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Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells
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Fluorescence cross-correlation spectroscopy using single wavelength laser.

Chao Xie1, Chaoqing Dong1, Jicun Ren1

  • 1College of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai, 200240 china.

Frontiers of Chemistry in China : Selected Publications From Chinese Universities
|April 15, 2020
PubMed
Summary

This study details a new fluorescence cross-correlation spectroscopy (FCCS) setup for analyzing molecular interactions. The optimized system accurately measures binding reactions, determining molecular concentration and size.

Keywords:
fluorescence cross-correlation spectroscopyquantum dotsingle laser excitationsingle-molecule detection

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Area of Science:

  • Biophysical Chemistry
  • Spectroscopy
  • Molecular Interactions

Background:

  • Fluorescence Cross-Correlation Spectroscopy (FCCS) is a powerful technique for studying molecular dynamics.
  • Accurate characterization of molecular interactions requires sensitive and optimized detection methods.
  • Previous FCCS setups often face challenges with cross-talk and detection volume.

Purpose of the Study:

  • To establish and optimize a single-wavelength laser-based Fluorescence Cross-Correlation Spectroscopy (FCCS) setup.
  • To validate the homebuilt FCCS system for studying molecular binding reactions.
  • To demonstrate the suppression of cross-talk effects using specific labeling probes.

Main Methods:

  • Development of a novel Fluorescence Cross-Correlation Spectroscopy (FCCS) system utilizing a single wavelength laser.
  • Systematic optimization of the FCCS setup to achieve a detection volume of approximately 0.7 fL.
  • Application of quantum dots (745 nm emission) and Rhodamine B (580 nm emission) as probes with a 532 nm laser to minimize cross-talk.

Main Results:

  • The optimized FCCS setup achieved a highly sensitive detection volume of ~0.7 fL.
  • Successful application of the system to study the binding reaction between human immunoglobulin G and goat antihuman immunoglobulin G.
  • Demonstrated near-complete suppression of cross-talk between fluorescent probes.

Conclusions:

  • The developed single-wavelength laser FCCS system is effective for studying molecular binding kinetics.
  • The system enables precise determination of molecule numbers, concentration, diffusion times, and hydrodynamic radii.
  • This optimized FCCS approach offers a robust tool for biophysical and biochemical analyses.