Centre for Energy Research, Nanobiosensorics Laboratory, Institute of Technical Physics and Materials Science, 1121 Budapest, Hungary.
Department of Biological Physics, Eötvös Loránd University, 1117 Budapest, Hungary.
Biosensors (Basel). 2021 Jan 24;11(2):32. doi: 10.3390/bios11020032.
The binding of integrin proteins to peptide sequences such as arginine-glycine-aspartic acid (RGD) is a crucial step in the adhesion process of mammalian cells. While these bonds can be examined between purified proteins and their ligands, live-cell assays are better suited to gain biologically relevant information. Here we apply a computer-controlled micropipette (CCMP) to measure the dissociation constant () of integrin-RGD-binding. Surface coatings with varying RGD densities were prepared, and the detachment of single cells from these surfaces was measured by applying a local flow inducing hydrodynamic lifting force on the targeted cells in discrete steps. The average behavior of the populations was then fit according to the chemical law of mass action. To verify the resulting value of = (4503 ± 1673) 1/µm, a resonant waveguide grating based biosensor was used, characterizing and fitting the adhesion kinetics of the cell populations. Both methods yielded a within the same range. Furthermore, an analysis of subpopulations was presented, confirming the ability of CCMP to characterize cell adhesion both on single cell and whole population levels. The introduced methodologies offer convenient and automated routes to quantify the adhesivity of living cells before their further processing.
整合素蛋白与精氨酸-甘氨酸-天冬氨酸（RGD）等肽序列的结合是哺乳动物细胞黏附过程中的关键步骤。虽然可以在纯化的蛋白质与其配体之间检查这些键合，但活细胞测定更适合获得具有生物学相关性的信息。在此，我们应用计算机控制的微管（CCMP）来测量整合素-RGD 结合的解离常数（）。通过在离散步骤中对靶向细胞施加局部流动诱导的流体提升力，制备了具有不同 RGD 密度的表面涂层，并测量了单个细胞从这些表面的脱离。然后，根据质量作用化学定律对群体的平均行为进行拟合。为了验证所得的 值 = (4503 ± 1673) 1/µm，使用了基于共振波导光栅的生物传感器来表征和拟合细胞群体的黏附动力学。两种方法都得到了在相同范围内的 值。此外，还提出了亚群分析，证实了 CCMP 能够在单细胞和整个群体水平上表征细胞黏附的能力。所提出的方法为在进一步处理之前量化活细胞的黏附性提供了便捷和自动化的途径。
