A hydrogel-based versatile screening platform for specific biomolecular recognition in a well plate format
A hydrogel-based versatile screening platform for specific biomolecular recognition in a well plate format
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DOI：
10.1007/s00216-012-5850-9
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发表时间：
2012-04-01
影响因子：
4.3
通讯作者：
Groll, Juergen
中科院分区：
文献类型：
--
作者：
Beer, Meike V.;Rech, Claudia;Groll, Juergen
高通量生物分子相互作用的精确测定关键取决于表面涂层技术，该技术允许在非相互作用环境中固定各种相互作用伙伴。我们提出了一种基于异氰酸酯官能六臂聚环氧乙烷星形聚合物的一步式水凝胶涂层系统，适用于市售的 96 孔微量滴定板，该系统将简单而强大的涂层应用与多功能生物功能化结合起来。该系统产生对非特异性蛋白质吸附和细胞粘附的抵抗力，如荧光标记的牛血清白蛋白和原代人真皮成纤维细胞 (HDF) 所证明的那样，并且当配体固定在该表面上时，对生物分子识别过程的评估具有高特异性。一个特别的优点是可以固定多种生物分子并将本身惰性的涂层转化为特定的相互作用表面。我们在这里演示了广泛的生化重要配体的固定和定量，例如肽序列GRGDS和GRGDSK-生物素、广泛适用的偶联分子生物胞素、蛋白纤连蛋白以及碳水化合物N-乙酰葡糖胺和N-乙酰乳糖胺。建立了酶联免疫吸附测定的简化方案，用于涂层表面配体的检测和定量。使用 HDF 评估肽和蛋白质修饰表面上的细胞粘附。所有涂层均采用一步制备技术（包括生物活化）进行涂覆，这使得该系统适合以与最常用的测试系统兼容的形式进行高通量筛选。
Precise determination of biomolecular interactions in high throughput crucially depends on a surface coating technique that allows immobilization of a variety of interaction partners in a non-interacting environment. We present a one-step hydrogel coating system based on isocyanate functional six-arm poly(ethylene oxide)-based star polymers for commercially available 96-well microtiter plates that combines a straightforward and robust coating application with versatile bio-functionalization. This system generates resistance to unspecific protein adsorption and cell adhesion, as demonstrated with fluorescently labeled bovine serum albumin and primary human dermal fibroblasts (HDF), and high specificity for the assessment of biomolecular recognition processes when ligands are immobilized on this surface. One particular advantage is the wide range of biomolecules that can be immobilized and convert the per se inert coating into a specifically interacting surface. We here demonstrate the immobilization and quantification of a broad range of biochemically important ligands, such as peptide sequences GRGDS and GRGDSK-biotin, the broadly applicable coupler molecule biocytin, the protein fibronectin, and the carbohydrates N-acetylglucosamine and N-acetyllactosamine. A simplified protocol for an enzyme-linked immunosorbent assay was established for the detection and quantification of ligands on the coating surface. Cell adhesion on the peptide and protein-modified surfaces was assessed using HDF. All coatings were applied using a one-step preparation technique, including bioactivation, which makes the system suitable for high-throughput screening in a format that is compatible with the most routinely used testing systems.