Synthesis, characterization and applications of tether supported biomembrane-microsphere assemblies

Our objective is to develop a highly miniaturized, solid-phase platform for assaying the function and inhibition of integral membrane proteins (IMPs). To functionalize materials with active membrane proteins, the challenge is to build stabilized, supported biomembranes in which the substrate to biomembrane spacing can be controlled to accommodate larger membrane protein. The silica microsphere (5 micron) surface was functionalized with DiNHS-PEG in order to make a tethering bridge designed to yield spacing. Confocal fluorescence microscopy was utilized to analyze the silica surface at different stages of surface modification and examine the passivation of the substrate to the formation of lipid bilayers. The lateral mobility and fluidity of the supported membranes was analyzed using fluorescence recovery after photobleaching (FRAP). Fluorescence Activated Cell Sorting (FACS) was used as a quality control step to purify the lipid tethered microsphere complexes. These structures have been employed as a route to functionally immobilize the yeast drug efflux pump PDR5, an important anti-fungal drug target. Another intention is to develop a new platform to present molecules to stem cells within a biomimetic architecture: as laterally-mobile molecules embedded in the context of a tailored biomembrane.