Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter.

Reconstitution of functionally active membrane protein into artificially made lipid bilayers is a challenge that must be overcome to create a membrane-based biomimetic sensor and separation device. In this study we address the efficacy of proteoliposome fusion with planar membrane arrays. We establish a protein incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein reconstitution in biomimetic membrane arrays may be quantified using the developed FomA assay. Specifically, we show that FomA vesicles are inherently fusigenic. Optimal FomA incorporation is obtained with a proteoliposome lipid-to-protein molar ratio (LPR)=50 more than 10(5) FomA proteins could be incorporated in a bilayer array with a total membrane area of 2mm(2) within 20 min. This novel assay for quantifying protein delivery into lipid bilayers may be a useful tool in developing biomimetic membrane applications.

Free-standing biomimetic polymer membrane imaged with atomic force microscopy.

Fluid polymeric biomimetic membranes are probed with atomic force microscopy (AFM) using probes with both normal tetrahedrally shaped tips and nanoneedle-shaped Ag(2)Ga rods. When using nanoneedle probes, the collected force volume data show three distinct membrane regions which match the expected membrane structure when spanning an aperture in a hydrophobic scaffold. The method used provides a general method for mapping attractive fluid surfaces. In particular, the nanoneedle probing allows for characterization of free-standing biomimetic membranes with thickness on the nanometer scale suspended over 300-µm-wide apertures, where the membranes are stable toward hundreds of nanoindentations without breakage.