From the Contents 1. Introduction 24369 2. Single Emulsions 24371 3. Double Emulsions 24378 4. Latest Developments and Label-Free Methods 24382

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.

We present an optimized protocol to encapsulate bacteria inside giant unilamellar lipid vesicles combined with a microfluidic platform for real-time monitoring of microbial growth and production. The microfluidic device allows us to immobilize the lipid vesicles and record bacterial growth and production using automated microscopy. Moreover, the lipid vesicles retain hydrophilic molecules and therefore can be used to accumulate products of microbial biosynthesis, which we demonstrate here for a riboflavin-producing bacterial strain. We show that stimulation as well as inhibition of bacterial production can be performed through the liposomal membrane simply by passive diffusion of inducing or antibiotic compounds, respectively. The possibility to introduce as well as accumulate compounds in liposomal cultivation compartments represents great advantage over the current state of the art systems, emulsion droplets, and gel beads. Additionally, the encapsulation of bacteria and monitoring of individual lipid vesicles have been accomplished on a single microfluidic device. The presented system paves the way toward highly parallel microbial cultivation and monitoring as required in biotechnology, basic research, or drug discovery.

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...

We present a new, general concept termed Hydrodynamic Thermal Confinement (HTC), and its implementation for the creation of microscale dynamic thermo-chemical microenvironments on biological surfaces. HTC is based on a scanning probe and operates under physiological conditions. The temperature can be regulated between 30° and 80 °C with ±0.2 °C precision and temperature ramps of 5 °C s over a footprint of ∼50 μm × 80 μm in a volume of ∼50 × 80 × 15 μm (∼50 pl).