Interdisciplinary Nanoscience Center (iNANO), Århus University, Gustav Wieds Vej 14, 8000 Århus, Denmark.
Lab Chip. 2015 Dec 21;15(24):4524-32. doi: 10.1039/c5lc00916b. Epub 2015 Nov 3.
A novel approach combining self-assembly-based colloidal lithography and polydimethylsiloxane (PDMS) micromolding to generate complex protein nanopatterns for studying the mechanisms of leukocyte extravasation within microchannels is presented. Nanostructured surfaces sealed onto PDMS-molded microchannels are chemically functionalized in situ in an all-aqueous process to generate bi-functional chemical nanopatterns. Subsequent co-immobilization with proteins makes use of common non-covalent coupling (e.g. HIS-tags, FC-tags and biotin-tags), giving nanopatterns of arbitrary combinations of oriented, functional proteins. Up to three different proteins were simultaneously co-immobilized into the microchannel with nanoscale precision, demonstrating the complex patterns. As a proof-of-principle, a mimic of an inflamed endothelium was constructed using a macro- and nanoscale pattern of intercellular adhesion molecule 1 (ICAM1) and P-selectin, and the response of leukocytes through live cell imaging was measured. A clear result on the rolling behavior of the cells was observed with rolling limited to areas where ICAM1 and P-selectin are present. This micro/nano-interface will open new doors to investigations of how spatial distributions of proteins control cellular activity.
提出了一种将基于自组装的胶体光刻和聚二甲基硅氧烷（PDMS）微成型相结合的新方法，以生成用于研究白细胞在微通道内渗出机制的复杂蛋白质纳米图案。通过在全水相中进行原位化学功能化，将密封在 PDMS 模制微通道上的纳米结构表面转化为双功能化学纳米图案。随后与蛋白质共固定化利用常见的非共价偶联（例如 HIS 标签、FC 标签和生物素标签），实现了具有任意定向、功能化蛋白质组合的纳米图案。高达三种不同的蛋白质可以以纳米级精度同时共固定到微通道中，从而展示了复杂的图案。作为原理验证，使用细胞间黏附分子 1（ICAM1）和 P 选择素的宏观和纳米图案构建了炎症内皮的模拟物，并通过活细胞成像测量了白细胞的反应。观察到细胞滚动行为的清晰结果，滚动仅限于存在 ICAM1 和 P 选择素的区域。这种微/纳米界面将为研究蛋白质的空间分布如何控制细胞活性开辟新的途径。
