Compartments for the spatially and temporally controlled assembly of biological processes are essential towards cellular life. Synthetic mimics of cellular compartments based on lipid-based protocells lack the mechanical and chemical stability to allow their manipulation into a complex and fully functional synthetic cell. Here, we present a high-throughput microfluidic method to generate stable, defined sized liposomes termed 'droplet-stabilized giant unilamellar vesicles (dsGUVs)'. The enhanced stability of dsGUVs enables the sequential loading of these compartments with biomolecules, namely purified transmembrane and cytoskeleton proteins by microfluidic pico-injection technology. This constitutes an experimental demonstration of a successful bottom-up assembly of a compartment with contents that would not self-assemble to full functionality when simply mixed together. Following assembly, the stabilizing oil phase and droplet shells are removed to release functional self-supporting protocells to an aqueous phase, enabling them to interact with physiologically relevant matrices.

Droplet-based microfluidics has emerged as a powerful tool in synthetic biology. For many applications, chemical functionalization of the droplets is a key process. Therefore, a straightforward and broadly applicable approach is developed to functionalize the inner periphery of microfluidic droplets with diverse reactive groups and components. Instead of covalent modification of the droplet-stabilizing surfactants, this method relies on cholesteroltagged DNA that self-assembles at the droplet periphery. The cholesteroltagged DNA serves as an attachment handle for the recruitment of complementary DNA. The complementary DNA can carry diverse functional groups. We exemplify our method by demonstrating the attachment of amine groups, DNA nanostructures, microspheres, a minimal actin cortex, and leukemia cells to the droplet periphery. It is further shown that the DNAmediated attachment to the droplet periphery is temperature-responsive and reversible. It is envisioned that droplet functionalization via DNA handles will help to tailor droplet interfaces for diverse applications-featuring programmable assembly, unique addressability, and stimuli-responsiveness.The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/adfm.201808647. not universally applicable-it depends on the success of the chemical synthesis and can interfere with the stability and the physical properties of the droplets. Moreover, the binding of the functional group to the droplet periphery is irreversible.DNA nanotechnology, [12] on the other hand can attain the programmable assembly of arbitrary nanoscale architectures like DNA-based lattices, [13] nanopores, [14][15][16] or lid-containing boxes. [17,18] DNA has also been used as a scaffold or linker to assemble secondary components including proteins, [19] gold nanoparticles [20] and liposomes. [21] In addition, networks of emulsion droplets [22,23] or colloid-coated droplets [24] have been created using DNA linkers. Yet in all cases, the linkage was based on biotinylated DNA, which requires additional efforts to graft streptavidin onto the droplet surface. Furthermore, it has never been demonstrated that it is possible to functionalize the interior of block-copolymer surfactantstabilized droplets with DNA.Here, we present a broadly applicable method for functionalizing microfluidic droplets utilizing the hydrophobic interaction of cholesterol-tagged DNA with the droplet-stabilizing surfactant. Notably, the interaction of cholesterol with perfluorinated chains has never been described or exploited before. We show that DNA handles can serve as reversible anchoring points for various components including reactive groups, DNA nanostructures, beads, proteins or even cells. The use of off-the-shelf available DNA holds considerable advantages compared to standard methods for droplet functionalization, including: the broad scope of options for site-directed chemical functionalization, the addressability and programmability due to specific base pairin...

Water-in-oil emulsion droplets created in droplet-based microfluidic devices have been tested and used recently as well-defined picoliter-sized 3D compartments for various biochemical and biomedical applications. In many of these applications, fluorescence measurements are applied to reveal the protein content, spatial distribution, and dynamics in the droplets. However, emulsion droplets do not always provide entirely sealed compartments, and partitioning of dyes or labeled molecules to the oil phase is frequently observed. Therefore, stable molecular retention in the droplets represents a challenge, and many physical and chemical key factors of microfluidic system components have to be considered. In this study, we investigated the retention of 12 commonly used water-soluble dyes in droplets having six different aqueous phase conditions. We demonstrate that the physicochemical properties of the dyes have a major influence on the retention level. In particular, hydrophilicity has a strong influence on retention, with highly hydrophilic dyes (LogD < -7) showing stable, buffer/medium independent retention. In the case of less hydrophilic dyes, we showed that retention can be improved by adjusting the surfactants physical properties, such as geometry, length, and concentration. Furthermore, we analyzed the retention stability of labeled biomolecules such as antibodies, streptavidin, and tubulin proteins and showed that stable retention can be strongly dependent on dye and surfactants selection.