银纳米粒子的局域表面等离子体共振散射在生化药物分析中的应用研究
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摘要
纳米技术已经在各个领域中发挥着独特的优势和潜力。将纳米技术与光散射技术相结合,开创光散射分析的新天地,是近年来光散射分析化学的发展方向和趋势。本文以银纳米粒子为研究对象,探讨了不同大小、形态的银纳米粒子的局域表面等离子体共振散射和吸收性质,并将银纳米粒子作为光散射探针用于生化和药物分析,建立了一系列分析方法。研究论文的主要内容概括如下:
1.合成了柠檬酸根包被的小粒径银纳米粒子,基于银纳米粒子在溶液中独特的局域表面等离子体共振吸收和散射特性,建立了一种简单、方便、快捷的半定量和定量检测盐酸黄连素的分析方法。银纳米粒子因表面包被有柠檬酸根,而处于静电排斥作用下,能均匀、稳定分散在溶液中,其独特的局域表面等离子体共振吸收性质使银胶溶液显黄色。在一定的pH环境中,当加入带正电的盐酸黄连素之后,由于破坏了银纳米之间的静电排斥作用,银粒子发生聚集,影响了银纳米粒子的局域表面等离子体共振,导致银胶的颜色发生从黄色到蓝色的变化。本文通过紫外可见吸收、光散射光谱等方法,研究了盐酸黄连素诱导下银纳米粒子的聚集过程,并且在优化的实验条件下,实现了浓度范围在0.05～0.4μM之间的盐酸黄连素的色度法检测以及紫外可见吸收定量检测。其中,根据银胶颜色变化而建立的色度检测方法是一种简单、不需要大型仪器、经济、快速的分析方法。
2.合成了具有强烈的光散射性质的大粒径银纳米粒子。利用银纳米粒子在不同蛋白质上吸附性质不同,建立了一种通过普通LED电筒和载玻片就可以进行可视化的、免标记的免疫分析方法。探讨了银纳米粒子在抗原、抗体固载的玻片上吸附的机理,并优化了银纳米粒子在玻片上的吸附条件,实现了银纳米粒子在抗体结合后的玻片上的选择性吸附。通过普通的荧光分光光度计测定光散射信号,实现了在10～160ng/ml浓度范围抗体的定量检测,检测下限达到5.6ng/ml。此外,在普通的白光LED电筒的照射下,吸附在玻片表面的银纳米粒子,由于具有强烈的局域表面等离子体共振散射性质,可以被肉眼观察到。因此,实现了使用银纳米粒子作为散射可视化探针的固相表面免疫分析法。
3.基于大粒径的银纳米粒子强烈的局域表面等离子体共振散射性质,我们提出了一种以玻片为固载相、银纳米粒子作为光散射探针、使用荧光分光光度计检测光散射信号的三明治免疫分析方法。该方法可以与高灵敏度的化学发光法媲美,且分析结果与临床检验结果相符合。此外,通过暗场光学显微镜,可以清楚的看到单个银纳米粒子的多色光散射现象,并结合显微光谱技术对单个银纳米粒子的光散射光谱进行了表征,这预示着银纳米粒子强烈的光散射特性可能成为一种新兴的光散射探针而应用于生化分析和标记中,为建立多色银纳米粒子标记的多通道分析方法打下了基础。
4.通过碘对银纳米粒子的腐蚀,研究了银纳米粒子的组成和形态发生变化后的光散射特性。研究表明,碘离子可以在银纳米粒子表面吸附,导致银纳米粒子局域表面等离子体共振散射和吸收降低;当溶液中加入氯化铜,使碘离子氧化成碘单质后,碘能与银纳米粒子发生明显的化学反应,导致银纳米粒子的局域表面等离子体共振散射和吸收峰完全消失,并生成新的散射峰。进一步通过扫描电子显微成像和暗场显微成像研究表明,银纳米粒子被碘腐蚀后生成了更大粒径的颗粒,并且具有与银纳米粒子明显不同的光散射特性。基于以上原理,建立了在普通荧光分光光度计的定量检测碘的光散射分析方法和可视化的检测手段。这说明碘与银纳米粒子作用后,所产生的强烈的光散射信号,将在生化分析和检测中有潜在的应用前景。
上述研究内容,探讨了不同大小、形状的银纳米粒子的局域表面等离子体共振散射及吸收性质以及药物、生物大分子与银纳米粒子之间的相互作用,建立了一系列基于银纳米粒子的光散射特性可视化分析方法。这将为银纳米粒子在光散射分析中的应用找到新途径。
The prosperities of nanoscience and nanotechnology have promoted the development of science in all fields. Light scattering technology in analytical chemistry supply much more opportunities and challenges in this evolution for analysts. Combining nanoscience in light scattering detection and establishing light scattering analytical methods based on nanotechnology would have new ways in analytical chemistry. In this thesis, silver nanoparticles (Ag-Nps), which have unique localized surface plasmon resonance scattering properties, have been investigated by interacting with drugs and biomolecules. Thus, new analytical methods based on the scattering of Ag-Nps have been established. The mainly points are as follows:
1. Citrate-capped silver nanopartilces with small size were synthesized, and a visual colorimetric method for the detection of berberine hydrochloride was proposed based on the color change caused by the aggregation of Ag-Nps. It was found that citrate-capped AgNps dispersed in water owing to the electrostatic repulsion from each other by the negative charged surface, presenting a bright yellow color. However, the presence of positively charged berberine could induce the aggregation of citrate-capped AgNps, resulting in color change from yellow to green, and even to blue depending on the concentration of berberine. The mechanism of color change and the effect of experiment condition were studied with UV-Vis absorption and light scattering spectrometry. Under the optimum condition, we can detect the berberine hydrochloride from 0.05μM to 0.4μM visually based on the color changes of the solution. It was identified that this colorimetric analytical method without use of expensive machines is very convenient, economy and speedy.
