【作者】 邹明静；

【导师】 蒋治良；

【作者基本信息】 广西师范大学 ， 分析化学， 2007， 硕士

【摘要】 第一部分:绪论综述了胶体金的制备、表征以及胶体金标记技术在生化分析中的应用。综述了近年来共振散射技术在分析化学中的应用。介绍了免疫球蛋白M与人绒毛膜促性腺激素的分析进展。第二部分:痕量免疫球蛋白M的金标记免疫共振散射光谱分析采用柠檬酸钠改良法制备了粒径为8 nm的金纳米微粒,并用于标记羊抗人免疫球蛋白M(IgM)。在适宜pH的KH2PO4-Na2HPO4缓冲溶液中,金标记羊抗人IgM与IgM发生特异性结合,导致体系在580 nm处的共振散射峰增强,cIgM在0.0015~2.00μg/mL范围内与共振散射强度的增强值ΔI580nm成线性关系。其回归方程为ΔI580nm=39.91c+1.95 (n =7, R=0.9989),检出限(3σ)为0.98 ng/mL。该方法简单、灵敏且选择性较好,用于定量分析人血清中的免疫球蛋白M,结果满意。第三部分:金银复合纳米微粒的共振散射效应及其在IgM免疫分析中的应用采用柠檬酸钠改良法制备粒径为8 nm的金纳米微粒标记羊抗人免疫球蛋白M(IgM)。在pH4.49的KH2PO4-Na2HPO4缓冲溶液中,金标羊抗人IgM与IgM发生特异性结合生成免疫金复合物,离心分离。取适量含金标羊抗人IgM的上层清液,在pH3.4柠檬酸-柠檬酸钠缓冲溶液中加入银增强试剂,生成金银复合纳米微粒,记录350 nm处的共振散射峰强度。在最佳条件下,IgM浓度cIgM在0.13~333.3 ng/mL范围内与350 nm处的共振散射强度降低值ΔI350 nm成良好线性关系,其回归方程为ΔI350 nm=0.1857cIgM + 2.10 (n =7, R=0.9982),检出限(3σ)为0.08 ng/mL。该方法灵敏且选择性较好,用于定量分析人血清中的免疫球蛋白M,结果满意。第四部分:金标记免疫共振散射光谱法检测痕量人绒毛膜促性腺激素采用柠檬酸钠改良法制备了粒径为8~50 nm的金纳米微粒,并用于标记兔抗人绒毛膜促性腺激素多克隆抗体。在适宜pH的柠檬酸-NaH2PO4缓冲溶液中,金标记兔抗hCG与hCG发生特异性结合,导致体系在580 nm处的共振散射峰强度减弱,hCG在一定的浓度范围内与共振散射强度的降低值呈良好的线性关系。本文对粒径为8,10,15 nm的金纳米微粒标记的兔抗hCG与hCG体系的共振散射光谱进行了研究,其线性范围分别为40~10000,40~10000,40~8000 mIU/mL,检出限分别为19.4, 15.1, 13.6 mIU/mL。该方法简便、灵敏且选择性较好,用于测定孕妇尿液中的hCG,结果满意。第五部分:银增强金标记免疫共振散射光谱法检测痕量人绒毛膜促性腺激素用粒径为8 nm的金纳米微粒标记兔抗人绒毛膜促性腺激素(hCG)多克隆抗体。在pH5.0的柠檬酸-Na2HPO4缓冲溶液中,hCG与金标兔抗hCG发生特异性结合生成免疫金复合物,离心分离。取适量含金标兔抗hCG的上层清液,在pH3.4柠檬酸-柠檬酸钠缓冲溶液中加入银增强试剂,所形成的金银复合纳米微粒在423 nm处有一个最强的共振散射峰。hCG浓度chCG在7.5~625.0 mIU/mL范围内与ΔI423 nm成良好的线性关系,其回归方程为ΔI423 nm= 0.2262c+0.17(n =7, R=0.9979),检出限(3σ)为2.7 mIU/mL。该方法灵敏且选择性较好,用于测定孕妇尿液中的hCG,结果与免疫分光光度法一致。更多还原

