新型氧化硅、金复合纳米材料的制备、组装及其在生物医学、催化上的应用
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摘要
新型金、氧化硅纳米材料和其复合纳米材料的制备、化学修饰、组装以及在化学、分子生物学、药学、医学等领域的应用研究是当前最热门的课题之一。本文对新型金、氧化硅纳米材料的制备、组装及在生物医学、化学催化上的应用进行了详细研究。主要开展的工作如下：
以3-巯基丙基三甲氧基硅烷(MPTMOS)代替3-氨基丙基三甲氧基硅烷作连接剂,合成了Au@SiO2核壳纳米粒,并对其进行了光谱、TEM、荧光显微镜成像等系列表征。实验结果表明,经改进后制得的Au@SiO2核壳纳米粒形貌呈球形、单分散性较好,金纳米粒位于其中心,分布均匀,无团聚的金纳米粒包覆在氧化硅壳中。荧光检测发现其有光致发光性质,荧光强度在连续扫描3600s后仍没有明显变化,表明该核壳纳米粒具有良好的抗漂白性能。在3-氨基丙基三甲氧基硅烷及戊二醛交联剂的作用下,把TPA抗体固定在Au@SiO2核壳纳米粒上,利用抗原和抗体的特异性识别作用成功实现了对CNE-1/亲鼻咽癌细胞的荧光成像。
以3-巯基丙基三甲氧基硅烷作连接剂,利用巯基能形成二硫键的性质把氧化硅纳米粒自组装成氧化硅纳米线,合成了金纳米粒子修饰的氧化硅纳米线,并对其进行了光谱、TEM、荧光显微镜成像等系列表征。实验结果表明,该方法可合成得到不同粗细的有金纳米粒包埋于其中或附在表面的氧化硅纳米线。所有实验都是在溶液中进行,不需要高温、真空及特殊的仪器设备,方法安全可行,成本低,有望放大生产以适应对这类新型氧化硅纳米线的大量需求。金纳米粒通过化学键负载在氧化硅纳米线上,表现出了较好的稳定性和较高的催化活性,在NaBH4相对大量过量的条件下,对硝基苯酚的催化还原反应符合准一级反应动力学,反应速率常数k=0.85min-1。此外,金纳米粒附在氧化硅纳米线的表面,目标分子(特别是生物分子)易接近,可潜在用于化学传感及生物传感的应用。
用普通光学玻璃做基底,用3-巯基丙基三甲氧基硅烷做粘结剂,先在玻璃芯片上成功制得了排布均匀可控的金纳米粒单层膜,然后依次用组织多肽抗原抗体(anti-TPA)、小牛血清蛋白(BSA)对其功能化修饰,制成了对组织多肽抗原(TPA)有特异性响应的TPA光学免疫传感芯片。该传感芯片性能稳定,选择性好,可用紫外可见分光光度计直接对复杂样品检测其中TPA含量。该方法技术上只需要通过测量最大吸收波长处的吸光度变化值来测定样品中TPA含量,操作简单快捷,容易普及。在优化条件下,传感芯片用样品培育一定时间后,其吸光度减小值(△A)与样品中TPA的浓度(C)在1-1000ng/L范围内呈良好的线性关系,检测限为1.2×10-3ng/L,灵敏度很高。通过对3种人工混合样品的回收试验,检测结果与真实值基本一致,回收率为95.7～103.0%,标准偏差RSD=1.6～2.8%,说明方法的准确度和精密度都较好。将制得的传感芯片初步应用于人的血清样品中TPA的检测,发现对癌症患者的血清响应比较明显,而对健康人的血清响应不明显。基于金纳米粒紫外可见吸收的免疫传感法是一种比较理想的分析方法,其在抗原定量检测上的应用是可行的。
将胶体金直接依次用anti-TPA、BSA、PEG修饰制成溶液基TPA光学生物传感探针,建立了一种快速、简单、有效的TPA定量分析方法。考察了反应时间、反应温度及工作溶液pH值等多种因素的影响。在优化条件下,探针吸光度的减少值与TPA的浓度在0.4-200ng/mL范围内近似呈线性关系,线性回归方程为-△A=0.02767C+0.00632,线性相关系数R=0.99271,检测限约为0.108ng/mL。把方法应用于真实血清样品的测定,检测结果与真实结果基本一致,相对误差在-4.0～8.7%范围内。
用3-氨基丙基三甲氧基硅烷作粘结剂,在金纳米粒单层膜基础上继续交替铺上氧化硅纳米粒、金纳米粒,成功制得了金纳米粒/氧化硅纳米粒交替排列的多层膜芯片,并对其进行了紫外可见吸收光谱、SEM等系列表征。实验结果表明,随金纳米粒层层数的增加,芯片在540nm处的吸收峰增高,而吸收峰的峰位、峰形基本保持不变。金纳米粒/氧化硅纳米粒多层复合膜的扫描电镜图显示,制备的多层膜芯片表面金纳米粒层金纳米粒的大小为20nm左右,分布比较均匀、规整,为进一步的应用研究奠定了坚实的基础。用anti-TPA对制得的多层复合膜进行修饰、用BSA封住余留的活性点,由此所制得的传感芯片对TPA分子有特异性识别响应,TPA分子通过对anti-TPA分子免疫反应使传感芯片在最大吸收波长处的吸光度值减小。实验结果显示,吸光度的减少值与TPA的浓度在1-10000ng/L范围内近似呈线性关系,线性回归方程为-△A=0.05266C+0.00758,线性相关系数R=0.99734,检测限约为0.027ng/L。把制得的TPA传感芯片应用于血清真实样品的检测,结果表明,3个血清样品的测试结果和当地医院测得值基本一致,标准偏差在允许范围之内。
Preparation, chemical functionalization and assembly of Au nanoparticles, silicon oxide nanomaterial and the Au/SiO2composite nanomaterials, and their applications are one of the most important subjects in the fields of chemistry, molecular biology, medicine and so on. The technologies of preparing and assembling Au, SiO2nanopartices, and their applications are reviewed in this dissertation. The main detail is as follows:
Au@SiO2core/shell nanoparticles were synthesized by using (3-mercaptopropyl)trimethoxysilane (MPTMOS) as cross-linker instead of (3-amino-propyl)trimethoxysilane. The nanoparticles were characterized by UV-visible spectroscopy, TEM and fluorescence microscope. Our results showed the nanocrystals were uniformly spherical and high monodisperse in water. Single gold nanoparticle was located at the center of silicon oxide shell. Moreover, no obvious change of fluorescence intensity of Au/SiO2composite nano-materials was observed after it was excited for3600seconds by successive intense irradiation, which demonstrates that these nanoparticles had excellent photostability. In addition, Au@SiO2core/shell nanoparticles were covalently conjugated with the TPA using (3-aminopropyl)trimethoxysilane and glutaraldehyde as the crosslinker and successfully completed the fluorescence imaging of nasopharyngeal carcinoma cells CNE-1.