2. We synthesized larger Ag-Nps, which have strong scattering properties considering that Ag-Nps have strong localized surface plasmon resonance scattering signals. A novel, label-free visual immunoassay method, based on the PRS signals of the Ag-NP electrostatic adsorbed on glass slides, on which antibody is bound, has been established to distinguish the immunoreactions on glass slides with a common LED torch. We discussed the mechanism of this method and investigated the effect of experimental conditions with the scattering signals of Ag- NPs measured on a common spectrofluorometer. Under optimal conditions, antibody over the range between 10 and 160 ng/mL with LOD of 5.6 ng/mL could be detected quantitatively with spectrofluorometer. If a white light-emitting diode (LED) torch is employed to illuminate the glass slides, we can make visual detection of the antibody by the naked eye, owing to the strong localized surface plasmon resonance signals scattered from the Ag-Nps.
3. We propose a localized surface plasmon resonance scattering immunoassay with common glass slides as a solid substrate by introducing Ag-Nps as scattering labels. The light scatting signals of silver nanoparticles could be measured with a common spectrofluorometer for clinical purposes. The quantitative study using human IgG as an antigen showed that the present immunoassay could have comparable high sensitivity with the new reported chemiluminescence immunoassays. On the other hand, the dark-field light scattering microscopic images showed that single silver nanoparticles can be clearly seen on the basis of its strong scattering light, indicating that silver nanoparticles as a light scattering probe may become a novel model in bioassay. Moreover, the scattering light from the AgNPs has different colors depending on the sizes and shapes, which has potential application in multiplexed assay using nanoparticles with different scattering colors. The localized surface plasmon resonance scattering features of a single AgNP, on the other hand, deserve further investigation and perhaps have potential applications in analytical chemistry. In addition, the visual immunoassay system could be constructed and easily observed by naked eyes with a common LED touch, supplying a new way for visual detection of immunoreactions on the basis of the light scattering signals.
4. The further investigation is on the localized surface plasmon resonance scattering features of Ag-Nps etched by iodine. We found that iodine ions can be adsorbed on the Ag-Nps surface in the presence of iodine ions in colloidal Ag-Nps suspension, and induce the localized surface plasmon resonance absorption and scattering quenching of Ag-NPs. However, if both iodine ions and copper ions were presented, iodine ions can be oxidized to iodine which can etch Ag-Nps causing the disappearance of localized surface plasmon resonance scattering. The features of Ag-Nps immobilized on glass slides before and after the interaction of iodine were further investigated with scanning electron microscopy and dark-field light scattering microscope. It was found that, the size of Ag-Nps gets enlarged and the surface of Ag-Nps gets roughness after the etching of iodine. The light scattered from the Ag-Nps after etching changed from blue light to white light. An analytical method for iodine thus can be established using a common spectrofluorometer based on the light scattering change of Ag-Nps etched by iodine. The strong scattering light induced from the etching of Ag-Nps by iodine will have potential application in biochemical analysis.
