分子印迹技术、量子点技术、和环境科学、食品安全,神经递质检测这几方面有机的结合。具体内容如下：
1绪论(第一章)
本部分内容主要包括系统地介绍纳米材料的性质、纳米材料的制备以及纳米材料的应用领域,化学修饰电极的概述,纳米材料与化学修饰电极相结合构筑电极,及纳米材料修饰电极的应用。纳米材料与分子印迹技术,包括纳米分子印迹材料的制备,以及分子印迹技术与量子点结合的应用以及量子点的制备和量子点的应用研究。最后阐述了本论文的目的和意义,指出论文的创新之处及主要研究内容。
2基于碳纳米管结合聚酰胺-胺型树状高分子PAMAM合纳米金电极固定乙酰胆碱酯酶修饰电极的构筑及其对农药卡巴呋喃的定量检测(第二章)
本章中我们主要的工作是基于层层自组装技术将电极表面修饰乙酰胆碱酯酶/PAMAM-Au/CNTs多层膜,并通过差分脉冲伏安法和循环伏安法来检测农药样品。这里用到的碳纳米管提供了一个在光电极和酶之间的界面，因为纳米管拥有高的电导率并且能够使酶的氧化还原中心集中。此外，树形分子PAMAM用于提供合适的微环境来保留修饰电极中乙酰胆碱酯酶的活性。在膜中的纳米金类似于导体的作用,便于电子传递的发生。从电化学性质研究中,可以看出实验对于农药卡巴呋喃有较好的相应,线性范围4.8×10-9M到0.9×10-7M；检测限4.0×10-9M(S/N=3)。,该传感器具有快捷、简便、灵敏等优点,为农药残留快速分析和监测提供了一种新的方法。
3聚酰胺-胺型树状高分子PAMAM合铂纳米粒子修饰电极固定谷氨酸氧化酶电极的构筑以及在谷氨酸在体检测中的应用(第三章)
本文通过构筑聚酰胺-胺型树状高分子PAMAM合铂纳米粒子修饰电极固定谷氨酸氧化酶电极,结合微渗析技术实现了谷氨酸的在体检测。从实验结果可以看出,此修饰电极对于谷氨酸与酶作用以后产生的过氧化氢的检测无需使用辣根过氧化酶,就可以实现,使体系更加简单,增加了测定的准确度。结合微渗析技术,实验中对线性扫描伏安法和电流时间曲线进行了研究,实验结果标明此电极对于过氧化氢的电化学催化功能良好,在-0.2V就有很好的响应,线性范围从1.0μM到50.0μM,检测限0.5μM(S/N=3)。该实验为神经递质在生物体中的在线检测提供了有力的检测手段。
4基于荧光量子点的纳米二氧化硅分子印迹材料的研制及其对农药甲基对硫磷的特异性检测(第四章)
本章通过表面分子印迹技术与荧光量子点的有机结合,农药对硫磷分子为模板分子,二氧化硅包裹的量子点材料表面印迹甲基对硫磷分子。实现了对甲基对硫磷的选择性检测,文中对几种主要的干扰物进行了检测,结果体现了材料较好的选择性。同时,论文对该分子印迹材料的吸附动力学与印迹因子等进行了研究,甲基对硫磷的线性范围是0.013mg Kg-1到2.63mg Kg-1,检测限4.0×10-3mgKg-1(SN)同时研究还发现,对一系列与对硫磷结构和性质相似的农药,该分子印迹传感器对甲基对硫磷的检测具有较高的选择性。由此可见本工作较好地将分子印迹技术特异性强、稳定性好和量子点高的灵敏度相结合。该印迹传感器用于蔬菜中甲基对硫磷农药的检测,取得良好的结果,有望进行痕量农药分析。
The nanomaterials have attracted more interest in the study of researchers from the early 1980s, because of the special physical, chemical properties and broad application. Nano materials can be divided into three categories:the nanoparticles. nano solid and nano assembly system. Nanomaterial is the particle with the scale in 1～100 nm. The unique structure makes it has four effects:surface effect, small size effect, quantum size effect and macroscopic quantum tunnel effect. Nanoparticles with unique chemical and physical properties can be applied in the field of electrical, optical, catalytical, magnetic materials, biological technology, drug delivery, etc. The Use of nanomaterials and nanotechnology modified electrodes and nano materials luminescent quantum dots and combination of molecular imprinting technique to analyze the testing substance made the measurement rapid and convenient.
In recent years, chemical modified electrode has become hotpot in the research. The most obvious advantage of modified electrode is that they can be modified with functional group as people needed. The application of the quantitative measurement is the combination system of separation, gathering and measurement to extend the range of electrochemical analysis. Because of its excellent selectivity, high sensitivity, rapid response, low cost and continuous on-line measurement in complex system. It has been widely used in the environment monitoring, biology, medical science, medicine, food and military and promising for these fields.