Based on the principle that the-SH could be oxidated to form a disulfide bond, we had synthesized the gold-nanoparticle-modified silica nanowires in solution by employing3-mercapto-propyltrimethoxysilane as the cross-linker under mild conditions. The nanowires were characterized by UV-visible spectroscopy, TEM and fluorescence microscope. The results demonstrate that the silica nanowires of various thicknesses that the gold nanoparticles were embedded in and/or attached on SiNWs were prepared. The method is simple, low cost, safe and feasible, so it is helpful for meeting the social great demand by synthesizing the SiNWs in large scale. Gold-nanoparticles were loaded on SiNWs by chemical bond that it showed the good stability and excellent catalytic activity. The Catalytic reduction of reaction of p-nitrophenol follows the first-order kinetic process when NaBH4is excessive, the reaction rate constant k=0.85min-1. Moreover, the composite nanoparticles were easy to covalently conjugate with the target molecule (i.e. biological molecule), and it made them become ideal candidates for use in chemical sensor and biosensor.
The monolayer membrane of gold nanoparticles was controllably prepared on the glass chip using optical glass as substrate and MPTMOS as agglomerant. Then, anti-TAP and BSA were used to functionalize this monolayer membrane of gold nanoparticles to prepare the optical immune sensing chip that could specifically response to TPA. This chip can directly detect the TPA in complex sample using ultraviolet visible spectrophotometer with good stabilization and selectivity. In addition, because this technique need only measure the absorbance change in the largest ultraviolet absorption wavelength of TPA to detect the content of TPA in samples, this method can be easily popularized for this kind of simple and fast format. Under the optimized conditions, the decreased absorbency (AA) was linear with TPA concentration (C) in the range of1～1000ng/L and the low detection limit was1.2×10-3ng/L. The results of determination for TPA in three synthetic samples were identical with the true values, the recovery (98.9%-102.4%) and the standard deviation (1.6～2.8%) was satisfactory. Moreover, the method was applied to the determination of the content of TPA in human serum samples. Compared to the serum of healthy person, the response value was improved obviously in cancer patients. The concept demonstrated that the application of Immunosensors based on the UV-Vis absorption of gold-nanoparticles to quantitative detecting the antigen was feasible.