In conclusion, localized surface plasmon resonance absorption and scattering features of Ag-Nps with different sizes were investigated in this thesis. The interactions between Ag-NPs and drugs/biomolecule were studied using light scattering spectral and microscopy methods. A series novel analytical methods based on the localized surface plasmon resonance scattering of Ag-Nps were established to detect drugs and biomolecules, and it is obviously that light scattering method on silver nanoparticles shows high promise in analytical chemistry.
1.合成了柠檬酸根包被的小粒径银纳米粒子,基于银纳米粒子在溶液中独特的局域表面等离子体共振吸收和散射特性,建立了一种简单、方便、快捷的半定量和定量检测盐酸黄连素的分析方法。银纳米粒子因表面包被有柠檬酸根,而处于静电排斥作用下,能均匀、稳定分散在溶液中,其独特的局域表面等离子体共振吸收性质使银胶溶液显黄色。在一定的pH环境中,当加入带正电的盐酸黄连素之后,由于破坏了银纳米之间的静电排斥作用,银粒子发生聚集,影响了银纳米粒子的局域表面等离子体共振,导致银胶的颜色发生从黄色到蓝色的变化。本文通过紫外可见吸收、光散射光谱等方法,研究了盐酸黄连素诱导下银纳米粒子的聚集过程,并且在优化的实验条件下,实现了浓度范围在0.05～0.4μM之间的盐酸黄连素的色度法检测以及紫外可见吸收定量检测。其中,根据银胶颜色变化而建立的色度检测方法是一种简单、不需要大型仪器、经济、快速的分析方法。
2.合成了具有强烈的光散射性质的大粒径银纳米粒子。利用银纳米粒子在不同蛋白质上吸附性质不同,建立了一种通过普通LED电筒和载玻片就可以进行可视化的、免标记的免疫分析方法。探讨了银纳米粒子在抗原、抗体固载的玻片上吸附的机理,并优化了银纳米粒子在玻片上的吸附条件,实现了银纳米粒子在抗体结合后的玻片上的选择性吸附。通过普通的荧光分光光度计测定光散射信号,实现了在10～160ng/ml浓度范围抗体的定量检测,检测下限达到5.6ng/ml。此外,在普通的白光LED电筒的照射下,吸附在玻片表面的银纳米粒子,由于具有强烈的局域表面等离子体共振散射性质,可以被肉眼观察到。因此,实现了使用银纳米粒子作为散射可视化探针的固相表面免疫分析法。
3.基于大粒径的银纳米粒子强烈的局域表面等离子体共振散射性质,我们提出了一种以玻片为固载相、银纳米粒子作为光散射探针、使用荧光分光光度计检测光散射信号的三明治免疫分析方法。该方法可以与高灵敏度的化学发光法媲美,且分析结果与临床检验结果相符合。此外,通过暗场光学显微镜,可以清楚的看到单个银纳米粒子的多色光散射现象,并结合显微光谱技术对单个银纳米粒子的光散射光谱进行了表征,这预示着银纳米粒子强烈的光散射特性可能成为一种新兴的光散射探针而应用于生化分析和标记中,为建立多色银纳米粒子标记的多通道分析方法打下了基础。
4.通过碘对银纳米粒子的腐蚀,研究了银纳米粒子的组成和形态发生变化后的光散射特性。研究表明,碘离子可以在银纳米粒子表面吸附,导致银纳米粒子局域表面等离子体共振散射和吸收降低;当溶液中加入氯化铜,使碘离子氧化成碘单质后,碘能与银纳米粒子发生明显的化学反应,导致银纳米粒子的局域表面等离子体共振散射和吸收峰完全消失,并生成新的散射峰。进一步通过扫描电子显微成像和暗场显微成像研究表明,银纳米粒子被碘腐蚀后生成了更大粒径的颗粒,并且具有与银纳米粒子明显不同的光散射特性。基于以上原理,建立了在普通荧光分光光度计的定量检测碘的光散射分析方法和可视化的检测手段。这说明碘与银纳米粒子作用后,所产生的强烈的光散射信号,将在生化分析和检测中有潜在的应用前景。
上述研究内容,探讨了不同大小、形状的银纳米粒子的局域表面等离子体共振散射及吸收性质以及药物、生物大分子与银纳米粒子之间的相互作用,建立了一系列基于银纳米粒子的光散射特性可视化分析方法。这将为银纳米粒子在光散射分析中的应用找到新途径。
The prosperities of nanoscience and nanotechnology have promoted the development of science in all fields. Light scattering technology in analytical chemistry supply much more opportunities and challenges in this evolution for analysts. Combining nanoscience in light scattering detection and establishing light scattering analytical methods based on nanotechnology would have new ways in analytical chemistry. In this thesis, silver nanoparticles (Ag-Nps), which have unique localized surface plasmon resonance scattering properties, have been investigated by interacting with drugs and biomolecules. Thus, new analytical methods based on the scattering of Ag-Nps have been established. The mainly points are as follows:
1. Citrate-capped silver nanopartilces with small size were synthesized, and a visual colorimetric method for the detection of berberine hydrochloride was proposed based on the color change caused by the aggregation of Ag-Nps. It was found that citrate-capped AgNps dispersed in water owing to the electrostatic repulsion from each other by the negative charged surface, presenting a bright yellow color. However, the presence of positively charged berberine could induce the aggregation of citrate-capped AgNps, resulting in color change from yellow to green, and even to blue depending on the concentration of berberine. The mechanism of color change and the effect of experiment condition were studied with UV-Vis absorption and light scattering spectrometry. Under the optimum condition, we can detect the berberine hydrochloride from 0.05μM to 0.4μM visually based on the color changes of the solution. It was identified that this colorimetric analytical method without use of expensive machines is very convenient, economy and speedy.