This paper are focusing on the research of modified method for electrode and probe the catalytic process of electro-active molecule and surface of the electrode to prepare the good selective, high sensitive biosensors. Meanwhile, we made use of the modified electrode to study the pesticide in vegetables and fruits and neurotransmitters in brains. The results of the tests are proved to be good. In this paper, we used the materials such as carbon nanotubes, redox enzyme, PAMAM preparared metal nanoparticles and silica molecularly imprinted materials with quantum dots as reaction platform. We characterized the surface topography by SEM. TEM to study the sensors. In the aspect of chemical modified electrodes. we used the methods of current-time curves, ac impedance, circular voltammetric to investigate electrochemical properties and catalytic response mechanism of the active substances on modified electrodes on the desighed functionally electrodes and thus build the effective new chemical modified electrodes for environmental and food safety detection. This paper also discussed the luminescent quantum dots and silicon ball of molecularly imprinted materials for detection of parathion-methyl. This thesis will strive to combination of nanotechnology, molecular imprinting technique, quantum dot technology, and environmental science, food safety, neurotransmitters detection The primary research work is as follows:
Chapter 1 Overview
In this part, a detailed outlines and reviews mainly on the characteristics, preparation and application of nano materials. It also contains the summary of the chemical modified electrode and the combination of nano materials and electrode.. After a review of CMEs. the development and application of nanotechnology and molecularly imprinted technology into biosensors and application of quantum dots with MIP technology were highlighted. Finally, we emphatically pointed out the purpose and significance of the dissertation, its innovation spot and content as well.
Chapter 2 Layer-by-Layer self-assembled acetylcholinesterase/ PAMAM- Au on CNTs modified electrode for sensing pesticides electrode for sensing pesticides
In this paper. an acetylcholinesterase (AChE)/dendrimers polyamidoamine (PAMAM)-Au/Carbon nanotubes (CNTs) multilayer modified electrode based on LbL self-assembled technique was employed in the detection of carbofuran in samples. The configuration of the nanostructure on the electrode provided a favorable environment to the immobilization of AChE. The modified films also improved the electrocatalytic characteristics and electron transfer speed between the films and the surface of electrode. The PAMAM-Au nanoparticless were characterized by SEM and UV-VIS methods. A set of experimental conditions were also optimized for the detection of the pesticides. A linear response over cabofuran concentration in the range of was exhibited with a detection limit of. The biosensor showed high sensitivity, good stability and reproducibility with promising application.
Chapter 3 On-line microdialysis system with poly (amidoamine)-encapsulated Pt nanoparticles biosensor for glutamate sensing in vivo
In this work, an amine-terminated poly (amidoamine) dendrimer containing Pt nanoparticles (PAMAM/Pt)nanocomposite was synthesized and a novel amperometric H2O2 biosensor based on PAMAM/Pt and MWCNTs was developed. The resulting film of MWCNTs/PAMAM/Pt was characterized by transmission electron microscopy (TEM), linear sweep voltammetry (LSV) and amperometric i-t curve. It demonstrates excellent electrocatalytic responses toward the reduction of H2O2 at -200 mV (vs.SCE) without HRP participation. Immobilized with glutamate oxidase (GlutaOx), an effective glutamate biosensor, was fabricated, and the in vivo detection for glutamate was realized combining with the on-line microdialysis system. The glutamate biosensor showed good linear range from1.0μM to 50.0μM with the detection limit of 0.5μM (S/N=3). The basal level of glutamate in the striatum of rat was detected continuously with this on-line system and was calculated to be 5.80±0.12μM (n=3). This method was proved to be sensitive and selective and may be feasible in the further application of physiology and pathology.
Chapter 4 Determination of Parathion-methyl in Vegetables by Fluorescent-Labeled Molecular Imprinted Polymer
A novel sensor for the determination of parathion-methyl based on couple grafting of functional molecular imprinted polymers (MIPs) was fabricated which is developed by anchoring the MIP layer on surfaces of silica particles embedded CdSe quantum dots by surface imprinting technology. The synthesized molecular imprinted silica nanospheres (CdSe@SiO2@MIP) allows a high selectivity and sensitivity of parathion-methyl via Fluorescence intensity decreasing when the M1P material rebinding the parathion-methyl molecule. Compared with the MIP fabricated in traditional method, the template of parathion-methyl was easier to remove from the CdSe@SiO2@MIP imprinted material. Under the optimal conditions, the Fluorescence intensity of parathion-methyl at the imprinted sensor was detected by spectrofluorophotometer. The relative fluorescence intensity of CdSe@SiO2@MIP decreased linearly with the increasing concentration of parathion-methyl range from 0.013 mg Kg-1 to 2.63 mg Kg-1 with a detection limit (3σ2) of 0.004 mg Kg-1 (S/N=3), which is lower than the MIP in tradition. The imprinted film sensor was applied to detect parathion-methyl in vegetable samples without the interference of other organophosphate pesticides and showed a prosperous application in the field of food safety.
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