Analytical Science Division, The Dow Chemical Company, 727 Norristown Rd., Spring House, PA 19477, USA.
Anal Bioanal Chem. 2013 Feb;405(6):1985-93. doi: 10.1007/s00216-012-6621-3. Epub 2012 Dec 14.
The immobilization of proteins on nanopatterned surfaces was investigated using in situ atomic force microscopy (AFM) and ex situ infrared reflectance-absorption spectroscopy (IRAS). The AFM-based lithography technique of nanografting provided control of the size, geometry, and spatial placement of nanopatterns within self-assembled monolayers (SAMs). Square nanopatterns of carboxylate-terminated SAMs were inscribed within methyl-terminated octadecanethiolate SAMs and activated using carbodiimide/succinimide coupling chemistry. Staphylococcal protein A was immobilized on the activated nanopatterns before exposure to rabbit immunoglobulin G. In situ AFM was used to monitor changes in the topography and friction of the nanopatterns in solution upon protein immobilization. Complementary studies with ex situ IRAS confirmed the surface chemistry that occurred during the steps of SAM activation and subsequent protein immobilization on unpatterned samples. Since carbodiimide/succinimide coupling chemistry can be used for surface attachment of different biomolecules, this protocol shows promise for development of other aqueous-based studies for nanopatterned protein immobilization.
采用原位原子力显微镜（AFM）和非原位红外反射吸收光谱（IRAS）研究了蛋白质在纳米图案表面上的固定化。基于 AFM 的纳米嫁接光刻技术可控制自组装单分子层（SAM）内纳米图案的大小、几何形状和空间位置。羧酸盐封端的 SAM 的正方形纳米图案在甲基封端的十八烷硫醇 SAM 内刻写，并使用碳二亚胺/琥珀酰亚胺偶联化学进行激活。在将兔免疫球蛋白 G 暴露于固定化的纳米图案之前，将金黄色葡萄球菌蛋白 A 固定在激活的纳米图案上。原位 AFM 用于监测蛋白质固定化过程中溶液中纳米图案形貌和摩擦的变化。非原位 IRAS 的补充研究证实了 SAM 激活过程中和随后在未图案化样品上固定蛋白质过程中发生的表面化学。由于碳二亚胺/琥珀酰亚胺偶联化学可用于不同生物分子的表面附着，因此该方案有望为基于纳米图案的蛋白质固定化的其他水相研究的开发提供参考。
