Eric E. Ross

Ultra-high vacuum surface analysis study of rhodopsin incorporation into supported lipid bilayers

Planar supported lipid bilayers that are stable under ambient atmospheric and ultra-high-vacuum conditions were prepared by cross-linking polymerization of bis-sorbylphosphatidylcholine (bis-SorbPC). X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) were employed to investigate bilayers that were cross-linked using either redox-initiated radical polymerization or ultraviolet photopolymerization. The redox method yields a more structurally intact bilayer; however, the UV method is more compatible with incorporation of transmembrane proteins. UV polymerization was therefore used to prepare cross-linked bilayers with incorporated bovine rhodopsin, a light-activated, G-protein-coupled receptor (GPCR). A previous study (Subramaniam, V.; Alves, I. D.; Salgado, G. F. J.; Lau, P. W.; Wysocki, R. J.; Salamon, Z.; Tollin, G.; Hruby, V. J.; Brown, M. F.; Saavedra, S. S. J. Am. Chem. Soc. 2005, 127, 5320-5321) showed that rhodopsin retains photoactivity after incorporation into UV-polymerized bis-SorbPC, but did not address how the protein is associated with the bilayer. In this study, we show that rhodopsin is retained in supported bilayers of poly(bis-SorbPC) under ultra-high-vacuum conditions, on the basis of the increase in the XPS nitrogen concentration and the presence of characteristic amino acid peaks in the ToF-SIMS data. Angle-resolved XPS data show that the protein is inserted into the bilayer, rather than adsorbed on the bilayer surface. This is the first study to demonstrate the use of ultra-high-vacuum techniques for structural studies of supported proteolipid bilayers.

Assembly of Lipid Bilayers on Silica and Modified Silica Colloids by Reconstitution of Dried Lipid Films

A method is presented for the assembly of lipid bilayers on silica colloids via reconstitution of dried lipid films solvent-cast from chloroform within packed beds of colloids ranging from 100 nm to 10 μm in diameter. Rapid solvent evaporation from the packed bed void volume results in uniform distribution of dried lipid throughout the colloidal bed. Fluorescence measurements indicate that significant, if not quantitative, retention of DOPC or DPPC films cast between sub-bilayer and multilayer quantities occurs when the colloids are redispersed in aqueous solution. Phospholipid bilayers assembled in this manner are shown to effectively passivate the surface of 250 nm colloids to nonspecific adsorption of bovine serum albumin. The method is shown to be capable of preparing supported bilayers on colloid surfaces that do not generally support vesicle fusion such as poly(ethylene glycol) (PEG) modified silica colloids. Bilayers of lipids that have not been reported to self-assemble by vesicle fusion, including gel-phase lipids and single-chain diacetylene amphiphiles, can also be formed by this method. The utility of the solid-core support is demonstrated by the facile assembly of supported lipid bilayers within fused silica capillaries to generate materials that are potentially suitable for the analysis of membrane interactions in a microchannel format.

Silica colloidal crystals as three-dimensional scaffolds for supported lipid films.

This report describes the assembly of laterally diffusive lipid layers within the pores of colloidal crystals for potential application in membrane-based sensing. The amount of lipid encapsulated within colloidal crystals depends upon the method used to introduce the lipid to the crystalline substrate. Relative to a planar supported lipid bilayer, lipid loading in a 6.6 microm thick crystal was 15-73 times greater, as observed by fluorescence microscopy. Protein adsorption studies indicate that the crystal pores are open and that the silica surface of the crystal is passivated with respect to adsorption of a model protein when coated with POPC. Furthermore, the mesoporous environment of the colloidal crystal is found to protect lipid films from drying and rehydration processes that destroy planar supported lipid bilayers. The potential of colloidal crystal encapsulated lipid films for chemical sensing is demonstrated by a model protein binding assay.

Site-isolated, intermolecularly photocrosslinkable and patternable dendritic quinacridones

Quinacridone-cored dendrimers with photocrosslinkable cinnamate moieties on the periphery can be patterned down to 5 micron features while retaining luminescence.

Metal ion binding to phospholipid bilayers evaluated by microaffinity chromatography.

Group I and II ion binding to phospholipid membranes was evaluated by affinity chromatography utilizing a new stationary phase system based on lipid bilayers supported within large-pore particles composed of Stöber silica spheres. Using an inductively coupled plasma mass spectrometer for detection, robust determination of binding selectivity within group II ions is achieved with capillary columns containing nanomole quantities of lipid and using picomoles of metal analyte. Columns with a unique lipid formulation can be prepared within three hours using a solvent-casting assembly method. The observable thermotropic phase behavior of dipalmitoylphosphatidylcholine has a significant effect on alkaline metal binding and demonstrates the dynamic nature of the supported bilayers. Of the group I ions, only lithium exhibits retention with neutral phosphatidylcholine bilayer stationary phases. A comparison of Stöber-based supports with two commercially available large-pore silicas reveals the effect that particle structure has on analyte accessibility to the bilayer surface as evaluated by retention per supported lipid mass.