Cao Ting, Wang Anfeng, Liang Xuemei, Tang Haiying, Auner Gregory W, Salley Steven O, Ng K Y Simon
Department of Chemical Engineering and Materials Science, Wayne State University, 5050 Anthony Wayne Drive, Detroit, Michigan 48202, USA.
Biotechnol Bioeng. 2007 Dec 15;98(6):1109-22. doi: 10.1002/bit.21503.
The immobilization of antibodies to sensor surfaces is critical in biochemical sensor development. In this study, Poly(ethylene glycol) (PEG) and Jeffamine spacers were employed to tether Escherichia coli K99 pilus antibody to silicon wafer surfaces for the purpose of improving the orientation of antibody as well as reducing the steric hindrance. To illustrate the effect of spacer length, a variety of linear polymers were used to covalently attach the antibodies to silicon surfaces. Atomic Force Microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) were used to characterize the surface morphology and chemical composition at each reaction step. The effect of spacer length in improving the specificity of immobilized antibody was investigated by attaching E. coli on the end of an AFM tip. The distribution of unbinding force and rupture distance from the force-distance curves obtained by AFM showed that the introduction of PEG spacer facilitates bacterial recognition which can improve the incidence of interactions by up to 90%. J600 proved to be the most effective spacer overcoming the steric hindrance seen with direct immobilization of antibody. In addition, the force spectroscopy reveals the elementary force quantum of E. coli-antibody to be 0.3 nN.
抗体固定于传感器表面在生化传感器开发中至关重要。在本研究中，聚乙二醇（PEG）和Jeffamine间隔臂被用于将大肠杆菌K99菌毛抗体连接到硅片表面，以改善抗体的取向并减少空间位阻。为说明间隔臂长度的影响，使用了多种线性聚合物将抗体共价连接到硅表面。原子力显微镜（AFM）和X射线光电子能谱（XPS）用于表征每个反应步骤的表面形态和化学成分。通过将大肠杆菌附着在AFM针尖末端，研究了间隔臂长度对提高固定化抗体特异性的影响。AFM获得的力-距离曲线中未结合力和断裂距离的分布表明，PEG间隔臂的引入促进了细菌识别，可将相互作用发生率提高多达90%。J600被证明是克服抗体直接固定时出现的空间位阻最有效的间隔臂。此外，力谱显示大肠杆菌-抗体的基本力量子为0.3 nN。
