LaGraff John R, Chu-LaGraff Quynh
Department of Physics, Applied Physics, and Astronomy, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
Langmuir. 2006 May 9;22(10):4685-93. doi: 10.1021/la0522303.
Unlabeled primary immunoglobulin G (IgG) antibodies and its F(ab')2 and Fc fragments were attached to oxygen-plasma-cleaned glass substrates using either microcontact printing (MCP) or physical adsorption during bath application from dilute solutions. Fluorescently labeled secondary IgGs were then bound to surface-immobilized IgG, and the relative surface coverage was determined by measuring the fluorescence intensity. Results indicated that the surface coverage of IgG increased with increasing protein solution concentration for both MCP and bath-applied IgG and that a greater concentration of IgG was transferred to a glass substrate using MCP than during physisorption during bath applications. Scanning force microscopy (SFM) showed that patterned MCP IgG monolayers were 5 nm in height, indicating that IgG molecules lie flat on the substrate. After incubation with a secondary IgG, the overall line thickness increased to around 15 nm, indicating that the secondary IgG was in a more vertical orientation with respect to the substrate. The surface roughness of these MCP patterned IgG bilayers as measured by SFM was observed to increase with increasing surface coverage. Physisorption of IgG to both unmodified patterned polydimethylsiloxane (PDMS) stamps and plasma-cleaned glass substrates was modeled by Langmuir adsorption kinetics yielding IgG binding constants of K(MCP) = 1.7(2) x 10(7) M(-1) and K(bath) = 7.8(7) x 10(5) M(-1), respectively. MCP experiments involving primary F(ab')2 and Fc fragments incubated in fluorescently labeled fragment-specific secondary IgGs were carried out to test for the function and orientation of IgG. Finally, possible origins of MCP stamping defects such as pits, pull outs, droplets, and reverse protein transfer are discussed.
未标记的原发性免疫球蛋白G（IgG）抗体及其F(ab')2和Fc片段通过微接触印刷（MCP）或在从稀溶液进行浴涂敷过程中的物理吸附，附着到经氧等离子体清洁的玻璃基板上。然后将荧光标记的二级IgG与表面固定的IgG结合，并通过测量荧光强度来确定相对表面覆盖率。结果表明，对于MCP和浴涂敷的IgG，IgG的表面覆盖率均随蛋白质溶液浓度的增加而增加，并且使用MCP转移到玻璃基板上的IgG浓度比浴涂敷过程中的物理吸附时更高。扫描力显微镜（SFM）显示，图案化的MCP IgG单层高度为5 nm，表明IgG分子平躺在基板上。与二级IgG孵育后，总线条厚度增加到约15 nm，表明二级IgG相对于基板处于更垂直的取向。通过SFM测量，观察到这些MCP图案化的IgG双层的表面粗糙度随表面覆盖率的增加而增加。通过Langmuir吸附动力学对IgG在未修饰的图案化聚二甲基硅氧烷（PDMS）印章和经等离子体清洁的玻璃基板上的物理吸附进行建模，得出IgG结合常数分别为K(MCP) = 1.7(2) x 10(7) M(-1)和K(浴) = 7.8(7) x 10(5) M(-1)。进行了涉及在荧光标记的片段特异性二级IgG中孵育的原发性F(ab')2和Fc片段以及MCP实验，以测试IgG的功能和取向。最后，讨论了MCP压印缺陷（如凹坑、拉出、液滴和反向蛋白质转移）的可能成因。
