功能化碳纳米颗粒的制备、性质及其分析应用研究
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摘要
碳材料是一种地球上比较普遍并且特殊的材料，它不仅可以形成硬度较大的金刚石，也可以形成较软的石墨。它的研究及其应用一直是科技创新的前沿领域，尤其是碳纳米管(CNTs)和石墨烯的出现，由于其优异的电磁学、光学、力学和热学等方面的性能，在化学、物理、材料科学、生物等诸多领域表现出诱人的应用前景，引起了科学界巨大的反响，兴起了在碳材料方面的研究热潮。碳纳米颗粒是碳材料中的一种，也具有类似的石墨结构，但是以前的研究是基于较小的碳纳米颗粒（碳量子点）的光学性质应用于成像方面，而基于较大的碳纳米颗粒的性质及应用研究还没有被报道。
本论文致力于结合我们在荧光传感方面的研究，对功能化碳纳米颗粒进行了深入的探讨。由于分析化学发展的一个重要方向是设计能够对特定目标分析物产生可测量信号的传感器。荧光传感器由于检测的灵敏度高、选择性好和使用方便等优点，近年来取得了较大发展。而在传感器中信号传导部分是影响荧光传感器传感性能的主要因素，因此研究与发展具有选择性好、灵敏度高又具有较好的稳定性和可逆性的传导材料一直是构建荧光传感器时值得探索和完善的重要方面。本论文应用对碳纳米颗粒的性质的研究，在碳纳米管和石墨烯作为构建传感平台的基础上，开拓了碳纳米颗粒作为荧光传感平台的应用，对碳纳米颗粒和核酸探针的作用机制进行了深入的研究，并进一步拓展了功能化碳纳米颗粒在催化性能方面的应用。主要完成了以下四个工作：
（1）利用蜡烛燃烧的方式得到不溶于水的碳灰，通过改进的方法得到水溶液分散的碳纳米颗粒，接着对其进行了SEM、AFM、DLS、XPS、Raman、FTIR和pH滴定等详细的表征分析。结果表明制得的碳纳米颗粒具有比较好的富氧含量，通过计算证明碳的氧化率为1/6，此方法可以简单、大量制备氧化碳纳米颗粒，并且有可能进一步实现工业化生产，同时也为我们的下一步应用奠定了坚实的理论基础。
（2）首次使用制备的碳纳米颗粒结合单标记荧光探针构建荧光传感平台，研究了单标记荧光探针和碳纳米颗粒的作用机制，通过数据分析提出了碳纳米颗粒和DNA之间是π-π堆积和静电排斥共同作用的机制，且碳纳米颗粒可以很好的对单链DNA和双链DNA进行区分，依据这些原理构建了基于碳纳米颗粒-荧光探针的传感平台实现了对DNA, Hg~(2+),三磷酸腺苷(ATP)和凝血酶(Thrombin)高灵敏度、高选择性检测，本荧光传感体系不需要双标记，同时排除了由于非标记使用的嵌入剂的不稳定性而造成检测器的不可重复性、检测灵敏度低的缺点，实现了荧光恢复的增强体系，消除了溶液中其他因素对荧光淬灭造成的影响。而且碳纳米颗粒和DNA之间的π-π堆积和静电排斥作用，以及碳纳米颗粒近似球形的结构，这些都有利于提高检测的灵敏度。
（3）核酸适配体结合小分子后的各向异性变化很小，很难达到灵敏的检测，由于DNA通过π-π堆积作用于碳纳米颗粒表面，而且碳纳米颗粒还可以对单双链DNA进行区分，依据这些理论和实验基础我们结合碳纳米颗粒和荧光各向异性技术发展了一种信号放大荧光各向异性的方法，对ATP和Apyraze实现了高灵敏度、高选择性的实时检测。这是首次基于荧光共振能量转移结合碳纳米材料和荧光各向异性技术应用于检测方面的报道，同时可以实现信号降低和信号增强检测，这种基于碳纳米颗粒放大各向异性方法的有效性、简易性和多样性会促使荧光各向异性技术的进一步发展。
（4）通过DNA和表面活性剂修饰(SDS、Tween20、DTAB)能够使未氧化的碳纳米颗粒在水溶液中具有很好的分散性，从而催化双氧水氧化3,3',5,5'-四甲基联苯二胺（3,3',5,5'-tetramethylbenzidine，TMB）显色，本方法相比于其他碳纳米材料催化TMB显色反应，具有材料便宜，显色时间短，催化效率高等特点。依据碳纳米颗粒的催化性能的比较，发现SDS修饰的碳纳米颗粒的催化活性最高，接着考察了SDS修饰的碳纳米颗粒对H_2O_2的检测。本实验原理可以对与H_2O_2有关的其他物质的分析检测提供新的思路。
Carbon material, a kind of relatively common and special materials in earth, cannot only form the hardest material, diamond, but also can form a flexible graphite. Theresearch and applications of carbon material have been the forefront of technologicalinnovation domain, especially carbon nanotubes (CNTs) and graphene, due to theirexcellent performance in electromagnetism, optical, thermodynamics and mechanics,have shown good prospect of application in chemistry, physics, materials science,biology, and many other fields, having caused a huge response to the scientificcommunity and risen in the research upsurge of carbon materials, and become thefocus of research in the various fields. Carbon nanoparticles also have the similarstructure of graphite, but most of the research is based on the smaller carbonnanoparticles (carbon quantum dot). However, the properties and application researchof the larger carbon nanoparticles have not been reported.
