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Quantifying Reversible Surface Binding via Surface-Integrated Fluorescence Correlation Spectroscopy.

Jonas Mücksch1, Philipp Blumhardt1, Maximilian T Strauss1,2

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Summary
This summary is machine-generated.

This study introduces a novel single-molecule method using fluorescence correlation spectroscopy to accurately measure binding rates of molecules to surfaces. The technique quantifies surface binding affinities and kinetics, advancing surface science research.

Keywords:
DNA hybridizationDNA-PAINTTotal internal reflection fluorescence correlation spectroscopy (TIR-FCS)binding ratessurface binding kinetics

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Area of Science:

  • Biophysics
  • Surface Chemistry
  • Molecular Interactions

Background:

  • Accurate quantification of molecular binding kinetics to surfaces is crucial for understanding biological processes and developing new materials.
  • Existing methods often face limitations in sensitivity, time resolution, or surface selectivity.

Purpose of the Study:

  • To develop a versatile and accurate single-molecule method for determining binding and dissociation rates of molecules to surfaces.
  • To adapt fluorescence correlation spectroscopy with total internal reflection illumination (TIR-FCS) for enhanced surface binding rate analysis.

Main Methods:

  • Modification of total internal reflection fluorescence correlation spectroscopy (TIR-FCS) with camera-based detection.
  • Spatially integrated signal correlation analysis, disregarding lateral spatial information to define the measurement volume.
  • Utilizing fluorescently labeled single-stranded DNA and surface-immobilized DNA origami structures as a model system.

Main Results:

  • The developed method accurately resolves association and dissociation rates in equilibrium over a wide range of time scales.
  • Demonstrated high surface selectivity for quantifying reversible surface attachment.
  • Successfully quantified surface affinities and binding rates for DNA duplexes with varying lengths.

Conclusions:

  • The presented single-molecule method offers a simple, versatile, and accurate approach for measuring surface binding kinetics.
  • This technique enhances the understanding of molecular interactions at surfaces and has broad applications in surface science and biophysics.