Andrew G. Richter

Using in situ X-ray reflectivity to study protein adsorption on hydrophilic and hydrophobic surfaces: benefits and limitations.

We have employed in situ X-ray reflectivity (IXRR) to study the adsorption of a variety of proteins (lysozyme, cytochrome c, myoglobin, hemoglobin, serum albumin, and immunoglobulin G) on model hydrophilic (silicon oxide) and hydrophobic surfaces (octadecyltrichlorosilane self-assembled monolayers), evaluating this recently developed technique for its applicability in the area of biomolecular studies. We report herein the highest resolution depiction of adsorbed protein films, greatly improving on the precision of previous neutron reflectivity (NR) results and previous IXRR studies. We were able to perform complete scans in 5 min or less with the maximum momentum transfer of at least 0.52 Å(-1), allowing for some time-resolved information about the evolution of the protein film structure. The three smallest proteins (lysozyme, cytochrome c, and myoglobin) were seen to deposit as fully hydrated, nondenatured molecules onto hydrophilic surfaces, with indications of particular preferential orientations. Time evolution was observed for both lysozyme and myoglobin films. The larger proteins were not observed to deposit on the hydrophilic substrates, perhaps because of contrast limitations. On hydrophobic surfaces, all proteins were seen to denature extensively in a qualitatively similar way but with a rough trend that the larger proteins resulted in lower coverage. We have generated high-resolution electron density profiles of these denatured films, including capturing the growth of a lysozyme film. Because the solution interface of these denatured films is diffuse, IXRR cannot unambiguously determine the film extent and coverage, a drawback compared to NR. X-ray radiation damage was systematically evaluated, including the controlled exposure of protein films to high-intensity X-rays and exposure of the hydrophobic surface to X-rays before adsorption. Our analysis showed that standard measuring procedures used for XRR studies may lead to altered protein films; therefore, we used modified procedures to limit the influence of X-ray damage.

Facile directed assembly of hollow polymer nanocapsules within spontaneously formed catanionic surfactant vesicles.

Surfactant vesicles containing monomers in the interior of the bilayer were used to template hollow polymer nanocapsules. This study investigated the formation of surfactant/monomer assemblies by two loading methods, concurrent loading and diffusion loading. The assembly process and the resulting aggregates were investigated with dynamic light scattering, small angle neutron scattering, and small-angle X-ray scattering. Acrylic monomers formed vesicles with a mixture of cationic and anionic surfactants in a broad range of surfactant ratios. Regions with predominant formation of vesicles were broader for compositions containing acrylic monomers compared with blank surfactants. This observation supports the stabilization of the vesicular structure by acrylic monomers. Diffusion loading produced monomer-loaded vesicles unless vesicles were composed from surfactants at the ratios close to the boundary of a vesicular phase region on a phase diagram. Both concurrent-loaded and diffusion-loaded surfactant/monomer vesicles produced hollow polymer nanocapsules upon the polymerization of monomers in the bilayer followed by removal of surfactant scaffolds.

Controlled Loading of Building Blocks into Temporary Self-Assembled Scaffolds for Directed Assembly of Organic Nanostructures

Using temporary self-assembled scaffolds to preorganize building blocks is a potentially powerful method for the synthesis of organic nanostructures with programmed shapes. We examined the underlying phenomena governing the loading of hydrophobic monomers into lipid bilayer interior and demonstrated successful control of the amount and ratio of loaded monomers. When excess styrene derivatives or acrylates were added to the aqueous solution of unilamellar liposomes made from saturated phospholipids, most loading occurs within the first few hours. Dynamic light scattering and transmission electron microscopy revealed no evidence of aggregation caused by monomers. Bilayers appeared to have a certain capacity for accommodating monomers. The total volume of loaded monomers is independent of monomer structure. X-ray scattering showed the increase in bilayer thickness consistent with loading monomers into bilayer interior. Loading kinetics is inversely proportional to the hydrophobicity and size of monomers. Loading and extraction kinetic data suggest that crossing the polar heads region is the rate limiting step. Consideration of loading kinetics and multiple equilibria are important for achieving reproducible monomer loading. The total amount of monomers loaded into the bilayer can be controlled by the loading time or length of hydrophobic lipid tails. The ratio of loaded monomers can be varied by changing the ratio of monomers used for loading or by the time-controlled replacement of a preloaded monomer. Understanding and controlling the loading of monomers into bilayers contributes to the directed assembly of organic nanostructures.

Transitions to a new chiral phase in a Langmuir monolayer

Isotherms and x-ray diffraction studies of eicosanoic acid Langmuir monolayers show a phase in which the molecular tilt is intermediate between nearest-neighbor (NN) and next-nearest-neighbor (NNN) directions. The transition from this “I” phase to an NN-tilted structure is first order, with a ∼60° change in the tilt direction, while the transition to an NNN-tilted structure is apparently continuous. These results can be explained using a Landau-type theory for uniaxially distorted lattices, which is a modification of an existing theory for hexagonal lattices [J. V. Selinger and D. R. Nelson, Phys. Rev. Lett. 61, 416 (1988)].

