Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia Pennsylvania 19104-6323, USA.
Langmuir. 2010 May 18;26(10):7117-25. doi: 10.1021/la904151h.
Tripod-shaped molecules were designed for chemical modification of the surface of probes used for atomic force microscopy (AFM). These chemically functionalized tips were used for chemical force spectroscopy (CFS) measurements of the ligand-protein receptor interaction in a biotin-NeutrAvidin model system. We demonstrate that by using this unique tripodal system, we can achieve significantly lower density of ligand on the AFM tip apex, which is optimal for true single molecule measurements. Furthermore, the molecular tripods form highly stable bonds to the AFM probes, leading to more robust and reproducible unbinding force data, thereby addressing one of the challenges in CFS studies. Histogram analysis of the hundreds of collected unbinding forces showed a specific distribution with a peak force maximum at approximately 165 pN, in good agreement with the previously reported data of single rupture events of biotin-avidin. We compared these molecular tripod tips with a molecular monopod. The results showed that the molecular tripods are more robust for repeated measurements. The distinct biotin-avidin force maximum was not observed in the control experiments. This indicated that the force distribution observed for molecular tripods corresponds to the specific rupture force between biotin and avidin. The improved robustness of molecular tripods for CFS will provide benefits in other ligand-receptor unbinding studies, including those of transmembrane receptor systems, which require high resolution, sensitivity, and reproducibility in force spectroscopy measurements.
三脚架分子被设计用于化学修饰原子力显微镜（AFM）探针的表面。这些化学功能化的尖端用于生物素-NeutrAvidin 模型系统中配体-蛋白受体相互作用的化学力谱（CFS）测量。我们证明，通过使用这种独特的三脚架系统，我们可以在 AFM 尖端达到明显更低的配体密度，这对于真正的单分子测量是最佳的。此外，分子三脚架与 AFM 探针形成高度稳定的键，从而导致更稳健和可重复的解键力数据，从而解决了 CFS 研究中的一个挑战。数百个收集的解键力的直方图分析显示出具有约 165 pN 的峰值力最大值的特定分布，与先前报道的生物素-亲和素单断裂事件的单个数据非常吻合。我们将这些分子三脚架与分子单足进行了比较。结果表明，分子三脚架在重复测量中更稳健。在对照实验中未观察到明显的生物素-亲和素力最大值。这表明观察到的分子三脚架的力分布对应于生物素和亲和素之间的特定断裂力。CFS 中分子三脚架的增强稳健性将为其他配体-受体解键研究提供益处，包括需要在力谱测量中具有高分辨率、灵敏度和可重复性的跨膜受体系统。
