Rapid Microfluidic Dilution for Single-Molecule Spectroscopy of Low-Affinity Biomolecular Complexes

Area of Science:

• Biophysics
• Biochemistry
• Analytical Chemistry

Background:

• Investigating low-affinity biomolecular complexes is crucial for understanding cellular processes.
• Confocal single-molecule spectroscopy requires precise control over sample concentration.

Purpose of the Study:

• To develop a microfluidic device for rapid, large-fold sample dilution.
• To enable the study of low-affinity biomolecular interactions using single-molecule spectroscopy.

Main Methods:

• Microfluidic device fabrication for rapid dilution (up to 10^5 fold in milliseconds).
• Single-molecule two-color and three-color Förster resonance energy transfer (FRET) spectroscopy.
• Analysis of dissociation kinetics and structural properties of protein complexes.

Main Results:

• The microfluidic device achieves dilution at the diffusion limit.
• Successfully studied dissociation kinetics and structural properties of low-affinity protein complexes.
• Demonstrated suitability for complexes with dissociation constants from low nanomolar to 10 μm.

Conclusions:

• The developed microfluidic device significantly enhances the study of low-affinity biomolecular interactions.
• The device offers versatility for a broad range of biomolecular binding affinities.
• The design ensures reliable operation and reproducible results for biophysical studies.