Measuring Receptor–Ligand Binding Kinetics on Cell Surfaces: From Adhesion Frequency to Thermal Fluctuation Methods

  • Wei Chen
  • Veronika I. Zarnitsyna
  • Krishna K. Sarangapani
  • Jun Huang
  • Cheng ZhuEmail author
Molecular Interactions


Interactions between surface-anchored receptors and ligands mediate cell–cell and cell–environment communications in many biological processes. Molecular interactions across two apposing cell membrane are governed by two-dimensional (2D) kinetics, which are physically distinct from and biologically more relevant than three-dimensional (3D) kinetics with at least one interacting molecular species in the fluid phase. Here we review two assays for measuring 2D binding kinetics: the adhesion frequency assay and the thermal fluctuation assay. The former measures the binding frequency as a function of contact duration and extracts the force-free 2D kinetics parameters by nonlinearly fitting the data with a probabilistic model. The latter detects bond formation/dissociation by monitoring the reduction/resumption of thermal fluctuations of a force sensor. Both assays are mechanically based and operate at the level of mostly single molecular interaction, which requires ultrasensitive force techniques. Characterization of one such technique, the biomembrane force probe, is presented.


Adhesion frequency assay Thermal fluctuation assay Micropipette Biomembrane force probe Kinetics Receptor–ligand interaction 



We thank our coworkers of references Chen et al. 1 and Huang et al. 5 who contributed the original data that are discussed here. This work was supported by National Institutes of Health Grants AI38282, AI44902, and HL091020. W.C. is a Predoctoral Fellowship recipient of the American Heart Association (Greater Southeast Affiliate).


  1. 1.
    Chen W., E. A. Evans, R. P. McEver, C. Zhu, Monitoring receptor-ligand interactions between surfaces by thermal fluctuations. Biophys. J. 94(2):694–701, 2008CrossRefGoogle Scholar
  2. 2.
    Chesla, S. E., P. Selvaraj, and C. Zhu. Measuring two-dimensional receptor-ligand binding kinetics by micropipette. Biophys. J. 75(3):1553–1572, 1998Google Scholar
  3. 3.
    Evans E., D. Berk, A. Leung, Detachment of agglutinin-bonded red blood cells. I. Forces to rupture molecular-point attachments. Biophys. J. 59(4):838–848, 1991Google Scholar
  4. 4.
    Evans E., V. Heinrich, A. Leung, K. Kinoshita, Nano- to microscale dynamics of P-selectin detachment from leukocyte interfaces. I. Membrane separation from the cytoskeleton. Biophys. J. 88(3):2288–2298, 2005CrossRefGoogle Scholar
  5. 5.
    Huang J., L. J. Edwards, B. D. Evavold, C. Zhu, Kinetics of MHC-CD8 interaction at the T cell membrane. J. Immunol. 179(11):7653–7662, 2007Google Scholar
  6. 6.
    Marshall B. T., M. Long, J. W. Piper, T. Yago, R. P. McEver, C. Zhu, Direct observation of catch bonds involving cell-adhesion molecules. Nature 423(6936):190–193, 2003CrossRefGoogle Scholar
  7. 7.
    Marshall B. T., K. K. Sarangapani, J. Wu, M. B. Lawrence, R. P. McEver, C. Zhu, Measuring molecular elasticity by atomic force microscope cantilever fluctuations. Biophys. J. 90(2):681–692, 2006CrossRefGoogle Scholar
  8. 8.
    Mehta P., R. Cummings, R. McEver, Affinity and kinetic analysis of P-selectin binding to P-selectin glycoprotein ligand-1. J. Biol. Chem. 273(49):32506–32513, 1998CrossRefGoogle Scholar
  9. 9.
    Nicholson M. W., A. N. Barclay, M. S. Singer, S. D. Rosen, P. A. van der Merwe, Affinity and kinetic analysis of L-selectin (CD62L) binding to glycosylation-dependent cell-adhesion molecule-1. J. Biol. Chem. 273(2):763–770, 1998CrossRefGoogle Scholar
  10. 10.
    Piper J. W., R. A. Swerlick, C. Zhu, Determining force dependence of two-dimensional receptor-ligand binding affinity by centrifugation. Biophys. J. 74(1):492–513, 1998CrossRefGoogle Scholar
  11. 11.
    Sarangapani K. K., T. Yago, A. G. Klopocki, M. B. Lawrence, C. B. Fieger, S. D. Rosen, R. P. McEver, C. Zhu, Low force decelerates L-selectin dissociation from P-selectin glycoprotein ligand-1 and endoglycan. J. Biol. Chem. 279(3):2291–2298, 2004CrossRefGoogle Scholar
  12. 12.
    Thoumine O., P. Kocian, A. Kottelat, J. J. Meister, Short-term binding of fibroblasts to fibronectin: optical tweezers experiments and probabilistic analysis. Eur. Biophys. J. 29(6):398–408, 2000CrossRefGoogle Scholar
  13. 13.
    Tolentino T. P., J. Wu, V. I. Zarnitsyna, Y. Fang, M. L. Dustin, C. Zhu, Measuring diffusion and binding kinetics by contact area FRAP. Biophys. J. 95(2):920–930, 2008CrossRefGoogle Scholar
  14. 14.
    Williams T. E., S. Nagarajan, P. Selvaraj, C. Zhu, Quantifying the impact of membrane microtopology on effective two-dimensional affinity. J. Biol. Chem. 276(16):13283–13288, 2001CrossRefGoogle Scholar
  15. 15.
    Wong W., K. Halvorsen, The effect of integration time on fluctuation measurements: calibrating an optical trap in the presence of motion blur. Opt. Express 14(25):12517–12531, 2006CrossRefGoogle Scholar
  16. 16.
    Wu J., Y. Fang, D. Yang, C. Zhu, Thermo-mechanical responses of a surface-coupled AFM cantilever. J. Biomech. Eng. 127(7):1208–1215, 2005CrossRefGoogle Scholar
  17. 17.
    Wu J., Y. Fang, V. I. Zarnitsyna, T. P. Tolentino, M. L. Dustin, C. Zhu, A coupled diffusion-kinetics model for analysis of contact area FRAP experiment. Biophys. J. 95(2):910–919, 2008CrossRefGoogle Scholar
  18. 18.
    Zarnitsyna V. I., J. Huang, F. Zhang, Y.-H. Chien, D. Leckband, C. Zhu, Memory in receptor-ligand-mediated cell adhesion. Proc. Natl. Acad. Sci. USA 104(46):18037–18042, 2007CrossRefGoogle Scholar
  19. 19.
    Zhu C., M. Long, S. E. Chesla, P. Bongrand, Measuring receptor/ligand interaction at the single-bond level: experimental and interpretative issues. Ann. Biomed. Eng. 30(3):305–314, 2002CrossRefGoogle Scholar

Copyright information

© Biomedical Engineering Society 2008

Authors and Affiliations

  • Wei Chen
    • 1
  • Veronika I. Zarnitsyna
    • 2
  • Krishna K. Sarangapani
    • 1
    • 2
  • Jun Huang
    • 2
  • Cheng Zhu
    • 1
    • 2
    Email author
  1. 1.Woodruff School of Mechanical EngineeringGeorgia Institute of TechnologyAtlantaUSA
  2. 2.Coulter Department of Biomedical EngineeringGeorgia Institute of TechnologyAtlantaUSA

Personalised recommendations