Microfluidic Construction of Synthetic Cellular Structures

Abstract

Among the multitudes of chemical structures in the cell, the membrane is the most difficult to synthesize. Microfluidic technology uniquely allows us to control the synthesis of cell membranes in a layer-by-layer fashion, and furthermore allows us control over the cargo of resultant vesicles. Vesicles were formed first by dispersing aqueous droplets into a phospholipid-containing oil phase, trapping the droplets in microfabricated cups, and finally flushing aqueous solution over the trapped droplets. In-plane valves were developed for computer control over the complex fluidic programming and capture cup geometry was optimized for droplet capture and complete phase exchange around trapped droplets. Membrane lamellarity was confirmed with α-hemolysin (αHL), an integral membrane protein that inserts into and selectively permeabilizes bilayers. The membranes were successfully permeabilized using purified αHL, as observed in confocal imaging microscopy. To demonstrate compartmentalized metabolism, the gene encoding αHL was expressed by an in-vitro transcription translation (IVTT) system within synthetic membranes. Activity was observed as before. After insertion of the gene and IVTT, only small-molecule cargo (fluorescein) dissipated from the interior of the vesicle, whereas large macromolecular cargo was retained. The lack of leakage of large cargo demonstrates both the internal metabolism as well as the presence of a only a single bilayer membrane. The computer-controlled membrane prototyping platform is now being used to reconstitute integral membrane protein signaling complexes (e.g. receptor kinases) and confirm their in vitro pharmacological behavior.