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Surface Analysis of Adsorbed Proteins: A Multi-Technique Approach to Characterize Surface Structure.

机译：吸附蛋白的表面分析：表征表面结构的多种技术方法。

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摘要

Adsorbed proteins on surfaces are important in many applications, including medical implants, sensors, marine materials, and in vitro substrates for cell culture and other uses. Understanding the protein structure on the surface would allow better control of the interaction of the material with the surrounding environment and a more reproducible system response. Protein adsorption is a complex process, and characterization requires the combination of multiple analysis techniques. In this thesis, adsorbed protein amounts were measured using x-ray photoelectron spectroscopy (XPS) and radiolabeled proteins. Protein conformation and orientation were measured using time-of-flight secondary ion mass spectrometry (ToF-SIMS), near edge x-ray absorption fine structure (NEXAFS), sum frequency generation (SFG), and enzyme-linked immunosorbent assays (ELISA). Surface type influenced the adsorbed surface concentration of albumin and fibronectin on surfaces of glass, polystyrene, titanium, and sulfonated polystyrene as measured by XPS and radiolabeled protein adsorption. More albumin adsorbed onto polystyrene than glass. More albumin and fibronectin adsorbed onto sulfonated polystyrene than titanium. ToF-SIMS also showed differences in structure of the proteins adsorbed onto the different surfaces. The A1 domain of von Willebrand Factor adsorbed in similar amounts onto glass, tissue culture polystyrene, and polystyrene surfaces, as measured by XPS. However, ToF-SIMS showed differences in solution exposure of A1 domain amino acids when adsorbed onto the three surfaces and NEXAFS showed the most ordered beta-sheet structure when A1 was adsorbed onto polystyrene. ELISA showed lowest binding of antibodies recognizing a nonlinear epitopes within A1 when A1 was adsorbed onto polystyrene. Functional studies using a parallel plate flow chamber measured platelet binding to A1 adsorbed onto the three surfaces. At high shear (20dyne/cm2), platelets showed most detachment from A1 adsorbed onto glass. At low shear (0.2dyne/cm 2), platelets showed most detachment from A1 adsorbed onto polystyrene. Surface analysis was also useful in characterizing collagen substrates created under different experimental conditions, including material source and pH. Collagen obtained from different sources exhibited altered adsorption behavior, both in amount adsorbed as measured by XPS and interaction with the A1 domain of von Willebrand Factor as measured by ToF-SIMS. SFG was used to identify differences in ordering of collagen adsorbed from solutions at different pH values. Collagen adsorbed at pH 8.0 showed higher SFG amide signal than collagen adsorbed at pH 6.5, suggesting greater ordering of the peptide backbone at pH 8.0. The differences in SFG signal were not due to the amount adsorbed protein, as XPS showed more collagen adsorbed onto tissue culture polystyrene at pH 6.5 than pH 8.0. These studies demonstrated that the surface type can have a large impact on adsorbed proteins, both in amount adsorbed and surface structure. These studies also showed that surface analysis is very useful in creating defined in vitro protein substrates. In all cases, it was crucial to use multiple analysis techniques to understand these systems.
机译：表面上吸附的蛋白质在许多应用中都很重要，包括医疗植入物，传感器，海洋材料以及用于细胞培养和其他用途的体外底物。了解表面上的蛋白质结构可以更好地控制材料与周围环境的相互作用，并获得更可重现的系统响应。蛋白质吸附是一个复杂的过程，其表征需要多种分析技术的结合。本文利用X射线光电子能谱（XPS）和放射性标记的蛋白质来测量蛋白质的吸附量。使用飞行时间二次离子质谱（ToF-SIMS），近边缘X射线吸收精细结构（NEXAFS），总频率生成（SFG）和酶联免疫吸附测定（ELISA）测量蛋白质的构象和方向。表面类型影响通过XPS和放射性标记蛋白质吸附测量的玻璃，聚苯乙烯，钛和磺化聚苯乙烯表面上白蛋白和纤连蛋白的吸附表面浓度。聚苯乙烯比玻璃吸附的白蛋白更多。与钛相比，更多的白蛋白和纤连蛋白吸附在磺化聚苯乙烯上。 ToF-SIMS还显示了吸附在不同表面上的蛋白质结构的差异。通过XPS测量，von Willebrand Factor的A1结构域以相似的量吸附在玻璃，组织培养聚苯乙烯和聚苯乙烯表面上。但是，ToF-SIMS在吸附到三个表面上时显示出A1域氨基酸溶液暴露的差异，而当NEXAFS在A1吸附到聚苯乙烯上时显示出最有序的β-折叠结构。 ELISA显示，当A1吸附到聚苯乙烯上时，识别A1内非线性表位的抗体的结合程度最低。使用平行板流动室的功能研究测量了血小板与吸附在三个表面上的A1的结合。在高剪切力（20达因/厘米2）下，血小板与吸附在玻璃上的A1分离最多。在低剪切（0.2dyne / cm 2）下，血小板显示出与吸附到聚苯乙烯上的A1的大部分脱离。表面分析还可用于表征在不同实验条件（包括材料来源和pH）下产生的胶原蛋白底物。从不同来源获得的胶原蛋白均表现出变化的吸附行为，无论是通过XPS测量的吸附量还是通过ToF-SIMS测量的与von Willebrand Factor的A1结构域的相互作用。 SFG用于鉴定在不同pH值下从溶液中吸附的胶原蛋白的顺序差异。在pH 8.0吸附的胶原蛋白显示出比在pH 6.5吸附的胶原蛋白更高的SFG酰胺信号，表明在pH 8.0时肽主链的有序性更高。 SFG信号的差异不是由于蛋白质吸附量引起的，因为XPS显示，在pH 6.5时，胶原蛋白吸附到组织培养物聚苯乙烯上的胶原蛋白比pH 8.0更多。这些研究表明，表面类型对吸附蛋白质的吸附量和表面结构都有很大影响。这些研究还表明，表面分析对于创建确定的体外蛋白质底物非常有用。在所有情况下，使用多种分析技术来理解这些系统都是至关重要的。

著录项

  • 作者

    Tronic, Elaine Hillenmeyer.;

  • 作者单位

    University of Washington.;

  • 授予单位 University of Washington.;
  • 学科 Engineering Biomedical.;Engineering Chemical.
  • 学位 Ph.D.
  • 年度 2012
  • 页码 213 p.
  • 总页数 213
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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