Measurement of Protein-Protein Interaction Dynamics Using Microfluidics and Particle Diffusometry

Area of Science:

• Biomolecular Sciences and Engineering
• Biophysics
• Biochemistry

Background:

• Protein-protein interactions are crucial for biotherapeutics, biosensors, and biomaterials.
• Existing methods for measuring these interactions are complex, requiring specialized facilities and expertise.
• There is a need for more accessible and convenient assays.

Purpose of the Study:

• To develop a novel, user-friendly method for measuring protein binding kinetics.
• To utilize microfluidics and particle diffusometry (PD) for this purpose.
• To provide an accurate and accessible alternative to current gold-standard assays.

Main Methods:

• Developed a label-free, particle-based assay using microfluidics and particle diffusometry (PD).
• Immobilized protein binding pairs on nanoparticles of different sizes.
• Measured changes in particle diffusivity in a microchannel using a fluorescence microscope and syringe pump.
• Quantified protein binding kinetics from diffusivity measurements over time.

Main Results:

• The method accurately quantifies protein binding kinetics using general laboratory equipment.
• Demonstrated feasibility by measuring the streptavidin-biotin association constant (1.74 ± 0.51 × 10^7 M^-1 s^-1).
• Results are comparable to established gold-standard methods in terms of accuracy.

Conclusions:

• The developed microfluidics and PD method is a simple, accurate, and accessible tool for measuring protein binding kinetics.
• This technique lowers the barrier for routine analysis of protein interactions in various research settings.
• Facilitates advancements in biotherapeutic design, biomolecular sensing, and protein-based biomaterials.