双偏振光干涉分析仪联用原子力显微镜分析蛋白质在固-液界面的吸附特性
文献类型：学位论文
| 作者 | 杜晓南 |
| 学位类别 | 硕士 |
| 答辩日期 | 2011-06-10 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 苏志国 |
| 关键词 | 双偏振光干涉分析仪 原子力显微镜 人血清白蛋白 血红蛋白 乙肝表面抗原 |
| 其他题名 | Characteristic Analysis of Protein Absorbed onto Solid-liquid Surface by Dual Polarization Interferometry (DPI) and Atomic Force Microscope (AFM) |
| 学位专业 | 生物化工 |
| 中文摘要 | 研究蛋白与界面的相互作用，是蛋白质纯化与复性、酶固定、蛋白药物剂型化等领域的重要内容，本论文选择双偏振光干涉分析仪（Dual Polarization Interferometry，DPI）和原子力显微镜（Atomic Force Microscope，AFM）作为研究固-液界面的主要方法，通过模拟层析介质的表面环境，研究蛋白质在固-液界面上的吸附行为与吸附形态。本论文通过表征人血清白蛋白（Human Serum Albumin，HSA）、血红蛋白和乙肝表面抗原（HBsAg）的固-液界面吸附行为，初步探究了不同界面性质带来的界面吸附差异，开展的研究工作如下： 1、选择结构清楚的单体蛋白HSA作为模型蛋白，初步探索蛋白质的界面行为。DPI结果表明，HSA在硅羟基表面及琼脂糖表面的吸附量分别为2.73ng/mm2、0.48ng/mm2，说明亲水性化合物减少了蛋白质的非特异性吸附。此外还考察了HSA在硅羟基表面和DEAE琼脂糖表面的高盐洗脱行为，结果显示，高盐对表面吸附的HSA洗脱效果明显。 2、血红蛋白为四亚基蛋白，DPI和AFM联用可以实现对同一界面上蛋白吸附量和吸附状态的检测。DPI结果表明，当溶液环境中血红蛋白的浓度为1.08mg/mL时蛋白在硅羟基表面的吸附厚度为3.54nm，AFM检测硅羟基表面的吸附血红蛋白直径在60-150nm之间，远高于血红蛋白的分子尺寸，说明在界面微环境的影响下，分子间出现了团聚。 3、联用DPI和AFM进一步分析了超大分子HBsAg的界面吸附行为和结构变化规律。DPI检测结果显示，0.67mg/mL的HBsAg在硅羟基表面及DEAE琼脂糖表面的吸附厚度分别为1.04nm、23.69nm。AFM扫描显示硅羟基表面的蛋白颗粒直径在288-401nm之间，DEAE琼脂糖表面的蛋白直径在520-971nm之间，说明HBsAg在表面形成大的团聚体，而DEAE琼脂糖表面的团聚现象更严重。此外还研究了表面的DEAE配基密度对吸附HBsAg的影响，DPI结果表明，随着配基密度的增加，HBsAg吸附量增加，AFM固相扫描结果也显示，随着配基密度的增加，HBsAg在表面的厚度和直径增加。 |
| 英文摘要 | The study of protein interfacial behavior is involved in a wide range of scientific endeavors, including protein purification, enzyme immobilization and protein formulation, etc. In this research, dual polarization interferometry (DPI) and atomic force microscope (AFM), novel methods to study solid-liquid surface, were employed for better understanding of chromatography phenomenon by reconstructing the simulant solid-liquid interface microenvironment. The adsorption behaviors of human serum albumin (HSA), hemoglobin and hepatitis B surface antigen onto solid-liquid surface were investigated. The following results were obtained: 1. The surfacial behavior of HSA, a model protein with clear structure, was investigated. The protein adsorption process as well as adsorbed mass, thickness and density on a SiN chip surface were monitored by DPI. Deposition of HSA on Si-OH and agarose surface reached the layer mass of 2.73ng/mm2 and 0.48ng/mm2, indicating that hydrophilic modification can reduce protein adsorption. In addition, high-salt elution turned out to be very effective since the amount of absorbed HSA on both Si-OH and DEAE agarose surface significantly decreased. 2. Hemoglobin (Hb) was attached to unmodified DPI sensor chip with an average thickness value of 3.54nm at a concentration of 1.08mg/mL, and then the chip surface was further characterized by AFM under the same condition. Aggregated Hb particles with a diameter of 60nm to 150nm were detected by AFM scanning, far larger than its molecular size. 3. DPI and AFM were employed to detect the surfacial behavior of gigantic molecular-HBsAg. The thickness of adsorbed HBsAg at a concentration of 0.67mg/mL was 1.04nm and 23.69nm on unmodified and DEAE agarose surface, respectively. Correspondingly, the diameter of absorbed HBsAg on unmodified and DEAE agarose surface were 288-401nm and 520-971nm, indicating aggregation of HBsAg into large particles. Ligand density of DEAE agarose had marked impact on absorbed HBsAg. The higher ligand density of DEAE agarose, the more HBsAg absorbed onto DEAE agarose surface. |
| 语种 | 中文 |
| 公开日期 | 2013-09-24 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1762]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 杜晓南. 双偏振光干涉分析仪联用原子力显微镜分析蛋白质在固-液界面的吸附特性[D]. 中国科学院研究生院. 2011. |
入库方式： OAI收割
来源：过程工程研究所
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