Direct Optical Detection of Protein-Ligand Interactions

  • Frank Gesellchen
  • Bastian Zimmermann
  • Friedrich W. Herberg
Part of the Methods in Molecular Biology™ book series (MIMB, volume 305)


Direct optical detection provides an excellent means to investigate interactions of molecules in biological systems. The dynamic equilibria inherent to these systems can be described in greater detail by recording the kinetics of a biomolecular interaction. Optical biosensors allow direct detection of interaction patterns without the need for labeling. An overview covering several commercially available biosensors is given, with a focus on instruments based on surface plasmon resonance (SPR) and reflectometric interference spectroscopy (RIFS). Potential assay formats and experimental design, appropriate controls, and calibration procedures, especially when handling low molecular weight substances, are discussed. The single steps of an interaction analysis combined with practical tips for evaluation, data processing, and interpretation of kinetic data are described in detail. In a practical example, a step-by-step procedure for the analysis of a low molecular weight compound interaction with serum protein, determined on a commercial SPR sensor, is presented.

Key Words

Optical biosensors surface plasmon resonance reflectometric interference spectroscopy biomolecular interaction analysis kinetics low molecular weight ligands 


  1. 1.
    Herberg F. W. and Zimmermann B. (1999) Analysis of protein kinase interactions using biomolecular interaction analysis. In Protein Phosphorylation-A Practical Approach (Hardie D. G., ed.), Vol. 2, pp. 335–371. Oxford University Press, Oxford.Google Scholar
  2. 2.
    Stenberg E., Persson B., Roos H. and Urbaniczky C. (1991) Quantitative determination of surface concentration of protein with surface plasmon resonance using radiolabeled proteins.J. Colloid Interface Sci. 143,513–526.CrossRefGoogle Scholar
  3. 3.
    Piehler J., Brecht A., Geckeler K. E., and Gauglitz G. (1996) Surface modification for direct immunoprobes. Biosens. Bioelectron. 11, 579–590.PubMedCrossRefGoogle Scholar
  4. 4.
    Piehler J., Brecht A., and Gauglitz G. (1996) Affinity detection of low molecular weight analytes. Analytical Chemistry 68, 139–143.CrossRefGoogle Scholar
  5. 5.
    Piehler J., Brecht A., Gauglitz G., Zerlin M., Maul C., Thiericke R., and Grabley S. (1997) Label-Free Monitoring of DNA-Ligand Interactions. Analytical Biochemistry 249, 94–102.PubMedCrossRefGoogle Scholar
  6. 6.
    Birkert O. and Gauglitz G. (2002) Development of an assay for label-free high-throughput screening of thrombin inhibitors by use of reflectometric interference spectroscopy. Anal. Bioanal. Chem. 372, 141–147.PubMedCrossRefGoogle Scholar
  7. 7.
    Birkert O., Tunnemann R., Jung G., and Gauglitz G. (2002) Label-free parallel screening of combinatorial triazine libraries using reflectometric interference spectroscopy. Anal. Chem. 74, 834–840.PubMedCrossRefGoogle Scholar
  8. 8.
    Kröger K., Bauer J., Fleckenstein B., Rademann J., Jung G., and Gauglitz G. (2002) Epitope-mapping of transglutaminase with parallel label-free optical detection. Biosensors and Bio electronics 17, 937–944.Google Scholar
  9. 9.
    Hanel C. and Gauglitz G. (2002) Comparison of reflectometric interference spectroscopy with other instruments for label-free optical detection. Anal. Bioanal. Chem. 372, 91–100.PubMedCrossRefGoogle Scholar
  10. 10.
    Wink T., de Beer J., Hennink W. E., Bult A., and van Bennekom W. P. (1999) Interaction between plasmid DNA and cationic polymers studied by surface plasmon resonance spectrometry. Analytical Chemistry 71, 801–805.CrossRefGoogle Scholar
  11. 11.
