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Correction: Yukhnovets et al. Impact of Molecule Concentration, Diffusion Rates and Surface Passivation on Single-Molecule Fluorescence Studies in Solution. <i>Biomolecules</i> 2022, <i>12</i>, 468.

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Macromolecular High-Affinity Binding Probed by Advanced Fluorescence Techniques.

Alida Meyer1, Benno Schedler1, Jörg Fitter1,2

  • 1RWTH Aachen University, I. Physikalisches Institut (IA), AG Biophysik, 52074, Aachen, Germany.

Chembiochem : a European Journal of Chemical Biology
|May 27, 2025
PubMed
Summary
This summary is machine-generated.

Advanced fluorescence microscopy techniques enable precise measurement of high-affinity biomolecular binding, even at picomolar concentrations. These methods offer broad applicability from living cells to complex sample environments.

Keywords:
FRETbiosensorsfluorescence spectroscopyprotein‐protein interactionssingle‐molecule studies

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Area of Science:

  • Biochemistry
  • Biophysics
  • Analytical Chemistry

Background:

  • Fluorescence techniques offer extreme sensitivity and selectivity for detecting molecular interactions.
  • Modern fluorescence microscopy has seen significant methodological advancements.
  • High-affinity binding can be measured at picomolar concentrations.

Purpose of the Study:

  • To describe the physical principles and experimental setups of fluorescence detection.
  • To present advanced fluorescence microscopy techniques for characterizing biomolecular binding.
  • To compare the advantages and limitations of various techniques.

Main Methods:

  • Confocal fluorescence microscopy for analyzing biomolecules in solution.
  • Single-molecule Förster resonance energy transfer (smFRET).
  • Two-color coincidence detection and fluorescence correlation spectroscopy (FCS).

Main Results:

  • Advanced techniques demonstrate broad applicability, including genetically encoded biosensors in living cells.
  • High controllability in measuring binding curves at very low molecule concentrations.
  • Characterization of binding in complex sample environments with minimal sample consumption.

Conclusions:

  • Fluorescence microscopy provides powerful tools for studying biomolecular interactions with high sensitivity and selectivity.
  • Advanced techniques like smFRET and FCS offer unique capabilities for single-molecule analysis.
  • These methods are crucial for understanding biological processes at the molecular level.