量子點熒光納米材料的制備、表征及其生物醫(yī)學檢測應用
發(fā)布時間：2018-08-07 08:01
【摘要】：生物醫(yī)學檢測領域,熒光標記分子是研究抗原-抗體、DNA鏈段、酶與底物等生物分子間相互作用的重要研究工具。熒光量子點(Quantum Dots)作為一種新型熒光納米材料,具有量子效率高、摩爾消光系數(shù)大、光穩(wěn)定性好、可控的熒光發(fā)射波長和寬的熒光激發(fā)波長范圍等優(yōu)異的光學性能,因而在生物分析、檢測等領域得到廣泛應用。本論文主要包含以下研究工作： (1)液體石蠟中合成高質量的CdSe量子點,并進一步在CdSe量子點表面包覆ZnSe/ZnS殼層,制備得到高質量的油溶性核殼量子點。首先在液體石蠟中以十八胺(octadecylamine, ODA)和三辛基氧膦(trioctylphosphine oxide, TOPO)為表面活性劑,高溫注射法合成CdSe量子點核。同時研究了配體、反應溫度、反應時間等試驗條件對合成的CdSe量子點的生長速度和熒光性質的影響,并將合成的CdSe量子點與以油酸為穩(wěn)定劑制備的CdSe量子點光譜性質做比較。制備得到的CdSe量子點熒光量子效率最高達到60%以上,熒光發(fā)射峰30nm,發(fā)光波長覆蓋490-650nm。 在制備得到的CdSe量子點核表面包覆ZnSe/ZnS殼層,研究了不同包覆殼層厚度、殼層前體和反應溫度等試驗條件,對制備得到的核殼量子點熒光性能的影響。通過投射電鏡、紫外-可見吸收和熒光分光光度計監(jiān)測包殼過程,結果表明采用該方法可以制備得到核殼結果量子點,并提供CdSe量子點的熒光量子效率和熒光穩(wěn)定性。 (2)將合成得到的油溶性核殼量子點通過谷胱甘肽分子轉移至水相,然后使用雙官能聚乙二醇(polyethylene glycol, PEG)交聯(lián)劑,將量子點表面配體交聯(lián),得到PEG化高分子包裹的量子點。動態(tài)光散射(dynamic light scattering, DSL)、凝膠電泳、傅里葉變換紅外光譜(Fourier Transform Infrared, FTIR)、核磁共振(nuclear magnetic resonance, NMR)和熱重分析(thermogravimetric analysis, TGA)等表征手段研究制備得到的水溶性量子點,證明PEG分子共價偶聯(lián)在量子點表面,并研究了偶聯(lián)劑、PEG濃度等工藝條件,優(yōu)化量子點交聯(lián)和PEG化的工藝參數(shù)。得到的PEG化高分子包裹的量子點,其pH穩(wěn)定性和化學交聯(lián)劑緩沖液中穩(wěn)定性有明顯提高,PEG化的量子點細胞毒性也有降低,可以明顯抑制量子點對細胞的非特異性吸附。 (3)將抗體分子共價偶聯(lián)在量子點表面,得到免疫熒光量子點,并對免疫量子點的熒光性能、免疫活性進行表征。使用EDC/Sulfo-NHS偶聯(lián)劑,將抗體與表面帶羧基的PEG化量子點共價偶聯(lián),超速離心分離,純化得到量子點-抗體(QD-Ab)復合物,對離心純化過程進行監(jiān)測,考察了不同封閉劑對制備得到的量子點-抗體復合物的免疫活性的影響,其中使用NH2-PEG為封閉劑制備得到的QD-Ab免疫活性較優(yōu),斑點膜免疫反應可以檢測到1.57ng的乙肝表面抗原(HBsAg)蛋白。使用自制的QD-Ab復合物,初步制備得到HBsAg免疫層析試紙。 (4)采用自制的簡易毛細管流體裝置(CFD)制備量子點編碼微珠。由聚四氟乙烯(PTFE)微管、針頭和玻璃毛細管組成簡易CFD,制備生成單分散的高分子溶液液滴,通過溶劑揮發(fā)法得到高分子微珠。調節(jié)CFD中流動相的流速可以控制液滴的直徑,得到粒徑從245到46.2微米的量子點微珠,進一步通過調節(jié)聚合物溶液中量子點的濃度研究微珠的編碼能力,可以得到5種不同熒光強度的單色量子點編碼微珠和25種雙色量子點編碼微珠,制備的微珠粒徑均一,形狀規(guī)則,并且制備結果重復性好,粒徑控制容易,具備大規(guī)模制備量子點編碼微珠的潛力。最后,將兔IgG分子通過羧基偶聯(lián)在微珠表面,使用熒光分子標記的抗體檢測微珠表面的生物分子,表明微珠在生物分子檢測方面的潛在應用。 通過上述幾方面關于量子點的合成、表面改性、生物功能化和免疫分析體系的構建的研究,初步探索了量子點相關材料在生物醫(yī)學檢測領域的應用,為量子點相關納米材料在實際檢測中的應用提供了研究基礎和研究模型。
[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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R318.08
本文編號：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.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R318.08
【參考文獻】
相關期刊論文 前2條
1 朱艷冰,李慶閣,王桂蘭,袁景利;新型銪絡合物用于HBsAg的時間分辨熒光免疫檢測[J];標記免疫分析與臨床;2002年03期
2 張振亞;梅興國;;現(xiàn)代熒光免疫分析技術應用及其新發(fā)展[J];生物技術通訊;2006年04期
，本文編號：2169393
本文鏈接：http://sikaile.net/yixuelunwen/swyx/2169393.html
