纳米粒子的制备及其在波长检测型表面等离子体子共振传感器中的应用研究
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摘要
半导体纳米粒子具有宽的激发光谱、窄的发射光谱、可准确调谐的发射波长、较高的荧光漂白阈值以及良好的化学稳定性等独特的光学和电子学性质,适用于荧光标记,可以成为一类理想的生物荧光探针。本论文介绍了一种在微波辅助条件下,采用摩尔比为2.5: 1的L-半胱氨酸盐酸盐和谷胱甘肽作稳定剂,合成了量子产率为65%的CdTe纳米粒子的新方法;提出了以微乳液为溶胀体系来实现不同大小的CdTe纳米粒子同时导入来制备荧光编码的聚苯乙烯微球的方法,不仅实现了纳米粒子标记数量的基本可控而且基本上避免了CdTe纳米粒子的泄漏;在反相微乳液中通过正硅酸乙酯的水解来实现把多个纳米粒子包覆在同一二氧化硅微球中,得到了具有高效发光的SiO2纳米微球,简化了反应步骤、降低了成本,并用此方法合成了Fe3O4@SiO2核壳式磁性微球;用分散聚合工艺合成了粒径分布均匀、表面光滑且具有超顺磁性的聚苯乙烯磁性微球,克服了微球内部磁性物质分布不均匀及微球形貌不规则等缺陷,在微球表面修饰了羧基,利于与生物分子的连接。
表面等离子体子共振(SPR)传感器具有实时监测反应动态过程、生物样品无需标记、灵敏度高、无背景干扰等特点,已在生命科学、环境科学等许多研究领域得到了广泛的应用。本论文简要介绍了自制的多波长同时检测型SPR传感器的光源、导光系统、传感元件、流通池、分光检测系统和数据处理系统。将半导体和磁性系列纳米微球与靶向DNA相连,实时监测了DNA探针的固定过程及DNA杂交反应的进行,并研究了荧光和磁性纳米微球对靶向DNA溶液浓度的影响。本研究对提高SPR传感器灵敏度有着重大的意义,揭示了纳米技术在生物传感器应用中新的发展方向。
Semiconductor nanoparticles, also known as quantum dots (QDs), provide extremely good fluorescent signal, and can be well used for fluorescent labeling. Also they are ideal bioconjugated fluorescent probes for bioanalysis, because of their unique optical and electronic properties, such as broad excitation spectrum, narrow emission spectrum, good tunability and photochemical stability, neglectable optical bleaching, etc. Hence, QDs have attracted considerable interest in biological and medical community. The basic knowledgement and characteristics of QDs,their synthesis and labeling approach,recent progress and prospect in potential application to immunoassay and rapid diagnostic analysis are reviewed in Part 1 Chapter 1, the basic properties, synthesis, applications such as bioseparation of magnetic nanoparticles are also reviewed.
In Part 1, Chapter 2, a method for hydrothermal synthesis of CdTe QDs under microwave-assisted condition is developed. Using L-cysteine hydrochloride and glutathione as stabilizing agents, the molar ratio is 2.5:1. CdTe QDs with controllable photoluminescence wavelength from 510 nm to 670 nm were prepared in 150 min, the photoluminescence quantum yield was shown to be 65%. Compared with CdTe QDs prepared with thiohydracrylic acid as stabilizing agent, as-prepared CdTe QDs show much narrower photoluminescence FWHM (Full Width at Half Maximum), more symmetrical emission peak and higher photoluminescence quantum yield. The as-prepared CdTe QDs contain–NH2 and–COOH groups, which have good biological compatibility and have almost no toxicity. The C. elegans were taken as detecting object to study the biological toxic effect of CdTe QDs. A novel technique of synthesizing fluorescence-encoded microspheres by dispersing polymerization method is then reported. By the following one-step swelling procedure, the different CdTe QDs were carried into the inner of polystyrene microspheres quantificationally. This procedure has the following outstanding advantaged: it can be operated easily and reproducibly. Besides, as-synthesized photoluminescent microspheres have better photostability, the CdTe QDs can not be isolated or leaked out easily which are carried into the inner of microspheres. Finally, the multiple CdTe QDs were embedded into one silica microsphere by reverse microemulsion method. The as-synthysized silica microspheres have high luminous efficiency and small side effects on cells or living beings.
In Part 1, Chapter 3, with oleic acid functionalized Fe3O4 nanoparticles as the magnetic carriers, styrene and crylic acid as the monomers, and divinylbenzene ( DVB ) as cross linker, the magnetic polystyrene microspheres were prepared by dispersion polymerization. A series of characterizations show that as-prepared magnetic polystyrene microspheres have narrower size distribution with smooth surface, fine morphology and structure. The factors that affect the size of microspheres were also discussed. The as-prepared microspheres have narrower size distribution with smooth surface, fine morphology and structure, pretty superparamagnetic behavior, the specific saturation magnetization of them reaches to 11.61 Am2/kg at room temperature, the method can overcome these shortcomings: nonhomogeneous distribution of the interior magnetic matericals, irregular morphology of microspheres. Fe3O4@SiO2 core-shell microspheres were synthesized by reverse microemulsion method.
