量子点荧光纳米材料的制备、表征及其生物医学检测应用
发布时间：2018-08-07 08:01
【摘要】：生物医学检测领域,荧光标记分子是研究抗原-抗体、DNA链段、酶与底物等生物分子间相互作用的重要研究工具。荧光量子点(Quantum Dots)作为一种新型荧光纳米材料,具有量子效率高、摩尔消光系数大、光稳定性好、可控的荧光发射波长和宽的荧光激发波长范围等优异的光学性能,因而在生物分析、检测等领域得到广泛应用。本论文主要包含以下研究工作： (1)液体石蜡中合成高质量的CdSe量子点,并进一步在CdSe量子点表面包覆ZnSe/ZnS壳层,制备得到高质量的油溶性核壳量子点。首先在液体石蜡中以十八胺(octadecylamine, ODA)和三辛基氧膦(trioctylphosphine oxide, TOPO)为表面活性剂,高温注射法合成CdSe量子点核。同时研究了配体、反应温度、反应时间等试验条件对合成的CdSe量子点的生长速度和荧光性质的影响,并将合成的CdSe量子点与以油酸为稳定剂制备的CdSe量子点光谱性质做比较。制备得到的CdSe量子点荧光量子效率最高达到60%以上,荧光发射峰30nm,发光波长覆盖490-650nm。 在制备得到的CdSe量子点核表面包覆ZnSe/ZnS壳层,研究了不同包覆壳层厚度、壳层前体和反应温度等试验条件,对制备得到的核壳量子点荧光性能的影响。通过投射电镜、紫外-可见吸收和荧光分光光度计监测包壳过程,结果表明采用该方法可以制备得到核壳结果量子点,并提供CdSe量子点的荧光量子效率和荧光稳定性。 (2)将合成得到的油溶性核壳量子点通过谷胱甘肽分子转移至水相,然后使用双官能聚乙二醇(polyethylene glycol, PEG)交联剂,将量子点表面配体交联,得到PEG化高分子包裹的量子点。动态光散射(dynamic light scattering, DSL)、凝胶电泳、傅里叶变换红外光谱(Fourier Transform Infrared, FTIR)、核磁共振(nuclear magnetic resonance, NMR)和热重分析(thermogravimetric analysis, TGA)等表征手段研究制备得到的水溶性量子点,证明PEG分子共价偶联在量子点表面,并研究了偶联剂、PEG浓度等工艺条件,优化量子点交联和PEG化的工艺参数。得到的PEG化高分子包裹的量子点,其pH稳定性和化学交联剂缓冲液中稳定性有明显提高,PEG化的量子点细胞毒性也有降低,可以明显抑制量子点对细胞的非特异性吸附。 (3)将抗体分子共价偶联在量子点表面,得到免疫荧光量子点,并对免疫量子点的荧光性能、免疫活性进行表征。使用EDC/Sulfo-NHS偶联剂,将抗体与表面带羧基的PEG化量子点共价偶联,超速离心分离,纯化得到量子点-抗体(QD-Ab)复合物,对离心纯化过程进行监测,考察了不同封闭剂对制备得到的量子点-抗体复合物的免疫活性的影响,其中使用NH2-PEG为封闭剂制备得到的QD-Ab免疫活性较优,斑点膜免疫反应可以检测到1.57ng的乙肝表面抗原(HBsAg)蛋白。使用自制的QD-Ab复合物,初步制备得到HBsAg免疫层析试纸。 (4)采用自制的简易毛细管流体装置(CFD)制备量子点编码微珠。由聚四氟乙烯(PTFE)微管、针头和玻璃毛细管组成简易CFD,制备生成单分散的高分子溶液液滴,通过溶剂挥发法得到高分子微珠。调节CFD中流动相的流速可以控制液滴的直径,得到粒径从245到46.2微米的量子点微珠,进一步通过调节聚合物溶液中量子点的浓度研究微珠的编码能力,可以得到5种不同荧光强度的单色量子点编码微珠和25种双色量子点编码微珠,制备的微珠粒径均一,形状规则,并且制备结果重复性好,粒径控制容易,具备大规模制备量子点编码微珠的潜力。最后,将兔IgG分子通过羧基偶联在微珠表面,使用荧光分子标记的抗体检测微珠表面的生物分子,表明微珠在生物分子检测方面的潜在应用。 通过上述几方面关于量子点的合成、表面改性、生物功能化和免疫分析体系的构建的研究,初步探索了量子点相关材料在生物医学检测领域的应用,为量子点相关纳米材料在实际检测中的应用提供了研究基础和研究模型。
[Abstract]:In the field of biomedical detection, fluorescent labeling molecules are important research tools to study the interaction between antigens and antibodies, DNA segments, enzymes and substrates. As a new kind of new fluorescent nanomaterials, fluorescence quantum dots (Quantum Dots) have high quantum efficiency, large Moorish extinction coefficient, good photostability, and controllable fluorescence emission wavelength and width. As a result of its excellent optical properties, such as excitation wavelength range, it has been widely used in biological analysis, detection and other fields.
