基于金纳米材料的光学传感方法设计及其在环境和生物分析中的应用

	基于金纳米材料的光学传感方法设计及其在环境和生物分析中的应用
	王莎莎
学位类型	博士
导师	陈令新
	2016-05
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	环境工程
关键词	金纳米材料局域表面等离子体共振光学传感比色分析荧光检测
其他摘要	随着工业的不断发展，环境污染已经达到严重危及生态平衡和人类身体健康的程度,因此发展简单、快速、灵敏的环境污染物检测技术和疾病诊断技术成为当今科研工作者研究的热点之一，而纳米材料的出现对检测技术的进步起到了显著的推动作用。由于具有优异的光学性能，金纳米材料可以在光学传感方法的设计当中作为良好的光学信号传导单元；再通过与对待测物有特异性识别作用的分子配体和化学、生物反应相结合，可以实现待测物高灵敏度、高选择性的检测。据此，我们发展了一系列基于金纳米材料的光学传感方法用于检测环境中的重金属离子、阴离子以及具有疾病指示功能的生物分子，并对它们在实际样品中的应用性能进行了探索，其具体内容如下： 1. 基于金纳米颗粒反聚集的比色分析法用于有机汞离子的检测。本方法采用金纳米颗粒作为信号传导单元，并利用纳米颗粒间的距离变化实现了有机汞离子的检测。单分散的金纳米颗粒在加入1-(2-吡啶偶氮)-2-萘酚（PAN）后会发生聚集，同时溶液颜色也会由红色变为蓝色。当有机汞离子存在时，由于PAN优先与有机汞离子发生络合作用，导致PAN引起金纳米颗粒发生聚集的能力得到削弱，此时反应溶液仍然可以保持红色。根据这个原理，建立了一种“蓝变红”（Blue to Red）型比色分析方法用于有机汞离子的检测。在掩蔽剂乙二胺四乙酸二钠（EDTA）的辅助作用下，所设计的分析方法表现出良好的灵敏度和选择性。 2. 基于金纳米棒的形貌变化比色检测铜离子（Cu²⁺）。在SCN^－的配位作用下，Au(SCN)₂⁻/Au的氧化还原电位低于H₂O₂/H₂O的氧化还原电位，此时金纳米棒会被双氧水氧化，导致其长径比的减小和溶液颜色由蓝色向红色的转变。由于Cu²⁺可以催化双氧水的分解，因此Cu²⁺的存在可以对双氧水氧化刻蚀金纳米棒起到一定的阻碍作用。据此，我们发展了一种免标记的Cu²⁺比色分析方法。这种在特异性氧化还原反应基础上建立起来的光学传感方法具有良好的选择性和抗干扰能力，同时由于所使用的纳米材料不需要经过复杂的修饰过程，使得操作步骤非常简单。此外，该方法在实际样品检测中表现出来的优良性能证明其具有一定的实际应用价值。 3. 辣根过氧化物酶辅助的金纳米棒形貌变化比色检测碘离子（I⁻）。在辣根过氧化物酶的辅助作用下，I⁻会被双氧水氧化生成I₂；而I₂可以致使十六烷基三甲基溴化铵稳定的金纳米棒发生轴向方向的氧化刻蚀，同时伴随着纵向表面等离子体共振吸收峰的蓝移和溶液的颜色变化。基于以上原理，建立了一种基于金纳米棒形貌变化的免标记的I⁻比色分析方法。这种方法简单、快速、灵敏，同时对I⁻具有良好的特异性识别能力，并在实际样品的检测中取得了令人满意的结果。 4. 基于异硫氰酸荧光素（FITC）修饰的金纳米颗粒荧光法检测铜离子（Cu²⁺）。本研究制备了FITC修饰的金纳米颗粒，通过两者之间荧光共振能量转移的发生导致FITC的荧光猝灭。当有半胱氨酸存在时，由于Au–S的结合能力强于Au–SCN的结合能力，FITC会被半胱氨酸取代重新分散到体系溶液中，此时反应体系的荧光增强；而Cu²⁺的加入可以催化半胱氨酸被溶解氧氧化形成具有二硫键的胱氨酸，此时FITC仍然可以吸附在金纳米颗粒表面，体系荧光不会恢复。根据这个现象，可以实现Cu²⁺高灵敏度、高选择性的分析检测。在最佳实验条件下，Cu²⁺的线性检出范围为1.0–17.0 nM，最低检出限为0.37 nM。 5. 基于金增强的金纳米颗粒模拟酶活性用于可视化免疫分析。纳米金免疫标记是一种应用广泛、技术成熟的免疫标记技术，可是由于金纳米颗粒自身的模拟酶活性较低，当其被抗体修饰之后，其催化活性几乎完全丧失。为了提高免疫标记的金纳米颗粒催化活性，本研究在“抗体–抗原–金标抗体”夹心结构形成之后，通过简单的金增强步骤使得金纳米颗粒继续生长，此时新生成的金层能够极大地提高金纳米颗粒的催化活性。在双氧水（H₂O₂）的存在下，增强后的金纳米颗粒能够将3,3’,5,5’-四甲基联苯胺（TMB）催化生成有色物质。基于这个原理，可以实现靶标分子（以Human IgG为例）高灵敏度、高选择性的可视化分析检测。在最佳实验条件下，该方法的线性检出范围为0.7–100 ng/mL，最低检出限为0.3 ng/mL，并在血清样品的检测中具有一定的应用价值。更重要的是，本方法的成功设计可以拓宽纳米材料在免疫分析检测中的应用范围。 ; With the continuous development of industry, the severity of the environmental pollution has threatened the ecological balance and human's health. As a results, it has become one of the research hotspots to design and develop the simple, rapid and sensitive measurement techniques for the detection of environmental pollutants and disease-related biomolecules. In recent decades, the emergence and usage of nanomaterials play significant roles in promoting the development of detection technology. Due to the unique optical properties, gold nanomaterials can be used as the units of signal transmission in the design of optical sensing methods. When combining ligands and chemical/biological reactions with the function of specific recognition, the detection of target molecules could be achieved with high sensitivity and selectivity. Accordingly, we developed a series of optical sensing