This thesis is devoted to the functionallization of carbon nanoparticles. Combinedwith our research in fluorescence sensing, an important design of the development ofanalytical chemistry is to a specific target analyte and produce a measurable signal.Due to high sensitivity, good selectivity and facility for detection, the fluorescencesensor has made great development in recent years. The part of signal conduction isone of the most important factors to the performance of fluorescent sensor, thereforethe research and development of conductive material with good selectivity, highsensitivity, stability and reversibility is worth exploring and perfecting. Based oncarbon nanotubes and graphene as the sensing platform, here we study the properties ofcarbon nanoparticle and the interaction mechanism between carbon nanoparticles andnucleic acid probe to design carbon nanoparticles as fluorescence sensing platform, atthe same time, we also explore the further development of the carbon nanoparticles incatalytic. The main works are as follows:
(1) Using a way of burning candle to get carbon ash of insoluble water, weobtain the carbon nanoparticles dispersion in water by our improved method, then thedetailed characterization analysis are carried on through SEM, AFM, DLS, XPS,Raman, FTIR and pH titration. The results show that the prepared carbon nanoparticleshave a relatively good content of oxygen, basically one of every six carbon is oxidizedthrough the calculation.This method is a simple, effective to prepare oxidized carbonnanoparticles, which may realize industrialized production, and it also lay a solid theoretical foundation for our next application.
(2) Combined with single-labeled fluorescent probes, we use prepared carbonnanoparticles to construct fluorescent sensing platform, Moreover, the interactionmechanism between fluorescent probes and carbon nanoparticles is studied. Theunderlying sensing mechanism, in contrast to those of common carbon nanostructuresand metal nanoparticles, is based on competition of electrostatic repulsion and π-πstacking interactions.Thus it can be very well to distinguish single-stranded DNA anddouble-stranded DNA. According to these principles, we design sensing platformwhich can realize high sensitivity, high selectivity detection of DNA, Hg~(2+), adenosinetriphosphate (ATP) and thrombin (Thrombin). Besides the sensing system need not thedouble mark. Meantime, it also ruled out instability caused by non-markers embeddedagent result in not repetitive and low detection sensitivity. Moreover it is a system offluorescence enhancement, thus the effect of solution and other factors on thefluorescence quenching are eliminated. The sensitivity and accuracy of detection areincreased. At the same time, the interaction mechanism of π-π stacking andelectrostatic repulsion between carbon nanoparticles and DNA, as well as theapproximation spherical structure of carbon nanoparticle, which are beneficial toimprove sensitivity of detection.
(3) After binding small molecule, the anisotropic of the aptamer changes a little,thus it is difficult to achieve a high sensitive detection. Here, according to theinteraction mechanism of DNA binding to the carbon nanoparticles on the surface by π-πstacking interactions in the third chapter, we combined carbon nanoparticles andfluorescence anisotropy technique to develop a method of signal amplifyingfluorescence anisotropy on the basis of the theory and experiment. The high sensitivityand selectivity detection for ATP and apyraze is realized. This is the first reported thebinding of carbon nano-materials and fluorescence anisotropy applied in detection. Atthe same time, the approach is extremely versatile in that both signal-off and signal-ondetections can be easily realized. The effectiveness, simplicity and diversity of thecCNPs-FA approach will enable the development of a class of probes for rapid, sensitive,and selective detection of biomolecule.
