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免疫球蛋白G的免疫纳米金催化共振散射光谱分析
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摘要
第一部分:绪论
     介绍了共振散射技术发展历史、分析应用以及发展前景;综述了金纳米微粒的制备、表征、以及胶体金标记技术在生化分析中的应用;介绍了免疫球蛋白G的分析进展。
     第二部分:氯金酸-羟胺-纳米金催化体系的共振散射光谱研究
     在柠檬酸钠-盐酸缓冲溶液中,金、银、铂、钯、四氧化三铁纳米粒子对氯金酸-盐酸羟胺生成金颗粒反应均具有催化作用。反应生成的大粒径金颗粒在796 nm处产生一个较强的共振散射峰。本文以共振散射光谱法等作为检测手段,详细研究了各种因素对纳米金催化反应的影响。随着催化剂纳米金浓度的增大,796 nm处的共振散射光强度线性增大。粒径为5 nm、10 nm、15 nm、30 nm、50 nm纳米金催化体系的线性范围分别为0.25~10.15 nM Au、1.27~50.76 nM Au、1.27~44.16 nM Au、1.83~50.76 nM Au、5.08~265.18 nM Au,其检出限分别为0.25 nM、0.99 nM、1.18 nM、1.19 nM、4.15 nM。据此建立了一个测定超痕量纳米金的催化共振散射光谱新方法,该法与纳米金标免疫反应结合将为超痕量免疫球蛋白、半抗原的分析提供了一种新技术。
     第三部分:免疫纳米金催化金增强共振散射光谱法检测超痕量免疫球蛋白G
     用粒径为10 nm的金纳米粒子标记羊抗人IgG抗体获得纳米金标记羊抗人IgG抗体(AuGIgG)。在pH 2.27的柠檬酸钠-盐酸缓冲溶液中,AuGIgG对氯金酸-盐酸羟胺生成较大粒径金颗粒这一慢反应具有较强的催化作用,该较大粒径金颗粒在796 nm处有一个较强的共振散射峰。在一定条件下,AuGIgG与IgG发生特异性结合生成纳米金免疫复合物,以16000 rpm速度离心分离获得未反应的AuGIgG,以它作催化剂催化氯金酸-盐酸羟胺反应生成较大粒径金颗粒,用共振散射光谱做检测技术,建立了测定IgG的免疫共振散射光谱新方法。结果表明,随着IgG浓度增大,离心溶液中AuGIgG浓度降低,I796nm线性降低,其降低值△I796nm与IgG浓度在0.08~16.0 ng·mL~(-1)范围内呈良好线性关系,检出限为0.02 ng·mL~(-1)。本法具有灵敏、快速和较高的特异性,用于定量分析人血清IgG,结果满意。
     第四部分:免疫纳米金催化铜增强共振散射光谱新方法及其生化分析应用在pH 4.2醋酸钠-醋酸缓冲溶液中,金、银、铂、钯、四氧化三铁、氧化亚铜纳米微粒对抗坏血酸还原硫酸铜生成铜颗粒反应均具有催化作用。反应生成的大粒径金-铜颗粒在610nm处产生一个较强的共振散射峰,发现纳米金的催化效果最好。本文以共振散射光谱法等作为检测手段,详细研究了各种因素对纳米金催化反应的影响。随着催化剂纳米金浓度的增大,610nm处的共振散射光强度线性增大。粒径为5 nm、10 nm、15 nm、纳米金催化体系的线性范围分别为0.02~1.63 nM Au、0.04~1.22 nM Au、0.12~4.71 nM Au,其检出限分别为0.013 nM、0.034 nM、0.100 nM。据此建立了一个测定超痕量纳米金的催化共振散射光谱新方法,该法与纳米金标免疫反应结合将为超痕量免疫球蛋白、半抗原的分析提供了一种新技术,利用此方法检测人IgG的线性范围为0.03~7.5 ng/mL,其检出限为0.015 ng/mL。
PartⅠIntroduction
     Basic knowledge of light scattering, resonance scattering and its application in analytical chemistry and nanoparticles were introduced. Summarized the preparation, identification and assembly of gold nanoparticles and its application in biochemical analysis in recent years. The analytical progress of IgG was also reviewed.
     PartⅡResonance Scattering Spectral Study of HAuCl4-NH2OH-nanogold catalytic system
     Au, Ag, Pt, Pd, Fe3O4 nanoparticles have catalytic effect on the particle reaction of HAuCl4-NH2OH to produce gold particles in big size, that exhibit strongest resonance scattering (RS) peak at 796 nm. The enhanced resonance scattering intensity at 796 nm is linear to nanogold concentration in the range of 0.25~10.15 nM, 1.27~50.76 nM, 1.27~44.16 nM, 1.83~50.76 nM and 5.08~265.18 nM nanogold in size of 5, 10, 15, 30 and 50 nm respectively, with a detection limits of 0.24 nM, 0.99 nM, 1.18 nM, 1.19 nM and 4.15 nM. Based on this, a new RS method was established to determine ultratrace nanogold.
     PartⅢA detection of ultratrace IgG using immunonanogold catalytic Gold-enhanced Resonance scattering spectral
     A 10 nm-nanogold was used to label goat anti-human IgG (GIgG) to obtain nanogold-labeled GIgG(AuGIgG). In pH 2.27 citrate-HCl buffer solution, AuGIgG showed strong catalytic effect on the reaction between HAuCl_4-NH_2OH to form big gold particles that exhibited a resonance scattering peak at 796 nm. Under the chosen conditions, AuGIgG combined with IgG to form immunocomplex of AuGIgG-IgG that can be separated by centrifuging at 16000 rpm. AuGIgG in centrifuging solution has also catalytic effect on the reaction. On those grounds, an immunonanogold catalytic resonance scattering assay for IgG was proposed. With addition of IgG, the AuGIgG in centrifuging solution decreased, the resonance scattering intensity at 796 nm I796nm decreased linearly. The decreased intensity△I796nm was linear to IgG concentration in the range of 0.08~16.0 ng.mL~(-1), with a detection limit of 0.02 ng.mL~(-1). This assay was applied to analysis of IgG in sera, with sensitivity, selectivity and rapidity.
     PartⅣA Novel and Highly Sensitive Immunonanogold Catalytic Copper-enhanced Resonance Scattering Spectral Bioassay
     In the NaAc-HAc buffer solution, Au, Ag, Pt, Pd, Fe3O4, and Cu2O nanoparticles have catalytic enhancement effect on the reduction of Cu2+ by ascorbic acid (AA) to form large size copper particles that exhibit a strong resonance scattering peak at 610nm. The new nanocatalytic reaction was studied firstly by resonance scattering (RS) spectral technique. Results showed that nanogold exhibited strongest catalytic enhancing effect. The RS intensity at 610nm I610 nm increased linearly with the concentration of 0.02~1.63 nM, 0.041~1.22 nM and 0.118~4.71 nM nanogold in size of 5 nm, 10 nm and 15 nm, with a detection limit of 0.013nM, 0.034nM and 0.10 nM, respectively. A new strategy of immunonanogold catalytic RS method was set up, combined the catalytic effect of immunonanogold on the reaction between CuSO4 and AA with the RS detection technique. As a model, 0.03 ng.mL~(-1) to 7.5 ng.mL~(-1) IgG can be assayed by this immunonanogold catalytic RS method, with a detection limit of 0.015 ng.mL~(-1).
引文
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