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Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects
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Biotin-avidin binding kinetics measured by single-molecule imaging.

Joshua R Wayment1, Joel M Harris

  • 1Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112-0850, USA.

Analytical Chemistry
|January 2, 2009
PubMed
Summary

Researchers developed a single-molecule fluorescence method to measure neutravidin binding kinetics to biotin. This technique offers insights into biotin-avidin complex formation and stability at liquid-solid interfaces for bioanalytical applications.

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Area of Science:

  • Biochemistry
  • Surface Chemistry
  • Biophysics

Background:

  • Avidin-biotin interaction is crucial for bioanalytical applications, enabling biomolecule immobilization.
  • Understanding binding kinetics at liquid-solid interfaces is vital for sensor and separation technologies.
  • Neutravidin, a deglycosylated avidin variant, is used for its specific binding properties.

Purpose of the Study:

  • To develop a single-molecule fluorescence method for measuring neutravidin-biotin binding kinetics.
  • To determine the affinity constant of neutravidin binding to surface-immobilized biotin.
  • To investigate the influence of immobilization chemistry on complex stability.

Main Methods:

  • Immobilization of biotin onto amine-functionalized glass surfaces using succinimidyl ester chemistry.
  • Controlled surface density of biotin via co-deposition with a silane.
  • Single-molecule fluorescence imaging using total-internal-reflection fluorescence (TIRF) microscopy.
  • In situ measurement of binding and unbinding events of labeled neutravidin.

Main Results:

  • Developed a robust single-molecule method for detecting and counting neutravidin binding events.
  • Quantified binding kinetics, revealing fast, diffusion-controlled association rates.
  • Observed that biotin immobilization chemistry influences the dissociation rate and complex stability.
  • Achieved high spatial resolution by creating widely spaced biotin binding sites.

Conclusions:

  • The developed single-molecule TIRF method accurately measures neutravidin-biotin binding kinetics and affinity.
  • Neutravidin binding is rapid and largely diffusion-limited.
  • Surface chemistry significantly impacts the stability and dissociation of the neutravidin-biotin complex.
  • This method provides a powerful tool for optimizing surface-based bioanalytical systems.