(4) The unburnt carbon nanoparticles modified by some DNA and surfactant (SDS,Tween20, DTAB) can dispersion in water easily. Carbon nanoparticle can catalyticperoxidase to oxidize the3,3',5,5'-tetramethylbbenzidine (TMB): thereby producing acolor change, in contrast to other carbon nanomaterial, carbon nanoparticles as catalystis not time-consuming, non-expensive, high catalytic efficiency and so on. On the basisof the catalytic properties of carbon nanoparticles modified by different surfactant, carbon nanoparticles modified SDS with the highest catalytic activity is selected forthe detection of H_2O_2. We also believe that this principle can be used to other analyticsassociated with H_2O_2for detection.
本论文致力于结合我们在荧光传感方面的研究，对功能化碳纳米颗粒进行了深入的探讨。由于分析化学发展的一个重要方向是设计能够对特定目标分析物产生可测量信号的传感器。荧光传感器由于检测的灵敏度高、选择性好和使用方便等优点，近年来取得了较大发展。而在传感器中信号传导部分是影响荧光传感器传感性能的主要因素，因此研究与发展具有选择性好、灵敏度高又具有较好的稳定性和可逆性的传导材料一直是构建荧光传感器时值得探索和完善的重要方面。本论文应用对碳纳米颗粒的性质的研究，在碳纳米管和石墨烯作为构建传感平台的基础上，开拓了碳纳米颗粒作为荧光传感平台的应用，对碳纳米颗粒和核酸探针的作用机制进行了深入的研究，并进一步拓展了功能化碳纳米颗粒在催化性能方面的应用。主要完成了以下四个工作：
（1）利用蜡烛燃烧的方式得到不溶于水的碳灰，通过改进的方法得到水溶液分散的碳纳米颗粒，接着对其进行了SEM、AFM、DLS、XPS、Raman、FTIR和pH滴定等详细的表征分析。结果表明制得的碳纳米颗粒具有比较好的富氧含量，通过计算证明碳的氧化率为1/6，此方法可以简单、大量制备氧化碳纳米颗粒，并且有可能进一步实现工业化生产，同时也为我们的下一步应用奠定了坚实的理论基础。
（2）首次使用制备的碳纳米颗粒结合单标记荧光探针构建荧光传感平台，研究了单标记荧光探针和碳纳米颗粒的作用机制，通过数据分析提出了碳纳米颗粒和DNA之间是π-π堆积和静电排斥共同作用的机制，且碳纳米颗粒可以很好的对单链DNA和双链DNA进行区分，依据这些原理构建了基于碳纳米颗粒-荧光探针的传感平台实现了对DNA, Hg~(2+),三磷酸腺苷(ATP)和凝血酶(Thrombin)高灵敏度、高选择性检测，本荧光传感体系不需要双标记，同时排除了由于非标记使用的嵌入剂的不稳定性而造成检测器的不可重复性、检测灵敏度低的缺点，实现了荧光恢复的增强体系，消除了溶液中其他因素对荧光淬灭造成的影响。而且碳纳米颗粒和DNA之间的π-π堆积和静电排斥作用，以及碳纳米颗粒近似球形的结构，这些都有利于提高检测的灵敏度。
（3）核酸适配体结合小分子后的各向异性变化很小，很难达到灵敏的检测，由于DNA通过π-π堆积作用于碳纳米颗粒表面，而且碳纳米颗粒还可以对单双链DNA进行区分，依据这些理论和实验基础我们结合碳纳米颗粒和荧光各向异性技术发展了一种信号放大荧光各向异性的方法，对ATP和Apyraze实现了高灵敏度、高选择性的实时检测。这是首次基于荧光共振能量转移结合碳纳米材料和荧光各向异性技术应用于检测方面的报道，同时可以实现信号降低和信号增强检测，这种基于碳纳米颗粒放大各向异性方法的有效性、简易性和多样性会促使荧光各向异性技术的进一步发展。
（4）通过DNA和表面活性剂修饰(SDS、Tween20、DTAB)能够使未氧化的碳纳米颗粒在水溶液中具有很好的分散性，从而催化双氧水氧化3,3',5,5'-四甲基联苯二胺（3,3',5,5'-tetramethylbenzidine，TMB）显色，本方法相比于其他碳纳米材料催化TMB显色反应，具有材料便宜，显色时间短，催化效率高等特点。依据碳纳米颗粒的催化性能的比较，发现SDS修饰的碳纳米颗粒的催化活性最高，接着考察了SDS修饰的碳纳米颗粒对H_2O_2的检测。本实验原理可以对与H_2O_2有关的其他物质的分析检测提供新的思路。
Carbon material, a kind of relatively common and special materials in earth, cannot only form the hardest material, diamond, but also can form a flexible graphite. Theresearch and applications of carbon material have been the forefront of technologicalinnovation domain, especially carbon nanotubes (CNTs) and graphene, due to theirexcellent performance in electromagnetism, optical, thermodynamics and mechanics,have shown good prospect of application in chemistry, physics, materials science,biology, and many other fields, having caused a huge response to the scientificcommunity and risen in the research upsurge of carbon materials, and become thefocus of research in the various fields. Carbon nanoparticles also have the similarstructure of graphite, but most of the research is based on the smaller carbonnanoparticles (carbon quantum dot). However, the properties and application researchof the larger carbon nanoparticles have not been reported.