(1) high quality CdSe quantum dots were synthesized in liquid paraffin, and the ZnSe/ZnS shell was coated on the surface of CdSe quantum dots. High quality oil soluble nuclear shell quantum dots were prepared. First, eighteen amine (octadecylamine, ODA) and three octyl phosphine (trioctylphosphine oxide, TOPO) were used as surface active agents in liquid paraffin, and high temperature injection method was used. CdSe quantum dots are nucleed. The effects of ligand, reaction temperature, reaction time and other test conditions on the growth rate and fluorescence properties of the synthesized CdSe quantum dots are also studied. The synthesized CdSe quantum dots are compared with the spectral properties of the CdSe quantum dots prepared with oleic acid as stabilizers. The fluorescence quantum efficiency of the prepared CdSe quantum dots is the highest. Above 60%, the fluorescence emission peak is 30nm, and the luminescence wavelength is covered by 490-650nm..
The ZnSe/ZnS shell was coated on the surface of the prepared CdSe quantum dots on the surface of the quantum dots. The effects of the coating thickness, the shell precursor and the reaction temperature on the fluorescence properties of the nuclear shell quantum dots were investigated. The shell process was monitored by the projective electron microscope, the UV visible absorption and the fluorescence spectrophotometer. The results showed that this formula was used. The core shell quantum dots can be prepared and the fluorescence quantum efficiency and fluorescence stability of CdSe quantum dots can be obtained.
(2) to transfer the synthesized oil soluble nuclear shell quantum dots through the glutathione molecule to the water phase, and then use the double functional polyethylene glycol (polyethylene glycol, PEG) crosslinking agent to cross link the ligands on the surface of the quantum dots to obtain the quantum dots of the PEG encapsulated polymer. Dynamic dispersion (dynamic light scattering, DSL), gel electrophoresis, Fourier transform. The Fourier Transform Infrared (FTIR), nuclear magnetic resonance (nuclear magnetic resonance, NMR) and thermogravimetric analysis (thermogravimetric analysis, TGA) were used to study the water soluble quantum dots. It was proved that the PEG molecules covalently coupled to the surface of the quantum dots, and studied the process conditions of coupling agent, concentration and so on. Quantum dots crosslinking and PEG process parameters. The quantum dots encapsulated by PEG, the stability of pH and the stability of the chemical crosslinker buffer are obviously improved, and the cytotoxicity of the PEG quantum dots can also be reduced, and the non-specific desorption of the quantum dots on the cells can be obviously inhibited.
(3) the antibody molecules are covalently coupled to the surface of the quantum dots to obtain the immunofluorescent quantum dots, and to characterize the fluorescence and immune activity of the immune quantum dots. The EDC/Sulfo-NHS coupling agent is used to covalently coupling the antibody with the PEG quantum dots with the carboxyl group on the surface, the ultra speed centrifugation is separated and the quantum dot antibody (QD-Ab) complex is purified, and the centrifugation is obtained. The purification process was monitored and the effects of different sealants on the immune activity of the prepared quantum dot antibody complex were investigated. The QD-Ab immunoreactivity of the NH2-PEG was better than that of the sealant. The dot membrane immunoreaction could detect the 1.57ng protein of the hepatitis B surface anti original (HBsAg). The initial QD-Ab complex was used initially. HBsAg immunochromatographic test paper was prepared step by step.
(4) a self-made simple capillary fluid device (CFD) was used to prepare a quantum dot encoded microbead. A simple CFD composed of PTFE microtubules, a needle and glass capillary was made to produce a monodisperse polymer solution of polymer solution, and a polymer microsphere was obtained by solvent evaporation. The diameter of the droplet could be controlled by adjusting the flow velocity of the liquid phase in the CFD. With the particle size from 245 to 46.2 microns, we can further study the coding ability of the microspheres by adjusting the concentration of the quantum dots in the polymer solution. 5 kinds of monochromatic quantum dots encoding microspheres and 25 dichroic quantum dots coding microspheres can be obtained with different fluorescence intensities. The prepared microspheres are uniform in size, shape rules, and the preparation results are repeated. It is easy to control the particle size, and has the potential for large-scale preparation of quantum dots. Finally, the rabbit IgG molecules are coupled to the surface of the microbeads by the carboxyl group. The biomolecules on the surface of the microbeads are detected by the antibody labeled by the fluorescent molecules, indicating the potential applications of microspheres in the detection of biomolecules.
Through the research on the synthesis of quantum dots, surface modification, biofunctionalization and the construction of immune analysis system, the application of quantum dots related materials in the field of biomedical detection is preliminarily explored, and the research foundation and research model are provided for the application of quantum dots related nanomaterials in actual detection.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.08
本文编号：2169393
[Abstract]:In the field of biomedical detection, fluorescent labeling molecules are important research tools to study the interaction between antigens and antibodies, DNA segments, enzymes and substrates. As a new kind of new fluorescent nanomaterials, fluorescence quantum dots (Quantum Dots) have high quantum efficiency, large Moorish extinction coefficient, good photostability, and controllable fluorescence emission wavelength and width. As a result of its excellent optical properties, such as excitation wavelength range, it has been widely used in biological analysis, detection and other fields.
