Microfluidic mixers for studying protein folding.

Area of Science:

• Biophysics and Structural Biology
• Chemical Engineering and Microfluidics

Background:

• Protein folding is crucial for biological function and human health but remains poorly understood due to its complex timescales.
• Conventional stopped-flow mixers are limited to studying folding kinetics starting around 1 millisecond.

Purpose of the Study:

• To develop and characterize a novel microfluidic mixer capable of achieving ultra-fast denaturant dilution for protein folding studies.
• To enable the investigation of protein folding dynamics on timescales faster than previously accessible.

Main Methods:

• A microfluidic device with micron-scale channels (5-10 μm width, 10 μm depth) was fabricated using silicon or fused silica.
• The mixer operates in the laminar flow regime (Reynolds number ≤100) to ensure predictable fluid dynamics and enable precise numerical simulations.
• Protein folding is initiated by diluting guanidine hydrochloride from 6 M to 0.06 M in approximately 8 microseconds using a unique channel geometry that constricts the protein flow.

Main Results:

• The microfluidic mixer achieves a ~100-fold denaturant dilution in ~8 microseconds, significantly faster than conventional methods.
• Precise numerical simulations of the fluid flow within the mixer showed excellent agreement with experimental observations.
• The device allows for the observation of protein folding dynamics using techniques like tryptophan fluorescence or FRET at high temporal resolution.

Conclusions:

• The developed microfluidic mixer provides a powerful new tool for studying protein folding kinetics at microsecond timescales.
• This technology overcomes the temporal limitations of traditional methods, opening new avenues for understanding protein dynamics and misfolding-related diseases.