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Detection and localization of single molecular recognition events using atomic force microscopy

Nature Methods volume 3, pages 347–355 (2006)Cite this article

Abstract

Because of its piconewton force sensitivity and nanometer positional accuracy, the atomic force microscope (AFM) has emerged as a powerful tool for exploring the forces and the dynamics of the interaction between individual ligands and receptors, either on isolated molecules or on cellular surfaces. These studies require attaching specific biomolecules or cells on AFM tips and on solid supports and measuring the unbinding forces between the modified surfaces using AFM force spectroscopy. In this review, we describe the current methodology for molecular recognition studies using the AFM, with an emphasis on strategies available for preparing AFM tips and samples, and on procedures for detecting and localizing single molecular recognition events.

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Figure 1: General principle of atomic force microscopy.
Figure 2: Schematics of surface chemistries commonly used for modifying AFM tips for single-molecule recognition studies.
Figure 3: Measurement of molecular recognition interaction forces.
Figure 4: Dynamic force spectroscopy of a single receptor-ligand bond.
Figure 5: Mapping molecular recognition sites on living cells.
Figure 6: Simultaneous topography and recognition imaging (TREC).

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References

  1. Turner, A.P. Biosensors—sense and sensitivity. Science 290, 1315–1317 (2000).

    CAS  PubMed  Google Scholar 

  2. Binnig, G., Quate, C.F. & Gerber, C. Atomic Force Microscope. Phys. Rev. Lett. 56, 930–933 (1986).

    CAS  PubMed  Google Scholar 

  3. Jena, B.P. & Hörber, J.K. Atomic Force Microscopy in Cell Biology, Methods in Cell Biology Vol. 68. (Academic Press, San Diego, 2002).

    Google Scholar 

  4. Engel, A. & Müller, D.J. Observing single biomolecules at work with the atomic force microscope. Nat. Struct. Biol. 7, 715–718 (2000).

    CAS  PubMed  Google Scholar 

  5. Clausen-Schaumann, H., Seitz, M., Krautbauer, R. & Gaub, H.E. Force spectroscopy with single bio-molecules. Curr. Opin. Chem. Biol. 4, 524–530 (2000).

    CAS  PubMed  Google Scholar 

  6. Fisher, T.E., Marszalek, P.E. & Fernandez, J.M. Stretching single molecules into novel conformations using the atomic force microscope. Nat. Struct. Biol. 7, 719–724 (2000).

    CAS  PubMed  Google Scholar 

  7. Florin, E.L., Moy, V.T. & Gaub, H.E. Adhesion forces between individual ligand-receptor pairs. Science 264, 415–417 (1994).

    CAS  PubMed  Google Scholar 

  8. Lee, G.U., Chrisey, L.A. & Colton, R.J. Direct measurement of the forces between complementary strands of DNA. Science 266, 771–773 (1994).

    CAS  PubMed  Google Scholar 

  9. Hinterdorfer, P., Baumgartner, W., Gruber, H.J., Schilcher, K. & Schindler, H. Detection and localization of individual antibody-antigen recognition events by atomic force microscopy. Proc. Natl. Acad. Sci. USA 93, 3477–3481 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J.M. & Gaub, H.E. Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276, 1109–1112 (1997).

    CAS  PubMed  Google Scholar 

  11. Oberhauser, A.F., Marszalek, P.E., Erickson, H.P. & Fernandez, J.M. The molecular elasticity of the extracellular matrix protein tenascin. Nature 393, 181–185 (1998).

    CAS  PubMed  Google Scholar 

  12. Rief, M., Clausen-Schaumann, H. & Gaub, H.E. Sequence-dependent mechanics of single DNA molecules. Nat. Struct. Biol. 6, 346–349 (1999).

    CAS  PubMed  Google Scholar 

  13. Benoit, M., Gabriel, D., Gerisch, G. & Gaub, H.E. Discrete interactions in cell adhesion measured by single-molecule force spectroscopy. Nat. Cell Biol. 2, 313–317 (2000).

    CAS  PubMed  Google Scholar 

  14. Lee, G.U., Kidwell, D.A. & Colton, R.J. Sensing discrete streptavidin-biotin interactions with atomic force microscopy. Langmuir 10, 354–357 (1994).