【Abstract】 PartⅠIntroductionThe preparation and identification of colloid gold and the application of gold-labeled technology in biochemical analysis were summarized. The application of resonance scattering technology in analytical chemistry in resent years was introduced. The analytical progress of immunoglobulin M and human chorionic gonadotrophin was also reviewed.PartⅡImmuno-nanogold Resonance Scattering Spectral Assay of Trance Immunoglobulin MGold nanoparticles of 8 nm, which were prepared by the improved method of trisodium citrate, were used to label goat anti-human immunoglobulin M (anti-IgM). In KH2PO4-Na2HPO4 buffer of suitable pH, the immune reaction between gold-labeled anti-IgM and IgM took place and the intensity of resonance scattering peak at 580 nm enhanced greatly. The intensityΔI580 nm is proportional to the concentration of IgM in the range of 0.0015-2.00μg/mL. The regression equation isΔI580nm= 39.91c+1.95 (n =7, R=0.9989), with the detection limits of 0.98 ng/mL (3σ). Owing to its simplicity, sensitivity and better selectivity, the method has been applied to the determination of IgM in serum of healthy human, with satisfactory results.PartⅢResonance Scattering Effect of Silver-gold Composite Nanoparticles and Its Application to Immunoassay of Trace Immunoglobulin MGold nanoparticles in the size of 8 nm were used to label goat anti-human immunoglobulin M (anti-IgM). In pH4.49 KH2PO4-Na2HPO4 buffer, the gold-labeled anti-IgM interacted with IgM, immunogold complex formed and its upper solution reacted with silver-enhanced reagents after centrifuging. The decreased intensity at 350 nm was recorded. Under the optimum condition, the decreased intensityΔI350nm was proportional to the concentration of IgM in the range of 0.13-333.3 ng/mL. The regression equation wasΔI350 nm=0.1857cIgM +2.10 (n =7, R=0.9982), with the detection limits of 0.08 ng/mL (3σ). The method has been also applied to the determination of IgM in serum of healthy human, with satisfactory results.PartⅣImmuno-nanogold Resonance Scattering Spectral Assay of Trance Human Chorionic GonadotrophinGold nanoparticles of 8-50 nm are used to label the polyclonal antibody of rabbit anti-human chorionic gonadotrophin(anti-hCG) . In citric acid-NaH2PO4 buffer of suitable pH, the immune reaction between gold-labeled rabbit anti-hCG and hCG took place and the intensity of resonance scattering peak at 580 nm decreased. The changed intensityΔI580 nm is proportional to the certain concentration of hCG .To the systems of gold nanoparticles of 8, 10 and 15 nm, the concentration of hCG in the range of 40-10000, 40-10000 and 40-8000 mIU/mL was proportional to the intensity with the detection limits of 19.4, 15.1 and 13.6 mIU/mL respectively. Owing to its simplicity, sensitivity and better selectivity, the method has been applied for the determination of hCG in urine of pregnant women, with satisfactory results.PartⅤA Silver-enhanced nanogold Labeling Immunoresonance Scattering Assay for Trace Human Chorionic GonadotrophinGold nanoparticles of 8 nm was used to label rabbit anti-human chorionic gonadotrophin(hCG). In pH5.0 citric acid-Na2HPO4 buffer, gold-labeled rabbit anti-hCG interacted with hCG, immunogold complex formed and its upper solution after centrifugation reacted with silver-enhanced reagents in pH3.4 citric acid-trisodium citrate buffer, while there was the strongest resonance scattering peak of gold-silver composite nanoparticles at 423 nm. Under the optimum condition, the decreased intensityΔI423nm was proportional to the concentration of hCG in the range of 7.5-625.0 mIU/mL. The regression equation wasΔI423 nm=0.2262c+0.17(n =7, R=0.9979), with the detection limits of 2.7 mIU/mL(3σ). The method has been also applied to the determination of hCG in urine samples of pregnant women, while immunospectrophotometry was used to compare, with satisfactory results.更多还原