The behavior of poly(ethylene glycol) (PEG) conjugated lipids was investigated in planar supported egg phosphatidylcholine bilayers as a function of lipopolymer density, chain length of the PEG moiety, and type of alkyl chains on the PEG lipid. Fluorescence recovery after photobleaching measurements verified that dye-labeled lipids in the membrane as well as the lipopolymer itself maintained a substantial degree of fluidity under most conditions that were investigated. PEG densities exceeding the onset of the mushroom-to-brush phase transition were found to confer air stability to the supported membrane. On the other hand, substantial damage or complete delamination of the lipid bilayer was observed at lower polymer densities. The presence of PEG in the membrane did not substantially hinder the binding of streptavidin to biotinylated lipids present in the bilayer. Furthermore, above the onset of the transition into the brush phase, the protein binding properties of these membranes were found to be very resilient upon removal of the system from water, rigorous drying, and rehydration. These results indicate that supported phospholipid bilayers containing lipopolymers show promise as rugged sensor platforms for ligand-receptor binding.

Supported lipid bilayers (SLBs) have been widely used as model systems to study cell membrane processes because they preserve the same 2D membrane fluidity found in living cells. One of the most significant limitations of this platform, however, is its inability to incorporate mobile transmembrane species. It is often postulated that transmembrane proteins reconstituted in SLBs lose their mobility because of direct interactions between the protein and the underlying substrate. Herein, we demonstrate a highly mobile fraction for a transmembrane protein, annexin V. Our strategy involves supporting the lipid bilayer on a double cushion, where we not only create a large space to accommodate the transmembrane portion of the macromolecule but also passivate the underlying substrate to reduce nonspecific protein-substrate interactions. The thickness of the confined water layer can be tuned by fusing vesicles containing polyethyleneglycol (PEG)conjugated lipids of various molecular weights to a glass substrate that has first been passivated with a sacrificial layer of bovine serum albumin (BSA). The 2D fluidity of these systems was characterized by fluorescence recovery after photobleaching (FRAP) measurements. Uniform, mobile phospholipid bilayers with lipid diffusion coefficients of around 3 × 10 −8 cm 2 /s and percent mobile fractions of over 95% were obtained. Moreover, we obtained annexin V diffusion coefficients that were also around 3 × 10 −8 cm 2 /s with mobile fractions of up to 75%. This represents a significant improvement over bilayer platforms fabricated directly on glass or using single cushion strategies.

Laser desorption/ionization (LDI) using silver nanoparticles (AgNPs) is shown to selectively ionize olefinic compounds, e.g., cholesterol, 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC), and carotenoids. Selective AgNP LDI can be carried out from complex mixtures without the addition of an organic matrix, sample cleanup, or prefractionation. Results presented in this report are the first to demonstrate the selective ionization of specific compounds from a complex mixture using metal nanoparticles.

A new method was developed to purify membrane bound species within a supported lipid bilayer (SLB) environment. SLBs consisting of phosphatidylcholine lipids and cholesterol were employed as the separation matrix. Cholesterol was used to reduce the diffusion of lipids within the bilayer and, therefore, substantially reduce mixing of the dye-conjugated lipids to be separated. These molecules were introduced into an SLB adjacent to the separations SLB and electrophoresis was employed to move these species through it. The method was powerful enough to completely resolve two isomers of Texas Red DHPE from each other. Moreover, these isomers could be separated from a BODIPY-conjugated lipid as well. Such procedures could be extended to the purification of peripheral and transmembrane proteins. Separation, purification, and detection of biomembrane species such as lipids and transmembrane proteins are difficult tasks. The processing conditions are often harsh, which can result in alteration of native structures or complete loss of material. 1,2 Furthermore, it is difficult to detect subtle post-translational changes in these molecules that occur on the cell surface. 3-5 Typical purification procedures often require one to dissolve the membrane in detergent, sonicate, filter through chromatographic columns, and separation into bands using gel electrophoresis. Procedures that circumvent such drawbacks would represent an attractive alternative and could significantly impact transmembrane proteomics.