    Melendez J., Carr R., Bartholomew D. U., Kukanskis K., Elkind J., Woodbury R., Furlong C., and Yee S. (1996) A commercial solution for surface plasmon sensing. Sensors and Actuators B: Chemical 35, 212–216.CrossRefGoogle Scholar
  12. 12.
    Cush R., Cronin J., Steward W., Maule C., Molloy J., and Goddard N. (1993) The resonant mirror: a novel optical biosensor for direct sensing of biomolecular interactions, Part I: Principle of operation and associated instrumentation. Biosens. Bioelectron. 8, 347–354.CrossRefGoogle Scholar
  13. 13.
    Cunningham B., Li P., Lin B., and Pepper J. (2002) Colorimetric resonant reflection as a direct biochemical assay technique. Sensors and Actuators B: Chemical 81, 316–328.CrossRefGoogle Scholar
  14. 14.
    Baird C. L. and Myszka D. G. (2001) Current and emerging commercial optical biosensors. J. Mol. Recognit. 14, 261–268.PubMedCrossRefGoogle Scholar
  15. 15.
    Rich R. L. and Myszka D. G. (2003) A survey of the year 2002 commercial optical biosensor literature. J. Mol. Recognit. 16, 351–382.PubMedCrossRefGoogle Scholar
  16. 16.
    Zimmermann B., Hahnefeld C., and Herberg F. W. (2002) Applications of biomolecular interaction analysis in drug development. TARGETS 1, 66–73.CrossRefGoogle Scholar
  17. 17.
    Engvall E. (1980) Enzyme immunoassay ELISA and EMIT. Methods Enzymol. 70,419–439.PubMedCrossRefGoogle Scholar
  18. 18.
    Karlsson R. (1994) Real-time competitive kinetic analysis of interactions between low-molecular-weight ligands in solution and surface-immobilized receptors. Anal. Biochem. 221, 142–151.PubMedCrossRefGoogle Scholar
  19. 19.
    Dillon P. P., Daly S. J., Manning B. M., and O'Kennedy R. (2003) Immunoassay for the determination of morphine-3-glucuronide using a surface plasmon resonance-based biosensor. Biosensors and Bioelectronics 18, 217–227.PubMedCrossRefGoogle Scholar
  20. 20.
    Tudos A. J., Lucas-van den Bos E. R., and Stigter E. C. (2003) Rapid surface plasmon resonance-based inhibition assay of deoxynivalenol. J. Agric. Food Chem. 51, 5843–5848.PubMedCrossRefGoogle Scholar
  21. 21.
    Deinum J., Gustavsson L., Gyzander E., Kullman-Magnusson M., Edström Å., and Karlsson R. (2002) A thermodynamic characterization of the binding of thrombin inhibitors to human thrombin, combining biosensor technology, stopped-flow spectrophotometry, and microcalorimetry. Analytical Biochemistry 300, 152–162.PubMedCrossRefGoogle Scholar
  22. 22.
    Day Y. S., Baird C. L., Rich R. L., and Myszka D. G. (2002) Direct comparison of binding equilibrium, thermodynamic, and rate constants determined by surface-and solution-based biophysical methods. Protein Sci. 11, 1017–1025.PubMedCrossRefGoogle Scholar
  23. 23.
    Roos H., Karlsson R., Nilshans H., and Persson A. (1998) Thermodynamic analysis of protein interactions with biosensor technology. J. Mol. Recognit. 11, 204–210.PubMedCrossRefGoogle Scholar
  24. 24.
    Markgren P. O., Schaal W., Hamalainen M., Karlen A., Hallberg A., Samuelsson B., and Danielson U. H. (2002) Relationships between structure and interaction kinetics for HIV-1 protease inhibitors. J. Med. Chem. 45,5430–5439.PubMedCrossRefGoogle Scholar
  25. 25.
    Karlsson R., Kullman-Magnusson M., Hamalainen M. D., Remaeus A., Andersson K., Borg P., Gyzander E., and Deinum J. (2000) Biosensor analysis of drug-target interactions: direct and competitive binding assays for investigation of interactions between thrombin and thrombin inhibitors. Anal. Biochem. 278, 1–13.PubMedCrossRefGoogle Scholar
  26. 26.
    Gestwicki J. E., Hsieh H. V., and Pitner J. B. (2001) Using receptor conforma-tional change to detect low molecular weight analytes by surface plasmon resonance. Anal. Chem. 73, 5732–5737.PubMedCrossRefGoogle Scholar
  27. 27.
