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Related Experiment Videos

Immunoglobulin surface-binding kinetics studied by total internal reflection with fluorescence correlation

N L Thompson, D Axelrod

    Biophysical Journal
    |July 1, 1983
    PubMed
    Summary

    Total internal reflection with fluorescence correlation spectroscopy (TIR/FCS) measures molecular binding and diffusion at surfaces. This technique quantifies adsorption/desorption kinetics, proving useful for studying solute-surface interactions.

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    Area of Science:

    • Surface science
    • Biophysical chemistry
    • Spectroscopy

    Background:

    • Understanding molecular interactions at surfaces is crucial in various scientific fields.
    • Existing techniques may require specific conditions or perturbations, limiting their applicability.
    • Accurate measurement of binding and diffusion kinetics is essential for characterizing surface phenomena.

    Purpose of the Study:

    • To present and validate total internal reflection with fluorescence correlation spectroscopy (TIR/FCS) as a novel technique.
    • To measure binding and unbinding rates and surface diffusion coefficients of fluorescent-labeled molecules at equilibrium.
    • To demonstrate the feasibility of TIR/FCS for quantifying solute-surface kinetic rates.

    Main Methods:

    • Utilized total internal reflection (TIR) to selectively excite surface-adsorbed fluorescent molecules.

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  • Employed fluorescence correlation spectroscopy (FCS) to analyze fluorescence fluctuations from a small surface area (~5 µm²).
  • Collected and autocorrelated fluorescence data using a photomultiplier and minicomputer to determine kinetic parameters.

    • Main Results:

    • TIR/FCS successfully measured binding and unbinding rates and surface diffusion coefficients.
    • Rhodamine-labeled immunoglobulin and insulin showed both reversible and irreversible adsorption to albumin-coated surfaces.
    • Antibody fragments demonstrated specific binding to DNP-coated surfaces, alongside significant nonspecific binding.

    Conclusions:

    • TIR/FCS is a feasible method for measuring equilibrium absorption/desorption kinetic rates at surfaces.
    • The technique is sensitive to molecular diffusion and binding dynamics.
    • TIR/FCS holds promise for quantifying specific solute-surface kinetic rates in systems with low nonspecific binding.