Multivesicular vesicles (MVVs) are artificial liposomal structures widely used as a platform to study the compartmentalisation of cells and as a scaffold for artificial cell/protocell models. Current preparation techniques for MVVs, however, offer poor control on the size, lamellarity, and loading of inner lipid vesicles. Here, we introduce a microfluidic device for the production of multivesicular droplets (MVDs): a novel model system combining the ease of use and control of droplet microfluidics with the biological relevance of MVVs. We use a perfluorinated carrier phase with a biocompatible surfactant to generate monodisperse droplets of an aqueous giant unilamellar lipid vesicle suspension. The successful on-chip formation and stability of MVDs is verified through high-speed microscopy. For bright field or fluorescence microscopy inspection, the MVDs are trapped in an array where the integrity of both lipid vesicles and droplets is preserved for up to 15 minutes. Finally, we show a two-step enzymatic reaction that takes place across the lipid vesicle membranes; the second reaction step occurs in the vesicle's interior, where the enzyme is encapsulated, while both the substrate and fluorescent product permeate across the membrane. Our approach opens the possibility to mimic artificial organelles with optimised reaction parameters (pH, ions, etc.) in each compartment.

The global surge in bacterial resistance against traditional antibiotics triggered intensive research for novel compounds, with antimicrobial peptides (AMPs) identified as a promising candidate. Automated methods to systematically generate and screen AMPs according to their membrane preference, however, are still lacking. We introduce a novel microfluidic system for the simultaneous cell-free production and screening of AMPs for their membrane specificity. On our device, AMPs are cell-free produced within water-in-oil-inwater double emulsion droplets, generated at high frequency. Within each droplet, the peptides can interact with different classes of co-encapsulated liposomes, generating a membrane-specific fluorescent signal. The double emulsions can be incubated and observed in a hydrodynamic trapping array or analyzed via flow cytometry. Our approach provides a valuable tool for the discovery and development of membrane-active antimicrobials.

for drug testing and discovery, directed evolution, antibody screening, sequencing, and many more. [2][3][4][5] Moreover, there are numerous examples where droplet microfluidics was employed for material studies and chemical synthesis. [6][7][8] However, droplet microfluidics has limitations; during long-term incubation, droplets suffer from volume reduction due to water diffusion out of the droplet into the oil, leading to changes in concentration of the encapsulated species. Furthermore, many applications require the addition of compounds to the droplet at a defined time point, which is achieved by droplet fusion, [9][10][11] triple emulsions, [12] or picoinjection. [13] These droplet manipulation steps require complicated setups or control by imaging and address each droplet individually, resulting in a decreased throughput. Moreover, the addition of molecules leads to a volume change and, hence, to the dilution of the encapsulated components. The encapsulation of cells within gel microbeads partially overcomes these limitations. [14] The use of gel microbeads makes it possible to address all entrapped cells at a selected time point, with massive parallelization capabilities. [15] Moreover, the gel matrix provides the necessary stability for flow cytometric analysis. [16,17] Nevertheless, more advanced systems for studying molecules secreted by cells or performing chemical reactions require additional means to prevent leakage of the studied molecules and cross contamination between microbeads. This can be achieved, for example, via chemical bonds [18,19] or by isolating beads using a hydrophobic phase, de facto forming an emulsion. [20,21] The flexibility of gel compartmentalization is therefore hindered by the necessity for supplementary immobilization protocols or the need for an additional immiscible phase, which has to be removed prior to flow cytometric analysis.Another promising approach for compartmentalization uses water-in-oil-in-water double emulsions (DEs). [22][23][24] In contrast to single emulsions, DEs are compatible with routine highthroughput screening instruments such as fluorescence-activated cell sorters (FACS). [25][26][27] This convenient use of standard instruments makes DEs an ideal tool for screening large variant libraries. [28][29][30][31] Compared to single emulsions, decreasing the oil phase to a thin shell surrounding the droplet also simplifies the access to individual DE vessels from the surrounding environment and the transfer of molecules through the oil shell can be partially regulated. [32] Previous studies show that small molecules such as fluorescein diacetate or anhydrotetracycline can pass the oil shell barrier and reach individual droplets, allowing for Microfluidic methods for the formation of single and double emulsion (DE) droplets allow for the encapsulation and isolation of reactants inside nanoliter compartments. Such methods have greatly enhanced the toolbox for high-throughput screening for cell or enzyme engineering and drug discovery. However, remaining challe...

The global surge in bacterial resistance against traditional antibiotics triggered intensive research for novel compounds, with antimicrobial peptides (AMPs) identified as a promising candidate. Automated methods to systematically generate and screen AMPs according to their membrane preference, however, are still lacking. We introduce a novel microfluidic system for the simultaneous cell-free production and screening of AMPs for their membrane specificity. On our device, AMPs are cell-free produced within water-in-oil-in-water double emulsion droplets, generated at high frequency. Within each droplet, the peptides can interact with different classes of co-encapsulated liposomes, generating a membrane-specific fluorescent signal. The double emulsions can be incubated and observed in a hydrodynamic trapping array or analysed via flow cytometry. Our approach provides a valuable tool for the discovery and development of membrane-active antimicrobials.

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.