Liquid TPA optical bio-sensing probe was constructed using the anti-TPA, BSA and PEG functionalized gold nanoparticles. This method provides a fast, simple and efficient character for quantitative analysis of TPA. In this work, a series of factors (i.e. reaction time, reaction temperature and pH) were investigated to find the better determination condition. Under the optimized conditions, a linear relationship of the decreased absorbency (△A) and TPA concentration (C) was established in the range of0.4-200ng/mL using the equation-△A=0.02767C+0.00632(R=0.99271) and the low detection limit was0.108ng/mL. The results of the determination for TPA in serum real samples were identical with the true value, and the relatively error is at-4.0～8.7%.
By using the (3-aminopropyl)trimethoxysilane as cross-linker, the multilayer chip was formed by alternating Au-nanoparticles and SiO2-nanoparticles on nano-Au single-layer. The multilayer chip was characterized by UV-visible spectroscopy, SEM and so on. The absorbing intensity at540nm of chip increased with enhancement of layer of Au-nanoparticles, but the position and shap of the absorption peak was nearly invariant. The TEM image of the multilayer film proved this method yielded the Au-nanopartices on the multilayer chip surface with20nm in diameter and uniform. The successful preparation of multilayer chip paved a way for further study and application. When the multilayer chip was modified by anti-TPA and the remaining active sites of multilayer chip were blocked by BSA, the as-prepared sensoring chip can specifically recognize TPA molecular and the absorbing intensity of sensoring chip at the maximum absorption wavelengths was decreased because of immune reaction between TPA and anti-TPA. The results showed a linear relationship of the decreased absorbency (△A) and TPA concentration (C) was established in the range of1～1000ng/L using the equation-△A=0.05266C+0.00758(R=0.99734) and the low detection limit was0.027ng/L. The results of the determination for TPA in three serum real samples were identical with the conventional method of hospital, and the standard deviation was satisfactory.
以3-巯基丙基三甲氧基硅烷(MPTMOS)代替3-氨基丙基三甲氧基硅烷作连接剂,合成了Au@SiO2核壳纳米粒,并对其进行了光谱、TEM、荧光显微镜成像等系列表征。实验结果表明,经改进后制得的Au@SiO2核壳纳米粒形貌呈球形、单分散性较好,金纳米粒位于其中心,分布均匀,无团聚的金纳米粒包覆在氧化硅壳中。荧光检测发现其有光致发光性质,荧光强度在连续扫描3600s后仍没有明显变化,表明该核壳纳米粒具有良好的抗漂白性能。在3-氨基丙基三甲氧基硅烷及戊二醛交联剂的作用下,把TPA抗体固定在Au@SiO2核壳纳米粒上,利用抗原和抗体的特异性识别作用成功实现了对CNE-1/亲鼻咽癌细胞的荧光成像。
以3-巯基丙基三甲氧基硅烷作连接剂,利用巯基能形成二硫键的性质把氧化硅纳米粒自组装成氧化硅纳米线,合成了金纳米粒子修饰的氧化硅纳米线,并对其进行了光谱、TEM、荧光显微镜成像等系列表征。实验结果表明,该方法可合成得到不同粗细的有金纳米粒包埋于其中或附在表面的氧化硅纳米线。所有实验都是在溶液中进行,不需要高温、真空及特殊的仪器设备,方法安全可行,成本低,有望放大生产以适应对这类新型氧化硅纳米线的大量需求。金纳米粒通过化学键负载在氧化硅纳米线上,表现出了较好的稳定性和较高的催化活性,在NaBH4相对大量过量的条件下,对硝基苯酚的催化还原反应符合准一级反应动力学,反应速率常数k=0.85min-1。此外,金纳米粒附在氧化硅纳米线的表面,目标分子(特别是生物分子)易接近,可潜在用于化学传感及生物传感的应用。