2. We synthesized larger Ag-Nps, which have strong scattering properties considering that Ag-Nps have strong localized surface plasmon resonance scattering signals. A novel, label-free visual immunoassay method, based on the PRS signals of the Ag-NP electrostatic adsorbed on glass slides, on which antibody is bound, has been established to distinguish the immunoreactions on glass slides with a common LED torch. We discussed the mechanism of this method and investigated the effect of experimental conditions with the scattering signals of Ag- NPs measured on a common spectrofluorometer. Under optimal conditions, antibody over the range between 10 and 160 ng/mL with LOD of 5.6 ng/mL could be detected quantitatively with spectrofluorometer. If a white light-emitting diode (LED) torch is employed to illuminate the glass slides, we can make visual detection of the antibody by the naked eye, owing to the strong localized surface plasmon resonance signals scattered from the Ag-Nps.
3. We propose a localized surface plasmon resonance scattering immunoassay with common glass slides as a solid substrate by introducing Ag-Nps as scattering labels. The light scatting signals of silver nanoparticles could be measured with a common spectrofluorometer for clinical purposes. The quantitative study using human IgG as an antigen showed that the present immunoassay could have comparable high sensitivity with the new reported chemiluminescence immunoassays. On the other hand, the dark-field light scattering microscopic images showed that single silver nanoparticles can be clearly seen on the basis of its strong scattering light, indicating that silver nanoparticles as a light scattering probe may become a novel model in bioassay. Moreover, the scattering light from the AgNPs has different colors depending on the sizes and shapes, which has potential application in multiplexed assay using nanoparticles with different scattering colors. The localized surface plasmon resonance scattering features of a single AgNP, on the other hand, deserve further investigation and perhaps have potential applications in analytical chemistry. In addition, the visual immunoassay system could be constructed and easily observed by naked eyes with a common LED touch, supplying a new way for visual detection of immunoreactions on the basis of the light scattering signals.
4. The further investigation is on the localized surface plasmon resonance scattering features of Ag-Nps etched by iodine. We found that iodine ions can be adsorbed on the Ag-Nps surface in the presence of iodine ions in colloidal Ag-Nps suspension, and induce the localized surface plasmon resonance absorption and scattering quenching of Ag-NPs. However, if both iodine ions and copper ions were presented, iodine ions can be oxidized to iodine which can etch Ag-Nps causing the disappearance of localized surface plasmon resonance scattering. The features of Ag-Nps immobilized on glass slides before and after the interaction of iodine were further investigated with scanning electron microscopy and dark-field light scattering microscope. It was found that, the size of Ag-Nps gets enlarged and the surface of Ag-Nps gets roughness after the etching of iodine. The light scattered from the Ag-Nps after etching changed from blue light to white light. An analytical method for iodine thus can be established using a common spectrofluorometer based on the light scattering change of Ag-Nps etched by iodine. The strong scattering light induced from the etching of Ag-Nps by iodine will have potential application in biochemical analysis.
In conclusion, localized surface plasmon resonance absorption and scattering features of Ag-Nps with different sizes were investigated in this thesis. The interactions between Ag-NPs and drugs/biomolecule were studied using light scattering spectral and microscopy methods. A series novel analytical methods based on the localized surface plasmon resonance scattering of Ag-Nps were established to detect drugs and biomolecules, and it is obviously that light scattering method on silver nanoparticles shows high promise in analytical chemistry.
引文
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