量子点和金簇纳米传感器制备及在药物检测中的应用研究

发布时间：2018-08-27 06:36

【摘要】：纳米传感器以其优异的光学性能以及出色的灵敏度,被广泛应用于化学、食品科学、临床医疗诊断、环境和医药科学等领域。纳米材料的选择对于传感器的灵敏度、特异性和稳定性等性能的影响至关重要,应用新型纳米材料(量子点和金纳米簇)与传感器相组合,构建特异性好和灵敏度高的纳米传感器,用于环境、农产品和中药材中农药残留快速检测以及临床重要药物分子的灵敏监测,不仅可推动纳米技术在人类科技发展史上的飞跃进步,更开拓了科研工作者的新思路。目前,农药百草枯、抗凝血药肝素、胰蛋白酶和叶酸等药物分子或蛋白质的检测方法种类繁多,如色谱技术、电化学法和酶联免疫法等,但这些方法在使用中或者易受实际样品其他成分的干扰、不易提取分析,或者检测步骤繁多、检测时间较长,或者稳定性较差、检测灵敏度偏低,或者检测费用昂贵、实际应用复杂。随着人类生活水平的不断提升,以及科学研究的层层深入,人们逐渐意识到环境安全、食品安全以及临床用药安全对人体健康的重要性,因此研究者迫切需要研制一些操作简便、检测快速、成本低廉、灵敏度较好的纳米传感器,用以方便快速检测与人类健康密切相关的农药分子,临床重要药物分子及重要蛋白质分子,以满足环境、食品安全评价和临床用药安全指南,从而为我国农产品和生药产业的快速安全发展及国际化走向奠定良好基础,也为临床用药和某些疾病快速诊断提供一定的参考价值,更为人类身体健康和生活质量指数提供可靠的评价指标。鉴于以上研究需求,本文基于量子点和金纳米颗粒的优良光学性能、高灵敏度和高选择性等优点,结合环境问题、农产品和生药生产中农药残留问题以及临床药物需求和检验等实际现状,针对一些重要的药物靶标分子,设计组装了一系列荧光纳米传感器,用以快速检测分析环境、农产品和药用植物中残留农药百草枯、临床抗凝血药肝素、叶酸以及胰蛋白酶,为建立农药残留检测和临床药物监测平台提供新思路。本文取得如下主要进展:第一章,我们综述了量子点和金属纳米簇的制备及两者在环境和农业污染物、蛋白质分析、生物成像等研究领域的应用情况,并阐述了本论文的研究意义和主要内容。第二章,为实现快速检测和筛查环境、农产品和药用植物样品中残留农药百草枯,我们应用光学性能良好的Cd S量子点作为荧光纳米材料建立简易灵敏检测百草枯的传感器。本章主要以谷胱甘肽为模板和保护剂,通过优化合成条件,实现在15min内快速合成水溶性的Cd S量子点。根据百草枯作为阳离子盐的特点,可依赖电子转移作用高效地猝灭GSH-Cd S量子点的荧光,从而根据荧光强度的变化实现对环境样品、农业样品和药用植物样品中百草枯的快速检测。此方法,检测百草枯的线性范围在0.025-1.50μg m L-1,与传统检测百草枯的方法相比,无需昂贵的酶或抗体等试剂,成本较低,而且具备较低的检测限(0.01μg m L-1)。该方法为环境、农产品和中药材中农药百草枯残留的特异检测提供一定的技术支持和可靠依据。第三章,为有效避免血液中蛋白质和离子等干扰物对抗凝血药肝素实时监测和检测的影响,我们构建了一个新颖的荧光“猝灭-恢复”纳米传感器,实现对肝素的高选择性快速检测。本章应用牛血清白蛋白(BSA)即作为模板物又充当保护剂来制备水溶性BSA-Cd S量子点,该量子点与金纳米粒子Au NPs会发生内滤作用从而造成BSA-Cd S荧光发生显著猝灭现象。鱼精蛋白可诱导金纳米粒子(Au NPs)团聚,结果引起了Au NPs吸收光谱发生明显改变,并伴随着溶液体系荧光恢复。由于肝素与鱼精蛋白通过静电作用优先结合,使得鱼精蛋白远离金纳米粒子,造成荧光内滤作用变强,从而导致体系荧光信号再次变化,以此来检测肝素含量,并在此基础上定量检测了实际样品血样中的肝素含量。此方法与传统方法相比,可有效屏蔽血液中常见蛋白、氨基酸和阳离子的干扰,特异快速检测其中的抗凝血药肝素的含量,此方法具有较高的灵敏度,检测限性范围是10-300 ng m L-1,最低检测限是2.2 ng m L-1。此方法的建立为临床密切监测和定量检测抗凝血药肝素在血清中的含量及其临床使用量具有一定的指导意义和参考价值。第四章,为快速检测人体尿液样品中的胰蛋白酶含量,为胰腺疾病诊断提供一定的依据,我们利用荧光金簇(GSH-Au NCs)构建了一种简单、灵敏且无标记的荧光纳米传感器,用于胰蛋白酶的快速检测。在本章主要根据细胞色素C具有强烈的吸电子作用,当与金纳米簇接触,即可有效的猝灭其荧光的原理。当体系内存在胰蛋白酶时,胰蛋白酶可催化水解细胞色素C(Cyt C),造成荧光恢复。所以,通过该体系荧光强度变化来快速测试胰蛋白酶的活性。此方法具有很好的选择性和抗干扰能力,而且快速简便,检测线性范围是0.001-0.2 mg m L-1,最低检测限为0.3μg m L-1,可用于实际尿液样品中胰蛋白酶含量的有效测定。为临床上胰蛋白酶的检测和分析提供一定技术平台。第五章,为安全有效检测影响胎儿发育的重要物质叶酸的含量,我们联合应用表面修饰的金纳米粒子(cyst-Au NPs)和金纳米簇(BSA-Au NCs)构建了一种新颖、选择性好且无标记的叶酸纳米传感器。本章基于金纳米颗粒通过表面等离子体共振能量转移作用猝灭金簇荧光的原理,向体系内加入叶酸后,叶酸可诱发金纳米粒子团聚,引起其紫外吸收峰发生红移,从而造成金纳米粒子(cyst-Au NPs)和金纳米簇(BSA-Au NCs)之间表面等离子体共振能量转移作用明显变弱,导致体系荧光信号变化,从而实现叶酸的快速检测。本方案不仅选择性好,而且具有吸光度和荧光双信号输出的功能,一方面可通过溶液颜色变化对叶酸进行半定量检测,更重要的是,可通过荧光方法准确测定血液中叶酸的含量。该方法检测叶酸的线性范围是0.11-2.27μmol L-1,最低检测限为0.065μmol L-1。本文通过以上五部分工作,设计制备了系列重要的识别不同靶标药物的量子点或金簇纳米传感器,并对这些传感器进行系统性的功能评价,从而在实际样品中包括环境样品、农业样品和药用植物样品中,人体血样和尿样中,实现各类药物和蛋白分子的快速灵敏检测。这类传感器的使用将有效缩短检测时间,提高检测灵敏度,降低检测成本,实现复杂样品中目标物的特异性检测,具有潜在的应用价值。这不仅为临床药物和蛋白质的快速灵敏检测提供可行方案和可靠依据,更为深入广泛地研究新型纳米材料的多种功能提供一定的新思路和方向。
[Abstract]:Nano-sensors are widely used in the fields of chemistry, food science, clinical diagnosis, environment and medicine because of their excellent optical properties and sensitivity. The selection of nano-materials is very important to the sensitivity, specificity and stability of the sensors. New nano-materials (quantum dots and Ginna) are used. Combining with sensors, nano-sensors with high specificity and sensitivity can be constructed for rapid detection of pesticide residues in environment, agricultural products and traditional Chinese medicines, and sensitive monitoring of clinical important drug molecules. It can not only promote the rapid development of nanotechnology in the history of human science and technology, but also open up new ideas for researchers. At present, there are many kinds of detection methods for pesticide paraquat, anticoagulant heparin, trypsin and folic