Scattering studies of hydrophobic monomers in liposomal bilayers: an expanding shell model of monomer distribution.

Hydrophobic monomers partially phase separate from saturated lipids when loaded into lipid bilayers in amounts exceeding a 1:1 monomer/lipid molar ratio. This conclusion is based on the agreement between two independent methods of examining the structure of monomer-loaded bilayers. Complete phase separation of monomers from lipids would result in an increase in bilayer thickness and a slight increase in the diameter of liposomes. A homogeneous distribution of monomers within the bilayer would not change the bilayer thickness and would lead to an increase in the liposome diameter. The increase in bilayer thickness, measured by the combination of small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS), was approximately half of what was predicted for complete phase separation. The increase in liposome diameter, measured by dynamic light scattering (DLS), was intermediate between values predicted for a homogeneous distribution and complete phase separation. Combined SANS, SAXS, and DLS data suggest that at a 1.2 monomer/lipid ratio approximately half of the monomers are located in an interstitial layer sandwiched between lipid sheets. These results expand our understanding of using self-assembled bilayers as scaffolds for the directed covalent assembly of organic nanomaterials. In particular, the partial phase separation of monomers from lipids corroborates the successful creation of nanothin polymer materials with uniform imprinted nanopores. Pore-forming templates do not need to span the lipid bilayer to create a pore in the bilayer-templated films.

Time-resolved loading of monomers into bilayers with different curvature.

Directed assembly of nanostructures within temporary and recyclable self-assembled scaffolds is emerging as an attractive method for the synthesis of nanomaterials with programmed properties. Understanding interactions of building blocks with amphiphilic scaffolds is critical for rational design of new nanostructures and nanodevices. Here we examine loading of hydrophobic monomers into bilayers with different curvatures. Time-resolved loading was studied by high performance liquid chromatography and dynamic light scattering. Despite differences in initial bilayer geometry, loading rates and maximum bilayer capacity are the same for liposomes with radii ranging from 25 to 100 nm. When using divinylbenzene (DVB) and dimyristoylphosphatidylcholine (DMPC), monomer/lipid loading ratio of 1.2 was achieved within 12 h. While accommodation of a large amount of monomers is likely to be accompanied with significant changes in bilayer structure, all liposomes in this study including those with smallest size and higher bilayer curvature retain encapsulated content and show no evidence of fusion during monomer loading. These results contribute to our understanding of interactions between hydrophobic molecules and lipid bilayers and expand the scope of the directed assembly method.

Calibrating an ellipsometer using x-ray reflectivity.

X-ray reflectivity has been used to find the optical refractive index of polymer thin film in order to calibrate a Stokes ellipsometer for film thickness measurements during the deposition procedure. A thin, spun-cast film of poly(tert-butyl acrylate) (PtBA) was made with a film thickness of ∼500 A. An x-ray reflectivity measurement was taken and the data were fit to determine the thickness of the PtBA film and the underlying silicon–oxide layer. This measurement was then used to calculate the optical refractive index for PtBA at the ellipsometer wavelength. Using this value for the refractive index subsequently allowed us to determine the film thickness for a series of PtBA films made by using a number of polymer solution concentrations resulting in film thickness ranging from 100 to 1300 A. These film thicknesses were found to be generally the same as those found using x-ray reflectivity. The success of this procedure suggests a useful method for calibrating an ellipsometer for fast in-lab measurements,...

Order in molecular liquids near solid-liquid interfaces

Abstract Much less is known about structure at solid–liquid interfaces than at solid or liquid surfaces, largely because it is more difficult for experimental probes to penetrate into an interface. In recent years, the availability of synchrotron radiation (which is intense and collimated, and whose energy and thus penetration depth can be varied) has made scattering experiments at such interfaces possible. This article reviews some recent studies of ordering at the liquid side of a solid–liquid interface.

Characterization of the Panoramic Annular Lens

The panoramic annular lens (PAL) consists of a single piece of glass, with spherical surfaces, that produces a flat annular image of the entire 360-deg surround of the optical axis of the lens. This paper describes the attributes of the PAL and shows that the lens maps elements from object to image space via a constant aspect ratio polar mapping. A panoramic video system (PVS) is described to illustrate how the characterization can be applied in experimental mechanics for cavity inspection and measurement.

Order in Langmuir-Blodgett films of lead and cadmium stearate: an X-ray diffraction study

Abstract We have studied monolayer and multilayer Langmuir-Blodgett films of lead stearate and cadmium stearate using grazing incidence X-ray diffraction. We find that although these two types of films have similar in-plane structures, lead stearate forms better ordered films: the correlation length increases with the number of layers in lead stearate but levels off at the three-layer value in cadmium stearate. The lattices of adjacent lead ion layers are correlated, but adjacent cadmium layers are not.