A double emulsion microfluidic platform for in vitro GFP expression

We have first developed a simple microfluidic based double emulsion system with potential applications in in vitro protein synthesis. The system involves a two-step process to make water-in-oil-in-water (W/O/W) emulsions. A PMMA microchip is used for the formation of water-in-oil (W/O) single emulsion droplets. Then the single emulsion droplets are transported to a PDMS/glass microchip to make the W/O/W double emulsion droplets. The system was first characterized by detecting fluorescein sodium salt as a model dye in the internal aqueous droplets using laser induced fluorescence. This allowed us to optimise the flow rates of the internal aqueous phase and outer continuous aqueous phase for the formation of the double emulsion droplets. On-chip storage of double emulsion droplets was then investigated to allow for protein synthesis from a PCR-generated DNA template using either commercial in vitro transcription and translation (IVTT) kits or crude E. coli S30 extracts. In vitro expression of the green fluorescent protein (GFP) was demonstrated successfully using this system.