[Rapid generation of double-layer emulsion droplets based on microfluidic chip]

Area of Science:

• Biotechnology
• Microfluidics
• Molecular Biology

Background:

• In vitro compartmentalization (IVC) with fluorescence-activated cell sorting (FACS) is crucial for high-throughput screening in protein engineering.
• Previous mechanical dispersion methods for droplet generation lacked homogeneity, hindering screening accuracy and efficiency.
• Advancements in microfluidic technology offer improved control over droplet generation.

Purpose of the Study:

• To develop a microfluidic chip-based method for generating uniform double emulsion droplets for biological screening.
• To overcome limitations in droplet size homogeneity and stability associated with traditional methods.
• To establish a foundation for advanced droplet-based high-throughput screening.

Main Methods:

• Fabrication of water-in-oil (W/O) single-layer and water-in-oil-in-water (W/O/W) double-layer droplet generation chips.
• Optimization of flow rates and phase ratios for precise control over droplet size and stability.
• Encapsulation and culture of Escherichia coli within double emulsion droplets for protein expression studies.

Main Results:

• Generation of monodisperse single-layer W1/O droplets (15.4–23.2 μm) with long-term stability.
• Production of uniform W1/O/W2 double-layer emulsion droplets (30–100 μm) at a rate of 1,000 droplets/s.
• Successful culture and protein expression of encapsulated E. coli within the generated droplets.

Conclusions:

• Microfluidic chip-based droplet generation provides superior control over droplet size and uniformity compared to mechanical methods.
• The developed double emulsion system supports cell encapsulation, culture, and protein expression, suitable for high-throughput screening.
• This technology advances droplet-based screening platforms, enhancing efficiency and accuracy in biological applications.