acid, such as chromatography, electrochemical method and enzyme-linked immunosorbent assay, but these methods are easy to be interfered by other components of the actual sample, difficult to extract and analyze, or a variety of detection steps, detection time, etc. With the continuous improvement of human living standards and the deepening of scientific research, people gradually realize the importance of environmental safety, food safety and clinical drug safety to human health, so researchers urgently need to study. Preparation of nano-sensors with simple operation, rapid detection, low cost and good sensitivity for rapid detection of pesticide molecules, clinical important drug molecules and important protein molecules closely related to human health in order to meet the environmental, food safety assessment and clinical drug safety guidelines, thus for China's agricultural products and pharmaceutical industry The rapid and safe development and the trend of internationalization have laid a good foundation for clinical medicine and rapid diagnosis of some diseases, and also provide a reliable evaluation index for human health and quality of life index. Considering the environmental problems, pesticide residues in agricultural products and raw medicines, clinical drug requirements and testing, a series of fluorescent nano-sensors were designed and assembled for the rapid detection and analysis of pesticide residues in environment, agricultural products and medicinal plants. Paraquat, clinical anticoagulants heparin, folic acid, and trypsin provide new ideas for the establishment of pesticide residue detection and clinical drug monitoring platforms. The following major advances have been made in this paper: In the first chapter, we reviewed the preparation of quantum dots and metal nanoclusters, and their applications in environmental and agricultural pollutants, protein analysis, bioimaging and other research areas. In the second chapter, in order to achieve rapid detection and screening of paraquat in the environment, agricultural products and medicinal plant samples, we use Cd S quantum dots with good optical properties as fluorescent nanomaterials to establish a simple and sensitive sensor for the detection of paraquat. The water-soluble Cd S quantum dots can be synthesized rapidly in 15 minutes by optimizing the synthesis conditions with glutathione as template and protectant. According to the characteristics of paraquat as cationic salt, the fluorescence of GSH-Cd S quantum dots can be quenched efficiently by electron transfer, and the fluorescence intensity of environmental and agricultural samples can be changed according to the fluorescence intensity. This method can be used to detect paraquat in the range of 0.025-1.50 UG m L-1. Compared with the traditional method, it does not need expensive enzymes or antibodies and other reagents. The cost is lower and the detection limit is lower (0.01 UG m L-1). This method is suitable for environment, agricultural products and Chinese herbal medicines. In Chapter 3, a novel fluorescent "quenching-recovery" nanosensor was constructed to effectively avoid the influence of protein and ion in blood on the real-time monitoring and detection of anticoagulant heparin. In this chapter, bovine serum albumin (BSA) was used as both a template and a protectant to prepare water-soluble BSA-Cd S quantum dots. The quantum dots interacted with gold nanoparticles Au NPs, resulting in significant quenching of BSA-Cd S fluorescence. Protamine could induce agglomeration of gold nanoparticles (Au