In the field of bottom-up synthetic biology, lipid membranes are the scaffold to create minimal cells and mimic reactions and processes at or across the membrane. In this context, we employ here a versatile microfluidic platform that enables precise positioning of nanoliter droplets with user-specified lipid compositions and in a defined pattern. Adjacent droplets make contact and form a droplet interface bilayer to simulate cellular membranes. Translocation of molecules across membranes are tailored by the addition of alpha-hemolysin to selected droplets. Moreover, we developed a protocol to analyze the translocation of non-fluorescent molecules between droplets with mass spectrometry. Our method is capable of automated formation of one- and two-dimensional droplet networks, which we demonstrated by connecting droplets containing different compound and enzyme solutions to perform translocation experiments and a multistep enzymatic cascade reaction across the droplet network. Our platform opens doors for creating complex artificial systems for bottom-up synthetic biology.

We investigated the permeation of molecules across lipid membranes on an open microfluidic platform. An array of droplet pairs was created by spotting aqueous droplets, dispersed in a lipid oil solution, onto a plate with cavities surrounded by a hydrophobic substrate. Droplets in two adjacent cavities come in contact and form an artificial lipid bilayer, called a droplet interface bilayer (DIB). The method allows for monitoring permeation of fluorescently tagged compounds from a donor droplet to an acceptor droplet. A mathematical model was applied to describe the kinetics and determine the permeation coefficient. We also demonstrate that permeation kinetics can be followed over a series of droplets, all connected via DIBs. Moreover, by changing the lipid oil composition after spotting donor droplets, we were able to create asymmetric membranes that we used to mimic the asymmetry of the cellular plasma membrane. Finally, we developed a protocol to separate and extract the droplets for label-free analysis of permeating compounds by liquid chromatography–mass spectrometry. Our versatile platform has the potential to become a new tool for the screening of drug membrane permeability in the future.