methods based on the use of gold nanomaterials for the detection of heavy metal ions, anions, and disease-related biomolecules, and their application in the actual sample were also be employed. The detail contents of this thesis are as follows: 1. Colorimetric detection of organic mercury based on anti-aggregation of gold nanoparticles. In this work, we used gold nanoparticles as the the unit of signal transmission, and took advantage of color change based on the distance change between nanoparticles to realize the detection of organic mercury. When introduced 1-(2-pyridylazo)-2-naphthol (PAN), the monodisperse gold nanoparticles became aggregated, along with the color change from red to blue. However, due to the prior coordination between organic mercury and PAN, the aggregation of gold nanoparticles was inhibited and the color of solution was still keeping red. According to this phenomenon, a “blue to red” colorimetric assay was developed for the determination of organic mercury, and the proposed method showed high sensitivity and selectivity with the assistance of ethylenediaminetetraacetic acid disodium salt (EDTA). 2. Label-free colorimetric sensing of Cu²⁺ based on accelerating decomposition of H₂O₂ using gold nanorods as an indicator. In this work, a novel label-free colorimetric strategy was reported for sensitive detection of Cu²⁺ by using the morphology transition of gold nanorods. H₂O₂ was employed as the oxidant for corrosion of gold nanorods, leading to the decrease of aspect ratio of gold nanorods and a distinct color change from bluish green to purplish red. By virtue of the strong and specific catalysis of Cu²⁺ to the decomposition of H₂O₂, the rate of redox corrosion can be decelerated. Based on this sensing strategy, we developed a colorimetric method for the detection of Cu²⁺. The proposed colorimetric sensor showed sensitive and selective response toward Cu²⁺ without any other labelling or modification steps, and it has been successfully applied to detect Cu²⁺ in shellfish samples. 3. Horseradish peroxidase (HRP) assisted colorimetric detection of I⁻ based on the morphology transition of gold nanorods. In this work, we utilized the color change of gold nanorods to design a colorimetric sensor for the detection of I⁻. In the presence of HRP, I⁻ could be oxidized to I₂ by H₂O₂, which could cause the etching of cetyltrimethyl ammonium bromide stabilized gold nanorods and lead to a blue shift of the longitudinal surface plasma resonance absorption peak and a color change from bluish green to purplish red. As a result, a label-free colorimetric method for the detection of I⁻ based on the morphology transition of gold nanorods was established. The designed method was simple, rapid and sensitive, and showed ability of specific recognition toward I⁻. Moreover, satisfactory results were obtained when detecting I⁻ in real water sample. 