Microfluidic methods to form single emulsion and double emulsion (DE) droplets have greatly enhanced the toolbox for high throughput screening for cell or enzyme engineering and drug discovery. However, remaining challenges in the supply of reagents into these enclosed nanoliter compartments limit the applicability of droplet microfluidics. Here, we introduce a strategy for on-demand delivery of reactants in DEs. We use lipid vesicles as transport carriers, which are co-encapsulated in double emulsions and release their cargo upon addition of an external trigger, here the anionic surfactant SDS. The reagent present inside the lipid vesicles stays isolated from the remaining content of the DE vessel until SDS enters the DE lumen and solubilizes the lipid bilayer. We demonstrate the versatility of the method with two critical applications, chosen as representative assays for high throughput screening. First, we trigger enzymatic reactions after releasing a reactant and second, we encapsulate bacteria and induce gene expression at a delayed time. The presented technique is compatible with the high throughput analysis of individual DE droplets using conventional flow cytometry as well as with microfluidic time-resolved studies. The possibility of delaying and controlling reagent delivery in current high throughput compartmentalization systems will significantly extend their range of applications e.g. for directed evolution, and further improve their compatibility with biological systems.
Double emulsion flow cytometry with high-throughput single droplet isolation and nucleic acid recovery
