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Enzyme immobilization on poly(methyl methacrylate) (PMMA) surfaces.

机译：将酶固定在聚甲基丙烯酸甲酯（PMMA）表面上。

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摘要

Microfabricated devices offer a number of important benefits for chemical analysis, and they have the potential for integrating several chemical processes, including sample preparation, chemical reactions, separation and detection, directly onto a single device. Polymer-based microfabricated devices offer a number of advantages as compared to silica-based devices. These advantages include lower cost, easier fabrication, and their potential to be mass-produced. We have been exploring the use of polymer-based microfabricated devices for the analysis of biological samples. Of particular interest to our laboratory is the coupling of these devices to mass spectrometry for protein identification. Here, I describe suitable approaches for covalent immobilization of biological analytes, such as enzymes, via amide linkage to poly(methyl methacrylate) (PMMA) surfaces. The results reveal that enzymes retain their activities after immobilization and can be used for the digestion of proteins and peptides. However, the kinetic parameters (Km and Vmax) of these enzymes are affected by the immobilization to PMMA. After the surface of PMMA is derivatized, the immobilization of enzymes is found to be extremely efficient with immobilized enzyme showing long-term stability. Enzymes can be immobilized to channel walls and PMMA microspheres, which are inserted in a reaction chamber, in PMMA microfabricated devices. These microfabricated devices are used for on-line enzymatic digestion of biological compounds and are interfaced with electrospray ionization-mass spectrometry (ESI-MS). Integration of on-chip enzymatic digestion and electrophoresis to separate components of a sample can be accomplished. It is envisioned that an integration of on-chip enzymatic digestion, separation by microchip electrophoresis and ESI-MS can be used to create a microchip-based proteomics sample preparation tool that will be available for the characterization of proteins.
机译：微型设备为化学分析提供了许多重要的好处，它们具有将多个化学过程（包括样品制备，化学反应，分离和检测）直接集成到单个设备上的潜力。与基于二氧化硅的装置相比，基于聚合物的微制造装置具有许多优势。这些优点包括较低的成本，更容易的制造以及它们的批量生产潜力。我们一直在探索使用基于聚合物的微细加工设备来分析生物样品。我们的实验室特别感兴趣的是将这些设备与质谱联用以进行蛋白质鉴定。在这里，我描述了通过酰胺键与聚甲基丙烯酸甲酯（PMMA）表面共价键合固定生物分析物（如酶）的合适方法。结果表明，酶在固定后仍保留其活性，可用于消化蛋白质和多肽。但是，这些酶的动力学参数（Km和Vmax）受固定在PMMA上的影响。在将PMMA的表面衍生化之后，发现酶的固定是极其有效的，其中固定的酶显示出长期稳定性。可以将酶固定在通道壁和PMMA微球中，该通道壁和PMMA微球被插入到PMMA微型设备中的反应室中。这些微型设备用于在线酶消化生物化合物，并与电喷雾电离质谱（ESI-MS）连接。可以将芯片上的酶消化和电泳集成到样品的各个成分中。可以预见，芯片上酶消化，通过微芯片电泳分离和ESI-MS分离的集成可用于创建基于微芯片的蛋白质组学样品制备工具，该工具可用于蛋白质的表征。

著录项

  • 作者

    Dominick, Wendy D.;

  • 作者单位

    University of Cincinnati.;

  • 授予单位 University of Cincinnati.;
  • 学科 Chemistry Analytical.
  • 学位 Ph.D.
  • 年度 2006
  • 页码 106 p.
  • 总页数 106
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 化学;
  • 关键词

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