High fidelity functional patterns of an extracellular matrix protein by electron beam-based inactivation
High fidelity functional patterns of an extracellular matrix protein by electron beam-based inactivation
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DOI：
10.1021/ja063698a
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发表时间：
2007-01-10
影响因子：
15
通讯作者：
Hoh, Jan H.
中科院分区：
文献类型：
--
作者：
Rundqvist, Jonas;Mendoza, Beatriz;Hoh, Jan H.
控制蛋白质在表面的组织结构为确定细胞如何解释局部信号分子的空间分布提供了强大的生物化学工具。在这里，我们描述了一个通用的高保真度的方法，基于电子束写入图案的蛋白质涂层表面的功能特性的长度范围从几十纳米到毫米。首先用细胞外基质蛋白纤连蛋白包被硅基质，然后通过暴露于高度聚焦的电子束使其局部失活。蛋白质的生物化学失活是通过抗体与纤连蛋白结合的丧失而建立的。功能性失活是由细胞无法在失活的基质上扩散或形成粘着斑决定的，导致细胞形状受限于图案，而它们在剩余的纤连蛋白上既扩散又形成粘着斑（并且不受约束）。这些蛋白质模式具有非常高的保真度，并且典型模式与模式的输入维度的一致性在2%以内。此外，特征边缘被很好地限定并且在粗糙度上接近分子尺寸。失活被证明是剂量依赖性的，在2 μ C cm（-2）处可观察到特异性结合的抑制，在50 μ C cm（-2）处可观察到生化活性的完全去除。失活的临界剂量也取决于加速电压，对于1 keV电子，在4-7 μ C cm（-2）时达到抗体结合的完全丧失，这相当于整个纤连蛋白二聚体的每个横截面积有50-90个电子，每个III型纤连蛋白结构域有2-4个电子。图案表面的AFM分析显示，电子束曝光不从表面去除可观的材料量，这表明图案化机制涉及局部失活，而不是在几个有机薄膜系统中观察到的烧蚀。
Controlling the organization of proteins on surfaces provides a powerful biochemical tool for determining how cells interpret the spatial distribution of local signaling molecules. Here, we describe a general high fidelity approach based on electron beam writing to pattern the functional properties of protein-coated surfaces at length scales ranging from tens of nanometers to millimeters. A silicon substrate is first coated with the extracellular matrix protein fibronectin, which is then locally inactivated by exposure to a highly focused electron beam. Biochemical inactivation of the protein is established by the loss of antibody binding to the fibronectin. Functional inactivation is determined by the inability of cells to spread or form focal adhesions on the inactivated substrate, resulting in cell shapes constrained to the pattern, while they do both (and are unconstrained) on the remaining fibronectin. These protein patterns have very high fidelity, and typical patterns agree with the input dimensions of the pattern to within 2%. Further, the feature edges are well defined and approach molecular dimensions in roughness. Inactivation is shown to be dose dependent with observable suppression of the specific binding at 2 mu C cm(-2) and complete removal of biochemical activity at similar to 50 mu C cm(-2) for 5 keV electrons. The critical dose for inactivation also depends on accelerating voltage, and complete loss of antibody binding was achieved at similar to 4-7 mu C cm(-2) for 1 keV electrons, which corresponds to similar to 50-90 electrons per cross-sectional area of a whole fibronectin dimer and similar to 2-4 electrons per type III fibronectin domain. AFM analysis of the pattern surfaces revealed that electron beam exposure does not remove appreciable amounts of material from the surface, suggesting that the patterning mechanism involves local inactivation rather than the ablation that has been observed in several organic thin film systems.