Since Liedberg developed the first surface plasmon resonance ( SPR ) sensor, the research and application of SPR technology have shown extensive growth and gradually become the research hot spot in the biosensor field in the world. The most important characteristics of SPR sensors are their versatility and capability for real time monitoring the association or dissociation of biomolecules on the surface of the sensor without the need for fluorescence or labeling of the biomolecules. These characteristics made the SPR technique an easy, convenient and reliable one for determining the concentration and molecular weight, monitoring change in structure, measuring kinetic constant and binding specificity of individual biomolecules. For above reasons, the SPR sensors grow as a new powerful technology in chemical and biological field. In Part 2, Chapter 1, the element theory, method and characteristic of SPR sensor are introduced. The categories and applications of wavelength modulation SPR sensors are discussed. The recent research progress of SPR sensor is also reviewed.
In Part 2, Chapter 2, a homemade wavelength modulation surface plasmon resonance sensor is introduced briefly. The setup consisted of white LED, light transmit system, sensing element, flow cell, detection system and data processing system. The light emitted from the white LED is polarized to obtain polarized light, and two lenses are employed to make the light parallel. The parallel polychromatic light beam passes through an optical prism and excites surface plasmon at the interface between the gold film and solution. The output light from the prism is guided into a charge coupled device ( CCD ) detector by fiber. The sensor was shown to relatively simple to use, rapid to respond, and cost-effective.
In Part 2, Chapter 3, the highly specific interaction of an avidin-biotin system was explored for immobilization of target DNA on the sensor memberane, CdTe@SiO2 core-shell microspheres coupled with target DNA, the wavelength modulation SPR instrument was developed for real-time determining the immobilizing process of biotinylated DNA and the performance of hybridization reaction. The target DNA coupling with CdTe@SiO2 core-shell microspheres was determined to be in the concentration range of 0.0262-14 nmol/L. In addition, the method of SPR sensor regeneration was investigated ( using a solution of 0.1 mol/L H3PO4, 0.1 mol/L NaOH, 0.1 mol/L HCl, 0.03 mol/L HNO3 or 0.3 mol/L citrate solution ). The citrate buffer ( 0.3 mol/L ) solution was chosen as regeneration solution.
In Part 2, Chapter 4, the 3-mercaptopropionic acid (MPA) was immobilized onto the gold film, and the association degrees of the target DNA coupled with different materials coated with magnetic nanoparticles with biotinylated DNA probes were investigated. Compared with target DNA coupled with magnetic polystyrene microspheres, the silica-coated magnetic nanoparticles conjugating with target DNA was validated to have faster association rate and smaller RSD. Besides, methods of enhancing SPR sensitivity were investigated by using magnetic microspheres. The target DNA was determined solely in the concentration range of 14-100 nmol/L, while target DNA conjugating with magnetic microspheres was tested in the concentration range of 24.3-120 pmol/L. It was shown that the association constant of biotinylated DNA probe with the target DNA coupling with magnetic microspheres was 3.31×107 mol-1 L, a three orders of magnitide improvement as compared with that obtained without magnetic microspheres. The 0.01 mol/L NaOH was chosen as regeneration solution. The research has greatly significance of enhancing sensitivity of SPR sensor, it reveals new direction of nanotechnology in biosensor application.
表面等离子体子共振(SPR)传感器具有实时监测反应动态过程、生物样品无需标记、灵敏度高、无背景干扰等特点,已在生命科学、环境科学等许多研究领域得到了广泛的应用。本论文简要介绍了自制的多波长同时检测型SPR传感器的光源、导光系统、传感元件、流通池、分光检测系统和数据处理系统。将半导体和磁性系列纳米微球与靶向DNA相连,实时监测了DNA探针的固定过程及DNA杂交反应的进行,并研究了荧光和磁性纳米微球对靶向DNA溶液浓度的影响。本研究对提高SPR传感器灵敏度有着重大的意义,揭示了纳米技术在生物传感器应用中新的发展方向。
Semiconductor nanoparticles, also known as quantum dots (QDs), provide extremely good fluorescent signal, and can be well used for fluorescent labeling. Also they are ideal bioconjugated fluorescent probes for bioanalysis, because of their unique optical and electronic properties, such as broad excitation spectrum, narrow emission spectrum, good tunability and photochemical stability, neglectable optical bleaching, etc. Hence, QDs have attracted considerable interest in biological and medical community. The basic knowledgement and characteristics of QDs,their synthesis and labeling approach,recent progress and prospect in potential application to immunoassay and rapid diagnostic analysis are reviewed in Part 1 Chapter 1, the basic properties, synthesis, applications such as bioseparation of magnetic nanoparticles are also reviewed.