    CAS  Google Scholar 

  15. Fritz, J., Katopidis, A.G., Kolbinger, F. & Anselmetti, D. Force-mediated kinetics of single P-selectin/ligand complexes observed by atomic force microscopy. Proc. Natl. Acad. Sci. USA 95, 12283–12288 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Grandbois, M., Beyer, M., Rief, M., Clausen-Schaumann, H. & Gaub, H.E. How strong is a covalent bond? Science 283, 1727–1730 (1999).

    CAS  PubMed  Google Scholar 

  17. Harada, Y., Kuroda, M. & Ishida, A. Specific and quantized antigen-antibody interaction measured by atomic force microscopy. Langmuir 16, 708–715 (2000).

    CAS  Google Scholar 

  18. Touhami, A., Hoffmann, B., Vasella, A., Denis, F.A. & Dufrêne, Y.F. Probing specific lectin-carbohydrate interactions using atomic force microscopy imaging and force measurements. Langmuir 19, 1745–1751 (2003).

    CAS  Google Scholar 

  19. Bustanji, Y. et al. Dynamics of the interaction between a fibronectin molecule and a living bacterium under mechanical force. Proc. Natl. Acad. Sci. USA 100, 13292–13297 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Dammer, U. et al. Binding strength between cell adhesion proteoglycans measured by atomic force microscopy. Science 267, 1173–1175 (1995).

    CAS  PubMed  Google Scholar 

  21. Grandbois, M., Dettmann, W., Benoit, M. & Gaub, H.E. Affinity imaging of red blood cells using an atomic force microscope. J. Histochem. Cytochem. 48, 719–724 (2000).

    CAS  PubMed  Google Scholar 

  22. Touhami, A., Hoffmann, B., Vasella, A., Denis, F.A. & Dufrêne, Y.F. Aggregation of yeast cells: direct measurement of discrete lectin-carbohydrate interactions. Microbiol. SGM 149, 2873–2878 (2003).

    CAS  Google Scholar 

  23. Kienberger, F. et al. Recognition force spectroscopy studies of the NTA-His6 bond. Single Mol. 1, 59–65 (2000).

    CAS  Google Scholar 

  24. Schmitt, L., Ludwig, M., Gaub, H.E. & Tampé, R. A metal-chelating microscopy tip as a new toolbox for single-molecule experiments by atomic force microscopy. Biophys. J. 78, 3275–3285 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Dupres, V. et al. Nanoscale mapping and functional analysis of individual adhesins on living bacteria. Nat. Methods 2, 515–520 (2005).

    CAS  PubMed  Google Scholar 

  26. Berquand, A. et al. Antigen binding forces of single antilysozyme Fv fragments explored by atomic force microscopy. Langmuir 21, 5517–5523 (2005).

    CAS  PubMed  Google Scholar 

  27. Lee, G. et al. Nanospring behaviour of ankyrin repeats. Nature 440, 246–249 (2006).

    CAS  PubMed  Google Scholar 

  28. Hinterdorfer, P., Schilcher, K., Baumgartner, W., Gruber, H.J. & Schindler, H. A mechanistic study of the dissociation of individual antibody-antigen pairs by atomic force microscopy. Nanobiology 4, 39–50 (1998).

    Google Scholar 

  29. Allen, S. et al. Spatial mapping of specific molecular recognition sites by atomic force microscopy. Biochemistry 36, 7457–7463 (1997).

    CAS  PubMed  Google Scholar 

  30. Ros, R. et al. Antigen binding forces of individually addressed single-chain Fv antibody molecules. Proc. Natl. Acad. Sci. USA 95, 7402–7405 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Strunz, T., Oroszlan, K., Schäfer, R. & Güntherodt, H.-J. Dynamic force spectroscopy of single DNA molecules. Proc. Natl. Acad. Sci. USA 96, 11277–11282 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Yersin, A. et al. Interactions between synaptic vesicle fusion proteins explored by atomic force microscopy. Proc. Natl. Acad. Sci. USA 100, 8736–8741 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Haselgrübler, T., Amerstorfer, A., Schindler, H. & Gruber, H.J. Synthesis and applications of a new poly(ethylene glycol) derivative for the crosslinking of amines with thiols. Bioconjugate Chem. 6, 242–248 (1995).

