Naval Research Laboratory, 4555 Overlook Avenue SW, Washington, DC 20375-5320, United States.
Nanocrine, Inc., Frederick, Maryland 21704, United States.
ACS Appl Bio Mater. 2021 Nov 15;4(11):7856-7864. doi: 10.1021/acsabm.1c00797. Epub 2021 Oct 26.
Surface ligand activity is a key design parameter for successfully interfacing surfaces with cells─whether in the context of investigations for understanding cellular signaling pathways or more applied applications in drug delivery and medical implants. Unlike other crucial surface parameters, such as stiffness and roughness, surface ligand activity is typically based on a set of assumptions rather than directly measured, giving rise to interpretations of cell adhesion that can vary with the assumptions made. To fill this void, we have developed a concurrent control technique for directly characterizing ligand surface activity. Pairs of gold-coated glass chips were biofunctionalized with RGD ligand in a parallel workflow: one chip for applications and the other for surface plasmon resonance (SPR)-based RGD activity characterization. Recombinant αβ integrins were injected over the SPR chip surface as mimics of the cellular-membrane-bound receptors and the resulting binding kinetics parameterized to quantify surface ligand activity. These activity measurements were correlated with cell morphological features, measured by interfacing MDA-MB-231 cells with the chip surfaces on the live cell microscope. We demonstrate how the interpretation of a cell phenotype based on direct activity measurements can vary markedly from interpretations based on assumed activity. The SPR concurrent control approach has multiple advantages due to the fact that SPR is a standardized technique and has the sensitivity to measure ligand activity across the most relevant range of extracellular surface densities, while the chip design can be used with all commonly used light microscopy modalities (e.g., phase contrast, DIC, and fluorescence) so that a wide range of phenotypic and molecular markers can be correlated to the ligand surface activity.
表面配体活性是成功将表面与细胞相互作用的关键设计参数——无论是在研究细胞信号通路的背景下，还是在药物输送和医疗植入物等更应用的应用中。与其他关键表面参数（如刚性和粗糙度）不同，表面配体活性通常基于一组假设，而不是直接测量，这导致对细胞黏附的解释可能因所做的假设而异。为了填补这一空白，我们开发了一种用于直接表征配体表面活性的并行控制技术。两对金涂覆的玻璃芯片在平行工作流中用 RGD 配体进行生物功能化：一个芯片用于应用，另一个用于基于表面等离子体共振（SPR）的 RGD 活性表征。重组 αβ 整合素被注射到 SPR 芯片表面上，作为细胞膜结合受体的模拟物，并且将由此产生的结合动力学参数化以量化表面配体活性。这些活性测量与通过将 MDA-MB-231 细胞与活细胞显微镜上的芯片表面接口测量的细胞形态特征相关联。我们展示了如何根据直接活性测量对细胞表型的解释与基于假设的活性的解释明显不同。由于 SPR 是一种标准化技术，并且具有在最相关的细胞外表面密度范围内测量配体活性的灵敏度，因此 SPR 并行控制方法具有多个优点，而 芯片设计可与所有常用的明场显微镜模式（例如相差、DIC 和荧光）一起使用，以便可以将广泛的表型和分子标记物与配体表面活性相关联。