用普通光学玻璃做基底,用3-巯基丙基三甲氧基硅烷做粘结剂,先在玻璃芯片上成功制得了排布均匀可控的金纳米粒单层膜,然后依次用组织多肽抗原抗体(anti-TPA)、小牛血清蛋白(BSA)对其功能化修饰,制成了对组织多肽抗原(TPA)有特异性响应的TPA光学免疫传感芯片。该传感芯片性能稳定,选择性好,可用紫外可见分光光度计直接对复杂样品检测其中TPA含量。该方法技术上只需要通过测量最大吸收波长处的吸光度变化值来测定样品中TPA含量,操作简单快捷,容易普及。在优化条件下,传感芯片用样品培育一定时间后,其吸光度减小值(△A)与样品中TPA的浓度(C)在1-1000ng/L范围内呈良好的线性关系,检测限为1.2×10-3ng/L,灵敏度很高。通过对3种人工混合样品的回收试验,检测结果与真实值基本一致,回收率为95.7～103.0%,标准偏差RSD=1.6～2.8%,说明方法的准确度和精密度都较好。将制得的传感芯片初步应用于人的血清样品中TPA的检测,发现对癌症患者的血清响应比较明显,而对健康人的血清响应不明显。基于金纳米粒紫外可见吸收的免疫传感法是一种比较理想的分析方法,其在抗原定量检测上的应用是可行的。
将胶体金直接依次用anti-TPA、BSA、PEG修饰制成溶液基TPA光学生物传感探针,建立了一种快速、简单、有效的TPA定量分析方法。考察了反应时间、反应温度及工作溶液pH值等多种因素的影响。在优化条件下,探针吸光度的减少值与TPA的浓度在0.4-200ng/mL范围内近似呈线性关系,线性回归方程为-△A=0.02767C+0.00632,线性相关系数R=0.99271,检测限约为0.108ng/mL。把方法应用于真实血清样品的测定,检测结果与真实结果基本一致,相对误差在-4.0～8.7%范围内。
用3-氨基丙基三甲氧基硅烷作粘结剂,在金纳米粒单层膜基础上继续交替铺上氧化硅纳米粒、金纳米粒,成功制得了金纳米粒/氧化硅纳米粒交替排列的多层膜芯片,并对其进行了紫外可见吸收光谱、SEM等系列表征。实验结果表明,随金纳米粒层层数的增加,芯片在540nm处的吸收峰增高,而吸收峰的峰位、峰形基本保持不变。金纳米粒/氧化硅纳米粒多层复合膜的扫描电镜图显示,制备的多层膜芯片表面金纳米粒层金纳米粒的大小为20nm左右,分布比较均匀、规整,为进一步的应用研究奠定了坚实的基础。用anti-TPA对制得的多层复合膜进行修饰、用BSA封住余留的活性点,由此所制得的传感芯片对TPA分子有特异性识别响应,TPA分子通过对anti-TPA分子免疫反应使传感芯片在最大吸收波长处的吸光度值减小。实验结果显示,吸光度的减少值与TPA的浓度在1-10000ng/L范围内近似呈线性关系,线性回归方程为-△A=0.05266C+0.00758,线性相关系数R=0.99734,检测限约为0.027ng/L。把制得的TPA传感芯片应用于血清真实样品的检测,结果表明,3个血清样品的测试结果和当地医院测得值基本一致,标准偏差在允许范围之内。
Preparation, chemical functionalization and assembly of Au nanoparticles, silicon oxide nanomaterial and the Au/SiO2composite nanomaterials, and their applications are one of the most important subjects in the fields of chemistry, molecular biology, medicine and so on. The technologies of preparing and assembling Au, SiO2nanopartices, and their applications are reviewed in this dissertation. The main detail is as follows:
Au@SiO2core/shell nanoparticles were synthesized by using (3-mercaptopropyl)trimethoxysilane (MPTMOS) as cross-linker instead of (3-amino-propyl)trimethoxysilane. The nanoparticles were characterized by UV-visible spectroscopy, TEM and fluorescence microscope. Our results showed the nanocrystals were uniformly spherical and high monodisperse in water. Single gold nanoparticle was located at the center of silicon oxide shell. Moreover, no obvious change of fluorescence intensity of Au/SiO2composite nano-materials was observed after it was excited for3600seconds by successive intense irradiation, which demonstrates that these nanoparticles had excellent photostability. In addition, Au@SiO2core/shell nanoparticles were covalently conjugated with the TPA using (3-aminopropyl)trimethoxysilane and glutaraldehyde as the crosslinker and successfully completed the fluorescence imaging of nasopharyngeal carcinoma cells CNE-1.