In Part 1, Chapter 2, a method for hydrothermal synthesis of CdTe QDs under microwave-assisted condition is developed. Using L-cysteine hydrochloride and glutathione as stabilizing agents, the molar ratio is 2.5:1. CdTe QDs with controllable photoluminescence wavelength from 510 nm to 670 nm were prepared in 150 min, the photoluminescence quantum yield was shown to be 65%. Compared with CdTe QDs prepared with thiohydracrylic acid as stabilizing agent, as-prepared CdTe QDs show much narrower photoluminescence FWHM (Full Width at Half Maximum), more symmetrical emission peak and higher photoluminescence quantum yield. The as-prepared CdTe QDs contain–NH2 and–COOH groups, which have good biological compatibility and have almost no toxicity. The C. elegans were taken as detecting object to study the biological toxic effect of CdTe QDs. A novel technique of synthesizing fluorescence-encoded microspheres by dispersing polymerization method is then reported. By the following one-step swelling procedure, the different CdTe QDs were carried into the inner of polystyrene microspheres quantificationally. This procedure has the following outstanding advantaged: it can be operated easily and reproducibly. Besides, as-synthesized photoluminescent microspheres have better photostability, the CdTe QDs can not be isolated or leaked out easily which are carried into the inner of microspheres. Finally, the multiple CdTe QDs were embedded into one silica microsphere by reverse microemulsion method. The as-synthysized silica microspheres have high luminous efficiency and small side effects on cells or living beings.
In Part 1, Chapter 3, with oleic acid functionalized Fe3O4 nanoparticles as the magnetic carriers, styrene and crylic acid as the monomers, and divinylbenzene ( DVB ) as cross linker, the magnetic polystyrene microspheres were prepared by dispersion polymerization. A series of characterizations show that as-prepared magnetic polystyrene microspheres have narrower size distribution with smooth surface, fine morphology and structure. The factors that affect the size of microspheres were also discussed. The as-prepared microspheres have narrower size distribution with smooth surface, fine morphology and structure, pretty superparamagnetic behavior, the specific saturation magnetization of them reaches to 11.61 Am2/kg at room temperature, the method can overcome these shortcomings: nonhomogeneous distribution of the interior magnetic matericals, irregular morphology of microspheres. Fe3O4@SiO2 core-shell microspheres were synthesized by reverse microemulsion method.
Since Liedberg developed the first surface plasmon resonance ( SPR ) sensor, the research and application of SPR technology have shown extensive growth and gradually become the research hot spot in the biosensor field in the world. The most important characteristics of SPR sensors are their versatility and capability for real time monitoring the association or dissociation of biomolecules on the surface of the sensor without the need for fluorescence or labeling of the biomolecules. These characteristics made the SPR technique an easy, convenient and reliable one for determining the concentration and molecular weight, monitoring change in structure, measuring kinetic constant and binding specificity of individual biomolecules. For above reasons, the SPR sensors grow as a new powerful technology in chemical and biological field. In Part 2, Chapter 1, the element theory, method and characteristic of SPR sensor are introduced. The categories and applications of wavelength modulation SPR sensors are discussed. The recent research progress of SPR sensor is also reviewed.
In Part 2, Chapter 2, a homemade wavelength modulation surface plasmon resonance sensor is introduced briefly. The setup consisted of white LED, light transmit system, sensing element, flow cell, detection system and data processing system. The light emitted from the white LED is polarized to obtain polarized light, and two lenses are employed to make the light parallel. The parallel polychromatic light beam passes through an optical prism and excites surface plasmon at the interface between the gold film and solution. The output light from the prism is guided into a charge coupled device ( CCD ) detector by fiber. The sensor was shown to relatively simple to use, rapid to respond, and cost-effective.
In Part 2, Chapter 3, the highly specific interaction of an avidin-biotin system was explored for immobilization of target DNA on the sensor memberane, CdTe@SiO2 core-shell microspheres coupled with target DNA, the wavelength modulation SPR instrument was developed for real-time determining the immobilizing process of biotinylated DNA and the performance of hybridization reaction. The target DNA coupling with CdTe@SiO2 core-shell microspheres was determined to be in the concentration range of 0.0262-14 nmol/L. In addition, the method of SPR sensor regeneration was investigated ( using a solution of 0.1 mol/L H3PO4, 0.1 mol/L NaOH, 0.1 mol/L HCl, 0.03 mol/L HNO3 or 0.3 mol/L citrate solution ). The citrate buffer ( 0.3 mol/L ) solution was chosen as regeneration solution.
In Part 2, Chapter 4, the 3-mercaptopropionic acid (MPA) was immobilized onto the gold film, and the association degrees of the target DNA coupled with different materials coated with magnetic nanoparticles with biotinylated DNA probes were investigated. Compared with target DNA coupled with magnetic polystyrene microspheres, the silica-coated magnetic nanoparticles conjugating with target DNA was validated to have faster association rate and smaller RSD. Besides, methods of enhancing SPR sensitivity were investigated by using magnetic microspheres. The target DNA was determined solely in the concentration range of 14-100 nmol/L, while target DNA conjugating with magnetic microspheres was tested in the concentration range of 24.3-120 pmol/L. It was shown that the association constant of biotinylated DNA probe with the target DNA coupling with magnetic microspheres was 3.31×107 mol-1 L, a three orders of magnitide improvement as compared with that obtained without magnetic microspheres. The 0.01 mol/L NaOH was chosen as regeneration solution. The research has greatly significance of enhancing sensitivity of SPR sensor, it reveals new direction of nanotechnology in biosensor application.
引文
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