Probing Shear Thinning Behaviors of IgG Molecules at the Air-Water Interface via Rheological Methods.

Shear thinning behavior, often observed in shear viscosity tests of IgG therapeutic molecules, could lead to significant disparities in the projections for the viscosity profile of a molecule. Despite its importance, molecular determinants of sheer thinning in protein suspensions are largely unknown. To better understand the factors influencing sheer thinning, viscosity profiles of IgG1 and IgG2 molecules were monitored over a wide range of bulk concentrations (0.007-70 mg/mL). The degree of shear-thinning of 70 and 0.007 mg/mL samples was minimal in comparison to the 0.7 mg/mL solution for both IgG molecules. These observations suggest that bulk concentration alone does not determine the degree of sheer thinning, and additional factors play a role. Additional data reveals, within a threshold range of concentrations, that a strong correlation exists between the degree of shear thinning and the surface area to volume (SA:V) ratio of an IgG sample exposed to the interface. The influence of the interface, however, diminishes when the bulk concentration falls outside this concentration window. Also revealed by interfacial oscillatory rheological testing, both IgG molecules showed solid-like behavior (G'i) at the air-water interface at 0.7 mg/mL, whereas liquid-like behavior (G″i) was dominant at 0.007 and 70 mg/mL concentrations. These observations imply that the lack of solid-like behavior was due to the absence of a network structure. Likewise the addition of polysorbate 20 (PS20) to the 0.7 mg/mL solutions decreased the degree of shear thinning by disrupting the network structure at the interface. Taken together, the results presented here suggest that, although shear thinning behavior is a manifestation of an interfacial, rather than a bulk, phenomenon, the extent of it depends on how susceptible the surface molecules are to the air-water interface, where the surface molecular structures are influenced by the bulk properties.

Preparation, characterization, and surface immobilization of native vesicles obtained by mechanical extrusion of mammalian cells.

Native vesicles or "reduced protocells" derived by mechanical extrusion concentrate selected plasma membrane components, while downsizing complexities of whole cells. We illustrate this technique, characterize the physical-chemical properties of these reduced configurations of whole cells, and demonstrate their surface immobilization and patternability. This simple detergent-free vesicularized membrane preparation should prove useful in fundamental studies of cellular membranes, and may provide a means to engineer therapeutic cells and enable high-throughput devices containing near-native, functional proteolipidic assemblies.

Detection of adulteration in acetonitrile.

To address the increasing concern that acetonitrile may be intentionally adulterated to meet the shortfall in global supplies resulting from a downturn in its manufacturing, three analytical techniques were examined in this study. Gas Chromatography with Thermal Conductivity Detection (GC-TCD), Near Infrared (NIR) spectroscopy and Fourier Transform Infrared (FT-IR) spectroscopy were assessed for their ability to detect and quantify potential adulterants including water, alternative organic solvents, and by-products associated with the production of acetonitrile. The results of the assessment of the three techniques for acetonitrile adulteration testing are discussed.

Cell attachment behavior on solid and fluid substrates exhibiting spatial patterns of physical properties.

The ability to direct proliferation and growth of living cells using chemically and topologically textured surfaces is finding many niche applications, both in fundamental biophysical investigations of cell-surface attachment and in developing design principles for many tissue engineering applications. Here we address cellular adhesion behavior on solid patterns of differing wettability (a static substrate) and fluid patterns of membrane topology (a dynamic substrate). We find striking differences in the cellular adhesion characteristics of lipid mono- and bilayers, despite their essentially identical surface chemical and structural character. These differences point to the importance of subtle variations in the physical properties of the lipid mono- and bilayers (e.g., membrane tension and out-of-plane undulations). Furthermore, we find that introducing phosphatidylserine into the patterned lipidic substrates causes a loss of cell-patterning capability. Implications of this finding for the mechanism by which phosphatidylserine promotes cellular adhesion are discussed.

Optical detection of ion-channel-induced proton transport in supported phospholipid bilayers.

The integration of ion-channel transport functions with responses derived from nanostructured and nanoporous silica mesophase materials is demonstrated. Patterned thin-film mesophases consisting of alternating hydrophilic nanoporous regions and hydrophobic nanostructured regions allow for spatially localized proton transport via selective dimerization of gramicidin in lipid bilayers formed on the hydrophilic regions. The adjoining hydrophobic mesostructure doped with a pH sensitive dye reports the transport. The ease of integrating functional membranes and reporters through the use of patterned mesophases should enable high throughput studies of membrane transport.

Lipid lateral mobility and membrane phase structure modulation by protein binding.

Using a combination of fluorescence correlation and infrared absorption spectroscopies, we characterize lipid lateral diffusion and membrane phase structure as a function of protein binding to the membrane surface. In a supported membrane configuration, cholera toxin binding to the pentasaccharaide headgroup of membrane-incorporated GM1 lipid alters the long-range lateral diffusion of fluorescently labeled probe lipids, which are not involved in the binding interaction. This effect is prominently amplified near the gel-fluid transition temperature, Tm, of the majority lipid component. At temperatures near Tm, large changes in probe lipid diffusion are measured at average protein coverage densities as low as 0.02 area fraction. Spectral shifts of the methylene symmetric and asymmetric stretching modes in the lipid acyl chain confirm that protein binding alters the fraction of lipid in the gel phase.

Direct photochemical patterning and refunctionalization of supported phospholipid bilayers.

A wet photolithographic route for micropatterning fluid phospholipid bilayers is demonstrated in which spatially directed illumination by short-wavelength ultraviolet radiation results in highly localized photochemical degradation of the exposed lipids. Using this method, we can directly engineer patterns of hydrophilic voids within a fluid membrane as well as isolated membrane corrals over large substrate areas. We show that the lipid-free regions can be refilled by the same or other lipids and lipid mixtures which establish contiguity with the existing membrane, thereby providing a synthetic means for manipulating membrane compositions, engineering metastable membrane microdomains, probing 2D lipid-lipid mixing, and designing membrane-embedded arrays of soluble proteins. Following this route, new constructs can be envisaged for high-throughput membrane proteomic, biosensor array, and spatially directed, aqueous-phase material synthesis.