期刊
BIOSENSORS-BASEL
卷 11, 期 2, 页码 -出版社
MDPI
DOI: 10.3390/bios11020032
关键词
adhesion; micropipette; waveguide; biosensor; two-dimensional dissociation constant; integrin-RGD-binding
资金
- Hungarian Academy of Sciences (Lendulet (Momentum) Program))
- National Research, Development and Innovation Office (NKFIH) [KH_17, PD 131543, KKP_19]
- National Research, Development and Innovation Office (NKFIH) (ERC_HU)
This study utilized a computer-controlled micropipette (CCMP) to measure the dissociation constant (K-d) of integrin-RGD binding, combined with techniques such as surface coating and local flow inducing hydrodynamic lifting force to study cell adhesion behavior. The results demonstrated that CCMP could effectively characterize cell adhesion at both single-cell and whole population levels, offering a convenient and automated approach for quantifying cell adhesivity before further processing.
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 (K-d) 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 K-d(2d) = (4503 +/- 1673) 1/mu m(2), a resonant waveguide grating based biosensor was used, characterizing and fitting the adhesion kinetics of the cell populations. Both methods yielded a K-d 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.
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