    Google Scholar 

  34. Riener, C.K. et al. Bioconjugation for biospecific detection of single molecules in atomic force microscopy (AFM) and in single dye tracing (SDT). Recent Res. Devel. Bioconj. Chem. 1, 133–149 (2002).

    CAS  Google Scholar 

  35. Raab, A. et al. Antibody recognition imaging by force microscopy. Nat. Biotechnol. 17, 902–905 (1999).

    CAS  Google Scholar 

  36. Zara, J.J. et al. A carbohydrate-directed heterobifunctional cross-linking reagent for the synthesis of immunoconjugates. Anal. Biochem. 194, 156–162 (1991).

    CAS  PubMed  Google Scholar 

  37. Carlsson, J., Drevin, H. & Axen, R. Protein thiolation and reversible protein-protein conjugation. N-Succinimidyl 3-(2-pyridyldithio)propionate, a new heterobifunctional reagent. J. Biochem. 173, 723–737 (1978).

    CAS  Google Scholar 

  38. Li, F., Redick, S.D., Erickson, H.P. & Moy, V.T. Force measurements of the α5β1 integrin-fibronectin interaction. Biophys. J. 84, 1252–1262 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Lower, S.K., Hochella, M.F. & Beveridge, T.J. Bacterial recognition of mineral surfaces: nanoscale interactions between Shewanella and α-FeOOH. Science 292, 1360–1363 (2001).

    CAS  PubMed  Google Scholar 

  40. Bowen, W.R., Lovitt, R.W. & Wright, C.J. Atomic force microscopy study of the adhesion of Saccharomyces cerevisiae. J. Coll. Interf. Sci. 237, 54–61 (2001).

    CAS  Google Scholar 

  41. Razatos, A., Ong, Y.-L., Sharma, M.M. & Georgiou, G. Molecular determinants of bacterial adhesion monitored by atomic force microscopy. Proc. Natl. Acad. Sci. USA 95, 11059–11064 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Scheuring, S. & Sturgis, J.N. Chromatic adaptation of photosynthetic membranes. Science 309, 484–487 (2005).

    CAS  PubMed  Google Scholar 

  43. Wagner, P., Hegner, M., Kernen, P., Zaugg, F. & Semenza, G. Covalent immobilization of native biomolecules onto Au(111) via N-hydroxysuccinimide ester functionalized self-assembled monolayers for scanning probe microscopy. Biophys. J. 70, 2052–2066 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Wagner, P. Immobilization strategies for biological scanning probe microscopy. FEBS Lett. 430, 112–115 (1998).

    CAS  PubMed  Google Scholar 

  45. Karrasch, S., Dolder, M., Schabert, F., Ramsden, J. & Engel, A. Covalent binding of biological samples to solid supports for scanning probe microscopy in buffer solution. Biophys. J. 65, 2437–2446 (1993).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Klein, D.C. et al. Covalent immobilization of single proteins on mica for molecular recognition force microscopy. ChemPhysChem 4, 1367–1371 (2003).

    CAS  PubMed  Google Scholar 

  47. Wagner, P., Hegner, M., Guntherodt, H.-J. & Semenza, G. Formation and in situ modification of monolayers chemisorbed on ultraflat template-stripped gold surfaces. Langmuir 11, 3867–3875 (1995).

    CAS  Google Scholar 

  48. Radmacher, M., Tillmann, R.W., Fritz, M. & Gaub, H.E. From molecules to cells: imaging soft samples with the atomic force microscope. Science 257, 1900–1905 (1992).

    CAS  PubMed  Google Scholar 

  49. LeGrimellec, C. et al. Imaging of the surface of living cells by low-force contact-mode atomic force microscopy. Biophys. J. 75, 695–703 (1998).

    CAS  Google Scholar 

  50. Almqvist, N. et al. Elasticity and adhesion force mapping reveals real-time clustering of growth factor receptors and associated changes in local cellular rheological properties. Biophys. J. 86, 1753–1762 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Stroh, C.M. et al. Detection of HSP60 on the membrane surface of stressed human endothelial cells (HUVECs) by atomic force and confocal microscopy. J. Cell Sci. 118, 1587–1594 (2005).

