Department of Micro- and Nanotechnology, DTU Nanotech, Technical University of Denmark , Ørsteds Plads 345E, DK-2800 Kgs. Lyngby, Denmark.
Biomacromolecules. 2014 Mar 10;15(3):894-9. doi: 10.1021/bm401745a. Epub 2014 Feb 19.
Immobilization of proteins onto polymer surfaces usually requires specific reactive functional groups. Here, we show an easy one-step method to conjugate protein covalently onto almost any polymer surface, including low protein-binding poly(ethylene glycol) (PEG), without the requirement for the presence of specific functional groups. Several types of proteins, including alkaline phosphatase, bovine serum albumin, and polyclonal antibodies, were photoimmobilized onto a PEG-coated polymer surface using a water-soluble benzophenone as photosensitizer. Protein functionality after immobilization was verified for both enzymes and antibodies, and their presence on the surface was confirmed by X-ray photoelectron spectroscopy (XPS) and confocal fluorescence microscopy. Conjugation of capture antibody onto the PEG coating was employed for a simplified ELISA protocol without the need for blocking uncoated surface areas, showing ng/mL sensitivity to a cytokine antigen target. Moreover, spatially patterned attachment of fluorescently labeled protein onto the low-binding PEG-coated surface was achieved with a projection lithography system that enabled the creation of micrometer-sized protein features.
蛋白质固定到聚合物表面通常需要特定的反应性功能基团。在这里，我们展示了一种简单的一步法，可将蛋白质共价固定到几乎任何聚合物表面，包括低蛋白结合的聚（乙二醇）（PEG），而不需要存在特定的功能基团。几种类型的蛋白质，包括碱性磷酸酶、牛血清白蛋白和多克隆抗体，使用水溶性二苯甲酮作为光引发剂，通过光固定到 PEG 涂覆的聚合物表面上。固定化后对酶和抗体的蛋白质功能进行了验证，并通过 X 射线光电子能谱（XPS）和共聚焦荧光显微镜确认了其在表面上的存在。通过将捕获抗体连接到 PEG 涂层上，简化了 ELISA 方案，无需阻断未涂覆的表面区域，对细胞因子抗原靶标具有 ng/mL 的灵敏度。此外，通过投影光刻系统实现了荧光标记的蛋白质在低结合 PEG 涂覆表面上的空间图案化附着，该系统能够创建微米级的蛋白质特征。
