Supported lipid bilayers at skeletonized surfaces for the study of transmembrane proteins.

Skeletonized zirconium phosphonate surfaces are used to support planar lipid bilayers and are shown to be viable substrates for studying transmembrane proteins. The skeletonized surfaces provide space between the bilayer and the solid support to enable protein insertion and avoid denaturation. The skeletonized zirconium octadecylphosphonate surfaces were prepared using Langmuir-Blodgett techniques by mixing octadecanol with octadecylphosphonic acid. After zirconation of the transferred monolayer, rinsing the coating with organic solvent removes the octadecanol, leaving holes in the film ranging from ∼50 to ∼500 nm in diameter, depending on the octadecanol content. Upon subsequent deposition of a lipid bilayer, either by vesicle fusion or by Langmuir-Blodgett/Langmuir-Schaefer techniques, the lipid assemblies span the holes providing reservoirs beneath the bilayer. The viability of the supported bilayers as model membranes for transmembrane proteins was demonstrated by examining two approaches for incorporating the proteins. The BK channel protein inserts directly into a preformed bilayer on the skeletonized surface, in contrast to a bilayer on a nonskeletonized film, for which the protein associates only weakly. As a second approach, the integrin α(5)ß(1) was reconstituted in lipid vesicles, and its inclusion in supported bilayers on the skeletonized surface was achieved by vesicle fusion. The integrin retains its ability to recognize the extracellular matrix protein fibronectin when supported on the skeletonized film, again in contrast to the response if the bilayer is supported on a nonskeletonized film.

Energy transfer between conjugated polyelectrolytes in layer-by-layer assembled films.

We present a study of Förster resonance energy transfer (FRET) between two emissive conjugated polyelectrolytes (CPEs) in layer-by-layer (LbL) self-assembled films as a means of examining their organization and architecture. The two CPEs are a carboxylic acid functionalized polyfluorene (PFl-CO(2)) and thienylene linked poly(phenylene ethynylene) (PPE-Th-CO(2)). The PFl-CO(2) presents a maximum emission at 418 nm, while the PPE-Th-CO(2) has an absorption λ(max) centered at 431 nm, in sufficient proximity for effective FRET. Several LbL films have been constructed using varied concentrations of the deposition solutions and identity of the buffer layers separating the two emissive layers, using a system of either weak polyelectrolytes, poly(allylamine hydrochloride) (PAH)/poly(sodium methacrylate) (PMA), or strong polyelectrolytes, poly(diallylammonium chloride) (PDDA)/poly(styrene sulfonate) sodium (PSS). The efficiency of FRET has been monitored using fluorescence spectroscopy. Initially, the fluorescence of the PFl-CO(2) (E(g) ∼ 3.0 eV), which emits at 420 nm, is quenched by the lower band gap PPE-Th-CO(2) (E(g) ∼ 2.5 eV). For films using the PAH/PMA system as buffer bilayers and deposited from 1 mM solutions, the PFl-CO(2) fluorescence is progressively recovered as the number of intervening buffer bilayers is increased. Ellipsometry measurements indicate that energy transfer between the two emissive layers is efficient to a distance of ca. 7 nm.

Bisphosphonate adaptors for specific protein binding on zirconium phosphonate-based microarrays.

Two bisphosphonate adaptors were designed to immobilize histidine-tagged proteins onto glass substrates coated with a zirconium phosphonate monolayer, allowing efficient and oriented immobilization of capture proteins, affitins directed to lysozyme, on a microarray format. These bifunctional adaptors contain two phosphonic acid anchors at one extremity and either one nitrilotriacetic acid (NTA) or two NTA groups at the other. The phosphonate groups provide a stable bond to the zirconium interface by multipoint attachment and allow high density of surface coverage of the linkers as revealed by X-ray photoelectron spectroscopy (XPS). Reversible high-density capture of histidine-tagged proteins is shown by real-time surface plasmon resonance enhanced ellipsometry and in a microarray format using fluorescence detection of AlexaFluor 647-labeled target protein. The detection sensitivity of the microarray for the target protein was below 1 nM, despite the monolayer arrangement of the probes, due to very low background staining, which allows high fluorescent signal-to-noise ratio. The performance of these Ni-NTA-modified zirconium phosphonate coated slides compared favorably to other types of microarray substrates, including slides with a nitrocellulose-based matrix, epoxide slides, and epoxide slides functionalized with Ni-NTA groups. This immobilization strategy has a large potential to fix any histidine-tagged proteins on zirconium or titanium ion surfaces.

Stable supported lipid bilayers on zirconium phosphonate surfaces.

Supported lipid bilayers that can fully represent biological cell membranes are attractive biomimetic models for biophysical and biomedical applications. In this study, we develop a new approach to engineering stable supported lipid membranes and demonstrate their utility for the study of protein-membrane interactions. This system uses a zirconium phosphonate monolayer to modify a substrate and generate a reactive surface that tethers the lipid membrane via a highly covalent bond between surface zirconium ions and divalent phosphate groups in the lipid assembly, for example, from phosphatidic acid. An advantage of the approach is that the zirconium phosphonate modifier can be applied to nearly any surface, allowing the same methods to be used on glass, gold, silicon, or plastic supports. The lipid bilayers are formed by vesicle fusion, either directly on the zirconated surface to form symmetric bilayers or following deposition of a Langmuir-Blodgett lipid layer to generate asymmetric bilayers. The membrane formation was studied by surface plasmon resonance enhanced ellipsometry (SPREE) as the phosphatidic acid composition was varied. We found that 10% of phosphatidic acid generates supported lipid bilayers stable to dehydration. The two-dimensional fluidity of these systems was characterized by fluorescence recovery after photobleaching (FRAP) measurements. Uniform, mobile supported lipid bilayers with lipid diffusion coefficients of approximately 4 mum(2)/s were obtained. SPREE was also used to measure kinetic parameters of the binding of melittin, a bee venom peptide, to asymmetric lipid bilayers with different electrostatic properties. The results are comparable to those obtained by other research groups, confirming that the model membranes behave as expected. Overall, the results of this study prove that supported lipid bilayers on zirconium phosphonate inorganic surfaces make up an attractive biomimetic system that is highly stable, can be used with multiple substrates, and does not require any biomolecule synthetic modifications.