This thesis is devoted to the functionallization of carbon nanoparticles. Combinedwith our research in fluorescence sensing, an important design of the development ofanalytical chemistry is to a specific target analyte and produce a measurable signal.Due to high sensitivity, good selectivity and facility for detection, the fluorescencesensor has made great development in recent years. The part of signal conduction isone of the most important factors to the performance of fluorescent sensor, thereforethe research and development of conductive material with good selectivity, highsensitivity, stability and reversibility is worth exploring and perfecting. Based oncarbon nanotubes and graphene as the sensing platform, here we study the properties ofcarbon nanoparticle and the interaction mechanism between carbon nanoparticles andnucleic acid probe to design carbon nanoparticles as fluorescence sensing platform, atthe same time, we also explore the further development of the carbon nanoparticles incatalytic. The main works are as follows:
(1) Using a way of burning candle to get carbon ash of insoluble water, weobtain the carbon nanoparticles dispersion in water by our improved method, then thedetailed characterization analysis are carried on through SEM, AFM, DLS, XPS,Raman, FTIR and pH titration. The results show that the prepared carbon nanoparticleshave a relatively good content of oxygen, basically one of every six carbon is oxidizedthrough the calculation.This method is a simple, effective to prepare oxidized carbonnanoparticles, which may realize industrialized production, and it also lay a solid theoretical foundation for our next application.
(2) Combined with single-labeled fluorescent probes, we use prepared carbonnanoparticles to construct fluorescent sensing platform, Moreover, the interactionmechanism between fluorescent probes and carbon nanoparticles is studied. Theunderlying sensing mechanism, in contrast to those of common carbon nanostructuresand metal nanoparticles, is based on competition of electrostatic repulsion and π-πstacking interactions.Thus it can be very well to distinguish single-stranded DNA anddouble-stranded DNA. According to these principles, we design sensing platformwhich can realize high sensitivity, high selectivity detection of DNA, Hg~(2+), adenosinetriphosphate (ATP) and thrombin (Thrombin). Besides the sensing system need not thedouble mark. Meantime, it also ruled out instability caused by non-markers embeddedagent result in not repetitive and low detection sensitivity. Moreover it is a system offluorescence enhancement, thus the effect of solution and other factors on thefluorescence quenching are eliminated. The sensitivity and accuracy of detection areincreased. At the same time, the interaction mechanism of π-π stacking andelectrostatic repulsion between carbon nanoparticles and DNA, as well as theapproximation spherical structure of carbon nanoparticle, which are beneficial toimprove sensitivity of detection.
(3) After binding small molecule, the anisotropic of the aptamer changes a little,thus it is difficult to achieve a high sensitive detection. Here, according to theinteraction mechanism of DNA binding to the carbon nanoparticles on the surface by π-πstacking interactions in the third chapter, we combined carbon nanoparticles andfluorescence anisotropy technique to develop a method of signal amplifyingfluorescence anisotropy on the basis of the theory and experiment. The high sensitivityand selectivity detection for ATP and apyraze is realized. This is the first reported thebinding of carbon nano-materials and fluorescence anisotropy applied in detection. Atthe same time, the approach is extremely versatile in that both signal-off and signal-ondetections can be easily realized. The effectiveness, simplicity and diversity of thecCNPs-FA approach will enable the development of a class of probes for rapid, sensitive,and selective detection of biomolecule.
(4) The unburnt carbon nanoparticles modified by some DNA and surfactant (SDS,Tween20, DTAB) can dispersion in water easily. Carbon nanoparticle can catalyticperoxidase to oxidize the3,3',5,5'-tetramethylbbenzidine (TMB): thereby producing acolor change, in contrast to other carbon nanomaterial, carbon nanoparticles as catalystis not time-consuming, non-expensive, high catalytic efficiency and so on. On the basisof the catalytic properties of carbon nanoparticles modified by different surfactant, carbon nanoparticles modified SDS with the highest catalytic activity is selected forthe detection of H_2O_2. We also believe that this principle can be used to other analyticsassociated with H_2O_2for detection.
引文
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