(1) high quality CdSe quantum dots were synthesized in liquid paraffin, and the ZnSe/ZnS shell was coated on the surface of CdSe quantum dots. High quality oil soluble nuclear shell quantum dots were prepared. First, eighteen amine (octadecylamine, ODA) and three octyl phosphine (trioctylphosphine oxide, TOPO) were used as surface active agents in liquid paraffin, and high temperature injection method was used. CdSe quantum dots are nucleed. The effects of ligand, reaction temperature, reaction time and other test conditions on the growth rate and fluorescence properties of the synthesized CdSe quantum dots are also studied. The synthesized CdSe quantum dots are compared with the spectral properties of the CdSe quantum dots prepared with oleic acid as stabilizers. The fluorescence quantum efficiency of the prepared CdSe quantum dots is the highest. Above 60%, the fluorescence emission peak is 30nm, and the luminescence wavelength is covered by 490-650nm..
The ZnSe/ZnS shell was coated on the surface of the prepared CdSe quantum dots on the surface of the quantum dots. The effects of the coating thickness, the shell precursor and the reaction temperature on the fluorescence properties of the nuclear shell quantum dots were investigated. The shell process was monitored by the projective electron microscope, the UV visible absorption and the fluorescence spectrophotometer. The results showed that this formula was used. The core shell quantum dots can be prepared and the fluorescence quantum efficiency and fluorescence stability of CdSe quantum dots can be obtained.
(2) to transfer the synthesized oil soluble nuclear shell quantum dots through the glutathione molecule to the water phase, and then use the double functional polyethylene glycol (polyethylene glycol, PEG) crosslinking agent to cross link the ligands on the surface of the quantum dots to obtain the quantum dots of the PEG encapsulated polymer. Dynamic dispersion (dynamic light scattering, DSL), gel electrophoresis, Fourier transform. The Fourier Transform Infrared (FTIR), nuclear magnetic resonance (nuclear magnetic resonance, NMR) and thermogravimetric analysis (thermogravimetric analysis, TGA) were used to study the water soluble quantum dots. It was proved that the PEG molecules covalently coupled to the surface of the quantum dots, and studied the process conditions of coupling agent, concentration and so on. Quantum dots crosslinking and PEG process parameters. The quantum dots encapsulated by PEG, the stability of pH and the stability of the chemical crosslinker buffer are obviously improved, and the cytotoxicity of the PEG quantum dots can also be reduced, and the non-specific desorption of the quantum dots on the cells can be obviously inhibited.
(3) the antibody molecules are covalently coupled to the surface of the quantum dots to obtain the immunofluorescent quantum dots, and to characterize the fluorescence and immune activity of the immune quantum dots. The EDC/Sulfo-NHS coupling agent is used to covalently coupling the antibody with the PEG quantum dots with the carboxyl group on the surface, the ultra speed centrifugation is separated and the quantum dot antibody (QD-Ab) complex is purified, and the centrifugation is obtained. The purification process was monitored and the effects of different sealants on the immune activity of the prepared quantum dot antibody complex were investigated. The QD-Ab immunoreactivity of the NH2-PEG was better than that of the sealant. The dot membrane immunoreaction could detect the 1.57ng protein of the hepatitis B surface anti original (HBsAg). The initial QD-Ab complex was used initially. HBsAg immunochromatographic test paper was prepared step by step.
(4) a self-made simple capillary fluid device (CFD) was used to prepare a quantum dot encoded microbead. A simple CFD composed of PTFE microtubules, a needle and glass capillary was made to produce a monodisperse polymer solution of polymer solution, and a polymer microsphere was obtained by solvent evaporation. The diameter of the droplet could be controlled by adjusting the flow velocity of the liquid phase in the CFD. With the particle size from 245 to 46.2 microns, we can further study the coding ability of the microspheres by adjusting the concentration of the quantum dots in the polymer solution. 5 kinds of monochromatic quantum dots encoding microspheres and 25 dichroic quantum dots coding microspheres can be obtained with different fluorescence intensities. The prepared microspheres are uniform in size, shape rules, and the preparation results are repeated. It is easy to control the particle size, and has the potential for large-scale preparation of quantum dots. Finally, the rabbit IgG molecules are coupled to the surface of the microbeads by the carboxyl group. The biomolecules on the surface of the microbeads are detected by the antibody labeled by the fluorescent molecules, indicating the potential applications of microspheres in the detection of biomolecules.
Through the research on the synthesis of quantum dots, surface modification, biofunctionalization and the construction of immune analysis system, the application of quantum dots related materials in the field of biomedical detection is preliminarily explored, and the research foundation and research model are provided for the application of quantum dots related nanomaterials in actual detection.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.08
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相关期刊论文 前2条
1 朱艳冰,李庆阁,王桂兰,袁景利;新型铕络合物用于HBsAg的时间分辨荧光免疫检测[J];标记免疫分析与临床;2002年03期
2 张振亚;梅兴国;;现代荧光免疫分析技术应用及其新发展[J];生物技术通讯;2006年04期
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