Microfluidic device for real-time formulation of reagents and their subsequent encapsulation into double emulsions

Emulsion drops are often employed as picoliter-sized containers to perform screening assays. These assays usually entail the formation of drops encompassing discrete objects such as cells or microparticles and reagents to study interactions between the different encapsulants. Drops are also used to screen influences of reagent concentrations on the final product. However, these latter assays are less frequently performed because it is difficult to change the reagent concentration over a wide range and with high precision within a single experiment. In this paper, we present a microfluidic double emulsion drop maker containing pneumatic valves that enable real-time formulation of different reagents using pulse width modulation and consequent encapsulation of the mixed solutions. This device can produce drops from reagent volumes as low as 10 mu L with minimal sample loss, thereby enabling experiments that would be prohibitively expensive using drop generators that do not contain valves. We employ this device to monitor the kinetics of the cell-free synthesis of green fluorescent proteins inside double emulsions. To demonstrate the potential of this device for real-time formulation, we perform DNA titration experiments to test the influence of DNA concentration on the amount of green fluorescence protein produced in double emulsions by a coupled cell-free transcription/translation system.