NPs), resulting in absorption of Au NPs. Because heparin and protamine bind preferentially through electrostatic interaction, protamine is far away from gold nanoparticles, and the fluorescence internal filtration is strengthened, which leads to the fluorescence signal change again, so as to detect the content of heparin, and on this basis, the quantitative detection of the real protamine is carried out. Compared with the traditional method, this method can effectively shield the interference of common proteins, amino acids and cations in blood and detect the content of anticoagulant heparin rapidly. This method has high sensitivity. The detection limit is 10-300 ng m L-1 and the minimum detection limit is 2.2 ng m L-1. In the fourth chapter, we constructed a fluorescent gold cluster (GSH-Au NCs) to detect the trypsin content in human urine samples and provide a basis for the diagnosis of pancreatic diseases. A simple, sensitive and unmarked fluorescent nanosensor was developed for the rapid detection of trypsin. In this chapter, the fluorescence of cytochrome C was effectively quenched by contact with gold nanoclusters due to the strong electron absorption of cytochrome C. When trypsin was present in the system, trypsin could catalyze the hydrolysis of cytochrome. C (Cyt C), resulting in fluorescence recovery. Therefore, the fluorescence intensity of the system changes to quickly test the activity of trypsin. This method has good selectivity and anti-interference ability, and fast and simple, detection linear range is 0.001-0.2 mg m L-1, the minimum detection limit is 0.3 UG m L-1, can be used for the actual urine samples of trypsin content. In the fifth chapter, we constructed a novel, selective and label-free gold nanoparticles (cyst-Au NPs) and gold nanoclusters (BSA-Au NCs) for the safe and effective detection of folic acid, an important substance affecting fetal development. Folic acid nanosensors. Based on the principle that gold nanoparticles quench the fluorescence of gold clusters by surface plasmon resonance energy transfer, folic acid can induce the agglomeration of gold nanoparticles and cause the red shift of their ultraviolet absorption peaks, resulting in the formation of gold nanoparticles (cyst-Au NPs) and gold nanoclusters (BSA-Au NCs). This scheme not only has good selectivity, but also has the function of absorbance and fluorescence dual-signal output. On the one hand, it can detect folic acid semi-quantitatively by changing the color of the solution. More importantly, it can detect folic acid semi-quantitatively by changing the color of the solution. The fluorescence method can accurately determine the content of folic acid in blood. The linear range of the method is 0.11-2.27 micromol L-1 and the minimum detection limit is 0.065 micromol L-1. In this paper, a series of important quantum dots or gold cluster nano-sensors for identifying different target drugs are designed and fabricated. The application of this kind of sensor will shorten the detection time, improve the detection sensitivity, reduce the detection cost and achieve the target in complex samples. The detection of the specificity of the substance has potential application value, which not only provides a feasible scheme and reliable basis for the rapid and sensitive detection of clinical drugs and proteins, but also provides a new idea and direction for the further and extensive study of the multiple functions of new nanomaterials.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TP212.3;R927

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