4. Highly sensitive fluorescent detection of Cu²⁺ based on fluorescein isothiocyanate functionalized gold nanoparticles In this work, an innovative fluorescence method for sensitive detection of Cu²⁺ was developed based on fluorescein isothiocyanate functionalized gold nanoparticles (FITC-Au NPs). Due to the strong binding affinity of isothiocyanate functional group to gold, FITC molecules could adsorb on the surfaces of Au NPs, forming a simple fluorescence resonance energy transfer (FRET) system, and the fluorescence intensity of FITC was remarkably quenched. Upon adding cysteine, FITC could be displaced from the surfaces of Au NPs because the formation constant (K_f) of Au-S linkage (K_f(AuS⁻)=4×10³⁵) was much higher than Au-SCN linkage (K_f(Au(SCN)²⁻)=10²³), leading to the recovery of fluorescence intensity. However, Cu²⁺ could catalyze O₂ to oxidize cysteine, and the generated disulfide cystine could not remove FITC from Au NPs’ surfaces. Therefore, the recovery of fluorescence intensity was much weaker when compared with that in the absence of Cu²⁺. Based on this strategy, the concentration of Cu²⁺ could be detected quantitatively. Under optimal conditions, our method exhibited high selectivity toward Cu²⁺ and provided a good linear relationship in the range of 1.0–17.0 nM with the detection limit of 0.37 nM calculated by 3σ/S. 5. Naked-eye sensitive ELISA-like assay based on gold-enhanced peroxidase-like immunogold activity. Gold nanoparticles have been mostly chosen as signal-producing tags to develop sensitive molecule-recognition-based immunoassays in the past decades, however, the relatively low peroxidase-like activity of gold nanoparticles could be almost inhibited when modified with proteins. In order to solve this problem, in this work, we added gold growth solution to cause the continuous enlargement of the immobilized gold nanoparticles, and the occurrence of new shell recovered their peroxidase-like activity evaluated by the catalytic oxidation of peroxidase enzyme substrate 3,3’,5,5’-tetramethylbenzidine (TMB) which could produce a bright blue color in the presence of H₂O₂. Under the optimal reaction conditions, the colorimetric immunoassay presented a good linear relationship in the range of 0.7–100 ng/mL and the limit of detection of 0.3 ng/mL calculated by 3σ/S for Human IgG detection. Furthermore, the developed colorimetric immunoassay was successfully applied to determinate Human IgG in diluted human serum and fetal bovine serum samples, and predicted the broad prospect for the wide use of peroxidase-like activity involved nanomaterials in bioassay and diagnostics.
语种	中文
文献类型	学位论文
条目标识符	http://ir.yic.ac.cn/handle/133337/13834
专题	中国科学院烟台海岸带研究所知识产出_学位论文
作者单位	中国科学院烟台海岸带研究所
第一作者单位	中国科学院烟台海岸带研究所
推荐引用方式 GB/T 7714	王莎莎. 基于金纳米材料的光学传感方法设计及其在环境和生物分析中的应用[D]. 北京. 中国科学院大学,2016.

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