    PubMed  Google Scholar 

  52. Schilcher, K., Hinterdorfer, P., Gruber, H.J. & Schindler, H. A non-invasive method for the tight anchoring of cells for scanning force microscopy. Cell Biol. Int. 21, 769–778 (1997).

    CAS  PubMed  Google Scholar 

  53. Le Grimellec, C. et al. High-resolution three-dimensional imaging of the lateral plasma membrane of cochlear outer hair cells by atomic force microscopy. J. Comp. Neurol. 451, 62–69 (2002).

    PubMed  Google Scholar 

  54. Schaer-Zammaretti, P. & Ubbink, J. Imaging of lactic acid bacteria with AFM - elasticity and adhesion maps and their relationship to biological and structural data. Ultramicroscopy 97, 199–208 (2003).

    CAS  PubMed  Google Scholar 

  55. Gad, M., Itoh, A. & Ikai, A. Mapping cell wall polysaccharides of living microbial cells using atomic force microscopy. Cell Biol. Int. 21, 697–706 (1997).

    CAS  PubMed  Google Scholar 

  56. Camesano, T.A., Natan, M.J. & Logan, B.E. Observation of changes in bacterial cell morphology using tapping mode atomic force microscopy. Langmuir 16, 4563–4572 (2000).

    CAS  Google Scholar 

  57. Kasas, S. & Ikai, A. A method for anchoring round shaped cells for atomic force microscope imaging. Biophys. J. 68, 1678–1680 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Dufrêne, Y.F., Boonaert, C.J., Gerin, P.A., Asther, M. & Rouxhet, P.G. Direct probing of the surface ultrastructure and molecular interactions of dormant and germinating spores of Phanerochaete chrysosporium. J. Bacteriol. 181, 5350–5354 (1999).

    PubMed  PubMed Central  Google Scholar 

  59. Bongrand, P., Capo, C., Mege, J.-L. & Benoliel, A.-M. Use of hydrodynamic flows to study cell adhesion. In Physical basis of cell adhesion (Bongrand, P., ed.) 125–156 (CRC Press, Boca Raton, Florida, 1988).

    Google Scholar 

  60. Leckband, D.E., Israelachvili, J.N., Schmitt, F.J. & Knoll, W. Long-range attraction and molecular rearrangements in receptor-ligand interactions. Science 255, 1419–1421 (1992).

    CAS  PubMed  Google Scholar 

  61. Merkel, R., Nassoy, P., Leung, A., Ritchie, K. & Evans, E. Energy landscapes of receptor-ligand bonds explored with dynamic force spectroscopy. Nature 397, 50–53 (1999).

    CAS  PubMed  Google Scholar 

  62. Ashkin, A. Optical trapping and manipulation of neutral particles using lasers. Proc. Natl. Acad. Sci. USA 94, 4853–4860 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Viani, M.B. et al. Small cantilevers for force spectroscopy of single molecules. J. Appl. Phys. 86, 2258–2262 (1999).

    CAS  Google Scholar 

  64. Burnham, N.A. et al. Comparison of calibration methods for atomic-force microscopy cantilevers. Nanotechnology 14, 1–6 (2003).

    CAS  Google Scholar 

  65. Evans, E. & Ritchie, K. Dynamic strength of molecular adhesion bonds. Biophys. J. 72, 1541–1555 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  66. Zhang, X.H., Bogorin, D.F. & Moy, V.T. Molecular basis of the dynamic strength of the sialyl Lewis X-selectin interaction. ChemPhysChem 5, 175–182 (2004).

    CAS  PubMed  Google Scholar 

  67. Bell, G.I. Models for the specific adhesion of cells to cells. Science 200, 618–627 (1978).

    CAS  PubMed  Google Scholar 

  68. Strunz, T., Oroszlan, K. & Schumakovitch, I. Güntherodt, H.-G. & Hegner, M. Model energy landscapes and the force-induced dissociation of ligand-receptor bonds. Biophys. J. 79, 1206–1212 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. Nevo, R. et al. A molecular switch between alternative conformational states in the complex of Ran and importin β1. Nat. Struct. Biol. 10, 553–557 (2003).

