剪切流动法研究表面固定化配基与目标分子的相互作用力

Investigation of Interaction Force Between Surface Immobilized Ligand and Objective Molecule Using Shear Flow Chamber

基于剪切流动腔技术,以微球作为受力载体,设计了一套可用于研究表面固定化配基与目标分子特异性相互作用力的实验和分析方法,并以人免疫球蛋白G(humanIgG)和羊抗人免疫球蛋白G(goatanti-humanIgG)分别作为模型配基和模型目标分子进行了研究.基于平面Poiseuille层流模型设计了流场参数,以数值计算结果验证了设计的合理性.使用牛血清白蛋白(BSA)作为非特异性对照,判断微球与基片表面的结合力来自配基和目标分子的生物特异性相互作用,并由进一步的目标分子灭活对比实验确认了这一结论.实验观察到微球与基片表面的结合力受到配基面密度的影响,说明发生结合的是多对而非单对蛋白质分子.将95%的微球被剥离时对应的壁面剪切率设定为临界剪切率,由大量实验结果拟合得到了临界剪切率与配基面密度间的定量关系.在受力分析模型中,考虑到多分子的结合,以及分子键位置不同造成的力臂长度的差异,最终计算得到单对配基与目标分子的平均结合力约为342pN.

Based on flow chamber technique and using carrier microspheres as force magnifiers, an investigation of the interaction force between surface immobilized ligand and objective molecule was carried out. Human immunoglobulin G （IgG） and goat anti-human IgG （anti-IgG） were employed as model ligand and model objective molecule respectively. The parameters of the flow field were designed based on Plane Poiseuille Flow and the design was validated by a numerical simulation. Using bovine serum albumin （BSA） as negative control, it was concluded that the adhesion force between the microspheres and the chip surface came from the specific interaction between the ligand and the objective molecule. And this conclusion was confirmed by an anti-IgG deactivation comparison. It was found that the adhesion force between the spheres and the chip surface was affected by the ligand surface concentration. The wall shear rate at which 95% microspheres were removed from the chip surface was set as the critical value, and the relationship between the critical shear rate and the ligand surface concentration was obtained. A mechanical analysis model considering both the ligand surface concentration and the difference of molecular bonds＇ position was proposed, which finally gave the result that the average interaction force between a single pair of ligand and objective molecule was about 342pN.