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Surface Plasmon Resonance Studies of the Specific Interactions of Hexamer Peptide Ligands with Human Immunoglobulin G.

机译：六聚体肽配体与人免疫球蛋白G特异性相互作用的表面等离子体共振研究。

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摘要

This study characterizes the human immunoglobulin G (IgG) binding on peptides grafted onto self-assembled monolayers (SAMs) and the binding events are studied primarily using surface plasmon resonance (SPR) technology. The dissertation also seeks to determine the optimum surface preparation and surface chemistry approaches for grafting the peptide so that the sensor surfaces demonstrate enhanced selectivity and sensitivity in both laboratory and industrial settings. Peptide covalent grafting was performed on pure and mixed SAMs, the surfaces were characterized and the peptide densities were quantified. Theoretical models were developed and implemented to describe the binding mechanism of IgG with grafted ligands.;Protein A was grafted onto SPR sensors and subsequent IgG binding characteristics were compared side-by-side to those of peptide-IgG binding. It was found that Protein A-based sensors showed much higher selectivities and higher binding capacities than their peptides based counterparts. Oligo(ethylene glycol) alkanethiol-based pure and mixed SAMs were grafted with peptides in order to determine the optimal surface among these, for enhanced selectivity. Among the mixed SAMs formed from different precursor solutions, a surface with peptides grafted onto mixed SAMs formulated from 10% amine-terminated/90% hydroxyl-terminated alkanethiols showed optimum selectivity. Studies were carried out to increase the peptide density via grafting of branched amines onto surfaces. The branched amine-based peptide surfaces displayed improved sensitivities and similar selectivities to the surfaces based on un-branched amine termini. Kinetic analyses were carried out to determine the characteristics of IgG binding to ligands grafted in the abovementioned methods.;Kinetic analysis of binding indicated that Protein A-IgG interactions have concentrationdependent affinity properties that could be attributed to the allosteric effects of the interaction. The lack of tertiary structure of the peptide contributed to a constant rate adsorption constant, ka, at all concentrations of IgG, regardless of flow rates, when peptides were grafted onto pure or mixed SAMs. However the adsorption rate constant, ka showed partial diffusion-controlled behavior when peptides were grafted onto branched amines on SAMs. This was attributed to the three dimensional matrix of branched amines introducing mass transport limitations when IgG binds to the peptides grafted onto the branched amines. It was also found that theoretical models must incorporate parameters to account for multiple-site binding of IgG on the branched amine-based surfaces.;The performances of the biosensing surfaces with peptides grafted onto pure SAMs, onto mixed SAMs and onto branched amines were analyzed in presence of complex mixtures and for their regeneration potentials. Complex mixture IgG binding gave the best results for branched amine-based peptide surfaces for a wide range of IgG concentrations. Regeneration behavior was optimum for the branched amine-based surfaces and peptide activity showed negligible decline after eight cycles of IgG binding and regeneration. Thus, among the peptide-based surfaces studied, the peptides grafted on branched amines were optimal with regards to peptide density, efficiency of amine terminus usage, sensitivity and regeneration potential. The theoretical models developed in this work helped to understand the binding mechanism of IgG to the peptide on various supports. The potential for commercial deployment of peptide-based IgG biosensors has been demonstrated in this research work. (Abstract shortened by UMI.).
机译：这项研究的特点是人类免疫球蛋白G（IgG）结合在嫁接到自组装单层（SAMs）上的肽上，并且主要使用表面等离子体共振（SPR）技术研究结合事件。本文还试图确定用于嫁接肽的最佳表面制备和表面化学方法，以使传感器表面在实验室和工业环境中均具有增强的选择性和灵敏度。在纯和混合SAM上进行肽共价接枝，表征表面并定量肽密度。建立并实现了理论模型来描述IgG与嫁接的配体的结合机理。;将蛋白A嫁接到SPR传感器上，并将随后的IgG结合特性与肽-IgG结合特性进行了比较。已经发现，基于蛋白质A的传感器比基于肽的传感器具有更高的选择性和更高的结合能力。为了确定更高的选择性，将低聚乙二醇乙二醇烷硫醇基纯和混合SAM与肽接枝，以确定其中的最佳表面。在由不同前体溶液形成的混合SAM中，肽接枝到由10％胺端基/ 90％羟基端基链烷硫醇配制的混合SAM上的表面表现出最佳的选择性。进行了通过将支链胺接枝到表面上以增加肽密度的研究。支链的基于胺的肽表面显示出改进的敏感性和与基于非支链的胺末端的表面相似的选择性。进行动力学分析以确定IgG与上述方法中嫁接的配体结合的特征。结合动力学分析表明蛋白A-IgG相互作用具有浓度依赖性亲和特性，这可归因于相互作用的变构作用。当将肽接枝到纯或混合SAM上时，无论流速如何，肽的三级结构的缺乏都会导致在所有IgG浓度下恒定速率的吸附常数ka。但是，当将肽接枝到SAM上的支链胺上时，吸附速率常数ka显示出部分扩散受控的行为。这归因于当IgG结合到嫁接到支链胺上的肽时，支链胺的三维矩阵引入了质量迁移限制。还发现理论模型必须包含参数，以说明IgG在支链胺基表面上的多位结合。;分析了将肽接枝到纯SAM，混合SAM和支链胺上的生物传感表面的性能存在复杂混合物及其再生潜力。对于很大范围的IgG浓度，复杂混合物IgG的结合对于支链胺基肽表面给出了最佳结果。再生行为对于基于支链胺的表面而言是最佳的，并且在八次IgG结合和再生循环后，肽的活性显示可忽略不计的下降。因此，在所研究的基于肽的表面中，在支链胺上接枝的肽在肽密度，胺末端使用效率，敏感性和再生潜力方面是最佳的。在这项工作中开发的理论模型有助于理解IgG与各种支持物上的肽的结合机制。这项研究工作证明了基于肽的IgG生物传感器在商业上的应用潜力。 （摘要由UMI缩短。）。

著录项

  • 作者

    Islam, Nafisa.;

  • 作者单位

    North Carolina State University.;

  • 授予单位 North Carolina State University.;
  • 学科 Engineering Chemical.;Engineering Materials Science.
  • 学位 Ph.D.
  • 年度 2013
  • 页码 193 p.
  • 总页数 193
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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