    Carrasco C., Facompre M., Chisholm J. D., Van Vranken D. L., Wilson W. D., and Bailly C. (2002) DNA sequence recognition by the indolocarbazole antitu-mor antibiotic AT2433-B1 and its diastereoisomer. Nucl. Acids. Res. 30, 1774–1781.PubMedCrossRefGoogle Scholar
  28. 28.
    Hendrix M., Priestley S. E., Joyve G. F., and Wong C.-H. (1997) Direct observation of aminoglycoside-RNA interactions by surface plasmon resonance. J. Am. Chem. Soc. 119, 3641–3648.PubMedCrossRefGoogle Scholar
  29. 29.
    Chapman R. L., Stanley T. B., Hazen R., and Garvey E. P. (2002) Small molecule modulators of HIV Rev/Rev response element interaction identified by random screening. Antiviral Res. 54, 149–162.PubMedCrossRefGoogle Scholar
  30. 30.
    Li K., Davis T. M., Bailly C., Kumar A., Boykin D. W., and Wilson W. D. (2001) A heterocyclic inhibitor of the REV-RRE complex binds to RRE as a dimer. Biochemistry 40, 1150–1158.PubMedCrossRefGoogle Scholar
  31. 31.
    Cheng X., Phelps C., and Taylor S. S. (2001) Differential binding of cAMP-dependent protein kinase regulatory subunit isoforms Ialpha and IIbeta to the catalytic subunit. J. Biol. Chem. 276, 4102–4108.PubMedCrossRefGoogle Scholar
  32. 32.
    Rich R. L., Day Y. S., Morton T. A., and Myszka D. G. (2001) High-resolution and high-throughput protocols for measuring drug/human serum albumin interactions using BIACORE. Anal. Biochem. 296, 197–207.PubMedCrossRefGoogle Scholar
  33. 33.
    Yaqub S., Abrahamsen H., Zimmerman B., Kholod N., Torgersen K. M., Mustelin T., Herberg F. W., Tasken K., and Vang T. (2003) Activation of C-terminal Src kinase (Csk) by phosphorylation at serine-364 depends on the Csk-Src homology 3 domain. Biochem. J. 372, 271–278.PubMedCrossRefGoogle Scholar
  34. 34.
    Myszka D. G. (2000) Kinetic, equilibrium, and thermodynamic analysis of mac-romolecular interactions with BIACORE. Methods Enzymol. 323, 325–340.PubMedCrossRefGoogle Scholar
  35. 35.
    Karlsson R. and Falt A. (1997). Experimental design for kinetic analysis of protein-protein interactions with surface plasmon resonance biosensors. J. Immunol. Methods 200, 121–133.PubMedCrossRefGoogle Scholar
  36. 36.
    Langmuir I. (1916) The constitution and fundamental properties of solids and liquids. Part I. Solids. J. Am. Chem. Soc. 38, 2221–2295.CrossRefGoogle Scholar
  37. 37.
    Langmuir I. (1918) The adsorption of gases on plane surfaces of glass, mica and platinum. J. Am. Chem. Soc. 40, 1361–1403.CrossRefGoogle Scholar
  38. 38.
    Hall D. R., Gorgani N. N., Altin J. G., and Winzor D. J. (1997) Theoretical and experimental considerations of the pseudo-first-order approximation in conventional kinetic analysis of IAsys biosensor data. Anal. Biochem. 253, 145–155.PubMedCrossRefGoogle Scholar
  39. 39.
    O’shannessy D. J. and Winzor D. J. (1996) Interpretation of deviations from pseudo-first-order kinetic behavior in the characterization of ligand binding by biosensor technology. Anal. Biochem. 236, 275–283.CrossRefGoogle Scholar
  40. 40.
    Hall D. R., Cann J. R., and Winzor D. J. (1996) Demonstration of an upper limit to the range of association rate constants amenable to study by biosensor technology based on surface plasmon resonance. Anal. Biochem. 235, 175–184.PubMedCrossRefGoogle Scholar
  41. 41.
    Schuck P. and Minton A. P. (1996) Analysis of mass transport-limited binding kinetics in evanescent wave biosensors. Anal. Biochem. 240, 262–272.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2005

Authors and Affiliations

  • Frank Gesellchen
    • 1
  • Bastian Zimmermann
    • 2
  • Friedrich W. Herberg
    • 1
  1. 1.Department of BiochemistryUniversity of KasselKasselGermany
  2. 2.Biaffin GmbH & Co KGKasselGermany

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