Lee Kyoung G, Pillai Shreekumar R, Singh Shree R, Willing Gerold A
Department of Chemical Engineering, University of Louisville, Louisville, Kentucky 40292, USA.
Biotechnol Bioeng. 2008 Mar 1;99(4):949-59. doi: 10.1002/bit.21644.
The investigation of Protein A and antibody adsorption on surfaces in a biological environment is an important and fundamental step for increasing biosensor sensitivity and specificity. The atomic force microscope (AFM) is a powerful tool that is frequently used to characterize surfaces coated with a variety of molecules. We used AFM in conjunction with scanning electron microscopy to characterize the attachment of protein A and its subsequent binding to the antibody and Salmonella bacteria using a gold quartz crystal. The rms roughness of the base gold surface was determined to be approximately 1.30 nm. The average step height change between the solid gold and protein A layer was approximately 3.0 +/- 1.0 nm, while the average step height of the protein A with attached antibody was approximately 6.0 +/- 1.0 nm. We found that the antibodies did not completely cover the protein A layer, instead the attachment follows an island model. Salt crystals and water trapped under the protein A layer were also observed. The uneven adsorption of antibodies onto the biosensor surface might have led to a decrease in the sensitivity of the biosensor. The presence of salt crystals and water under the protein A layer may deteriorate the sensor specificity. In this report, we have discussed the application and characterization of protein A bound to antibodies which can be used to detect bacterial and viral pathogens.
在生物环境中研究蛋白A和抗体在表面的吸附是提高生物传感器灵敏度和特异性的重要基础步骤。原子力显微镜（AFM）是一种强大的工具，经常用于表征涂覆有各种分子的表面。我们将AFM与扫描电子显微镜结合使用，以表征蛋白A在金石英晶体上的附着及其随后与抗体和沙门氏菌的结合。基底金表面的均方根粗糙度测定为约1.30nm。固体金与蛋白A层之间的平均台阶高度变化约为3.0±1.0nm，而附着有抗体的蛋白A的平均台阶高度约为6.0±1.0nm。我们发现抗体并未完全覆盖蛋白A层，相反，附着遵循岛状模型。还观察到蛋白A层下捕获的盐晶体和水。抗体在生物传感器表面的不均匀吸附可能导致生物传感器灵敏度降低。蛋白A层下盐晶体和水的存在可能会降低传感器的特异性。在本报告中，我们讨论了与抗体结合的蛋白A的应用和表征，其可用于检测细菌和病毒病原体。
