Groll Jürgen, Moeller Martin
DWI eV and Institute of Technical and Macromolecular Chemistry, RWTAachen University, Aachen, Germany.
Methods Enzymol. 2010;472:1-18. doi: 10.1016/S0076-6879(10)72019-X.
Poly(ethylene oxide) (PEO) is known as an excellent coating material to minimize nonspecific protein adsorption. For an examination of biomolecules attached to surfaces with sensitivities down to the single-molecule level, demands on the surface additionally comprise low-intrinsic fluorescence of the coating material and a possibility to immobilize biomolecules in their functional conformation. One strategy that combines the protein-resistant properties of PEO with chemical functionality is the use of star-shaped PEOs that allow for interpolymer cross-linking. Our system consists of six-arm PEO-based star polymers functionalized with reactive isocyanate groups at the ends of the polymer chain. The isocyante groups allow intermolecular cross-linking so that high grafting densities may be achieved, which render the surfaces extremely resistant to protein adsorption. Application by spin coating offers a simple procedure for the preparation of minimally interacting surfaces. The reactive end groups may be further biofunctionalized to recognize specific biomolecules such as streptavidin or His-tagged proteins in specific geometries or as single isolated molecules. These properties, together with the advantageous chemical properties of PEO, render the surfaces ideal for immobilizing proteins with detection limits down to the single molecule level. This chapter focuses on the preparation of substrates that are suitable for single-molecule experiments. Besides a detailed description of surface preparation, two examples for the single-molecule detection of immobilized proteins, nucleosomes and RNase H, are presented that demonstrate the advantages of the star-polymer derived coatings over linear-grafted PEO.
聚环氧乙烷（PEO）是一种出色的涂层材料，可最大限度地减少非特异性蛋白质吸附。为了检测附着在表面的生物分子，灵敏度要低至单分子水平，对表面的要求还包括涂层材料的低固有荧光以及将生物分子固定在其功能构象中的可能性。将PEO的抗蛋白质特性与化学功能性相结合的一种策略是使用允许聚合物间交联的星形PEO。我们的系统由基于六臂PEO的星形聚合物组成，在聚合物链的末端用反应性异氰酸酯基团进行功能化。异氰酸酯基团允许分子间交联，从而可以实现高接枝密度，使表面对蛋白质吸附具有极高的抗性。通过旋涂进行应用为制备最小相互作用表面提供了一种简单的方法。反应性端基可以进一步进行生物功能化，以识别特定几何形状或单个分离分子中的特定生物分子，如链霉亲和素或带有His标签的蛋白质。这些特性与PEO的有利化学性质相结合，使这些表面成为固定蛋白质的理想选择，检测限低至单分子水平。本章重点介绍适用于单分子实验的底物的制备。除了对表面制备的详细描述外，还给出了两个固定化蛋白质、核小体和核糖核酸酶H的单分子检测示例，展示了星形聚合物衍生涂层相对于线性接枝PEO的优势。