Based on the principle that the-SH could be oxidated to form a disulfide bond, we had synthesized the gold-nanoparticle-modified silica nanowires in solution by employing3-mercapto-propyltrimethoxysilane as the cross-linker under mild conditions. The nanowires were characterized by UV-visible spectroscopy, TEM and fluorescence microscope. The results demonstrate that the silica nanowires of various thicknesses that the gold nanoparticles were embedded in and/or attached on SiNWs were prepared. The method is simple, low cost, safe and feasible, so it is helpful for meeting the social great demand by synthesizing the SiNWs in large scale. Gold-nanoparticles were loaded on SiNWs by chemical bond that it showed the good stability and excellent catalytic activity. The Catalytic reduction of reaction of p-nitrophenol follows the first-order kinetic process when NaBH4is excessive, the reaction rate constant k=0.85min-1. Moreover, the composite nanoparticles were easy to covalently conjugate with the target molecule (i.e. biological molecule), and it made them become ideal candidates for use in chemical sensor and biosensor.
The monolayer membrane of gold nanoparticles was controllably prepared on the glass chip using optical glass as substrate and MPTMOS as agglomerant. Then, anti-TAP and BSA were used to functionalize this monolayer membrane of gold nanoparticles to prepare the optical immune sensing chip that could specifically response to TPA. This chip can directly detect the TPA in complex sample using ultraviolet visible spectrophotometer with good stabilization and selectivity. In addition, because this technique need only measure the absorbance change in the largest ultraviolet absorption wavelength of TPA to detect the content of TPA in samples, this method can be easily popularized for this kind of simple and fast format. Under the optimized conditions, the decreased absorbency (AA) was linear with TPA concentration (C) in the range of1～1000ng/L and the low detection limit was1.2×10-3ng/L. The results of determination for TPA in three synthetic samples were identical with the true values, the recovery (98.9%-102.4%) and the standard deviation (1.6～2.8%) was satisfactory. Moreover, the method was applied to the determination of the content of TPA in human serum samples. Compared to the serum of healthy person, the response value was improved obviously in cancer patients. The concept demonstrated that the application of Immunosensors based on the UV-Vis absorption of gold-nanoparticles to quantitative detecting the antigen was feasible.
Liquid TPA optical bio-sensing probe was constructed using the anti-TPA, BSA and PEG functionalized gold nanoparticles. This method provides a fast, simple and efficient character for quantitative analysis of TPA. In this work, a series of factors (i.e. reaction time, reaction temperature and pH) were investigated to find the better determination condition. Under the optimized conditions, a linear relationship of the decreased absorbency (△A) and TPA concentration (C) was established in the range of0.4-200ng/mL using the equation-△A=0.02767C+0.00632(R=0.99271) and the low detection limit was0.108ng/mL. The results of the determination for TPA in serum real samples were identical with the true value, and the relatively error is at-4.0～8.7%.
By using the (3-aminopropyl)trimethoxysilane as cross-linker, the multilayer chip was formed by alternating Au-nanoparticles and SiO2-nanoparticles on nano-Au single-layer. The multilayer chip was characterized by UV-visible spectroscopy, SEM and so on. The absorbing intensity at540nm of chip increased with enhancement of layer of Au-nanoparticles, but the position and shap of the absorption peak was nearly invariant. The TEM image of the multilayer film proved this method yielded the Au-nanopartices on the multilayer chip surface with20nm in diameter and uniform. The successful preparation of multilayer chip paved a way for further study and application. When the multilayer chip was modified by anti-TPA and the remaining active sites of multilayer chip were blocked by BSA, the as-prepared sensoring chip can specifically recognize TPA molecular and the absorbing intensity of sensoring chip at the maximum absorption wavelengths was decreased because of immune reaction between TPA and anti-TPA. The results showed a linear relationship of the decreased absorbency (△A) and TPA concentration (C) was established in the range of1～1000ng/L using the equation-△A=0.05266C+0.00758(R=0.99734) and the low detection limit was0.027ng/L. The results of the determination for TPA in three serum real samples were identical with the conventional method of hospital, and the standard deviation was satisfactory.
引文
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