    CAS  PubMed  Google Scholar 

  70. Simons, K. & Ikonen, E. Functional rafts in cell membranes. Nature 387, 569–572 (1997).

    CAS  PubMed  Google Scholar 

  71. Cabeen, M.T. & Jacobs-Wagner, C. Bacterial cell shape. Nat. Rev. Microbiol. 3, 601–610 (2005).

    CAS  PubMed  Google Scholar 

  72. Ludwig, M., Dettmann, W. & Gaub, H.E. Atomic force microscope imaging contrast based on molecular recognition. Biophys. J. 72, 445–448 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  73. Lehenkari, P.P., Charras, G.T., Nykänen, A. & Horton, M.A. Adapting atomic force microscopy for cell biology. Ultramicroscopy 82, 289–295 (2000).

    CAS  PubMed  Google Scholar 

  74. Raab, A. et al. Antibody recognition imaging by force microscopy. Nat. Biotechnol. 17, 902–905 (1999).

    CAS  Google Scholar 

  75. Han, W., Lindsay, S.M. & Jing, T. A magnetically driven oscillating probe microscope for operation in liquid. Appl. Phys. Lett. 69, 1–3 (1996).

    Google Scholar 

  76. Han, W., Lindsay, S.M., Dlakic, M. & Harrington, R.E. Kinked DNA. Nature 386, 563 (1997).

    CAS  PubMed  Google Scholar 

  77. Stroh, C.M. et al. Simultaneous topography and recognition imaging using force microscopy. Biophys. J. 87, 1981–1990 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  78. Stroh, C. et al. Single-molecule recognition imaging microscopy. Proc. Natl. Acad. Sci. USA 101, 12503–12507 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  79. Ebner, A. et al. Localization of single avidin-biotin interactions using simultaneous topography and molecular recognition imaging. ChemPhysChem 6, 897–900 (2005).

    CAS  PubMed  Google Scholar 

  80. Wong, S.S., Joselevich, E., Woolley, A.T., Cheung, C.L. & Lieber, C.M. Covalently functionalized nanotubes as nanometre-sized probes in chemistry and biology. Nature 394, 52–55 (1998).

    CAS  PubMed  Google Scholar 

  81. Fritz, J. et al. Translating biomolecular recognition into nanomechanics. Science 288, 316–318 (2000).

    CAS  PubMed  Google Scholar 

  82. Wu, G. et al. Bioassay of prostate-specific antigen (PSA) using microcantilevers. Nat. Biotechnol. 19, 856–860 (2001).

    CAS  PubMed  Google Scholar 

  83. Ando, T. et al. A high-speed atomic force microscope for studying biological macromolecules. Proc. Natl. Acad. Sci. USA 98, 12468–12472 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  84. Humphris, A.D., Hobbs, J.K. & Miles, M.J. Ultrahigh-speed scanning near-field optical microscopy capable of over 100 frames per second. Appl. Phys. Lett. 83, 6–8 (2003).

    CAS  Google Scholar 

  85. Janovjak, H. Struckmeier & Müller, D.J. Hydrodynamic effects in fast AFM single-molecule force measurements. Eur. Biophys. J. 34, 91–96 (2005).

    CAS  PubMed  Google Scholar 

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Acknowledgements

Our work is supported by Belgian Funds (National Foundation for Scientific Research (FNRS), Fonds Spéciaux de Recherche (Université Catholique de Louvain), Interuniversity Poles of Attraction Programme (Federal Office for Scientific, Technical and Cultural Affairs), Région wallonne), by the Austrian National Science Fund, by the Austrian Nano and GENAU initiative from the Austrian Ministery of education, science and culture, and by the FP6 of the European Union. Y.F.D. is a Research Associate of the FNRS.

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  1. Institute for Biophysics, Johannes Kepler University of Linz, Altenbergerstr. 69, Linz, A-4040, Austria

    Peter Hinterdorfer

  2. Laboratory of Interfaces Chemistry, Catholic University of Louvain, Croix du Sud 2/18, Louvain-la-Neuve, B-1348, Belgium

    Yves F Dufrêne

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Correspondence to Peter Hinterdorfer or Yves F Dufrêne.

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Hinterdorfer, P., Dufrêne, Y. Detection and localization of single molecular recognition events using atomic force microscopy. Nat Methods 3, 347–355 (2006). https://doi.org/10.1038/nmeth871

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