A wide range of important biological processes occur at phospholipid membranes including cell signaling, where a peptide or small molecule targets a membrane-localized receptor protein. In this work, we report the adaptation of confocal Raman microscopy to quantify populations of unlabeled glucagon-like peptide-1 (GLP-1), a membrane-active 30-residue incretin peptide, in supported phospholipid bilayers deposited on the interior surfaces of wide-pore porous silica particles. Quantification of lipid bilayer-associated peptide is achieved by measuring the Raman scattering intensity of the peptide relative to that of the supported lipid bilayer, which serves as an internal standard. The dependence of the bilayer-associated GLP-1 population on the solution concentration of GLP-1 produces an isotherm used to determine the equilibrium constant for peptide-bilayer association and the maximum peptide surface coverage. The maximum coverage of GLP-1 in the lipid bilayer was found to be only 1/5th of a full monolayer based on its hydrodynamic radius. The saturation coverage, therefore, is not limited by the size of GLP-1 but by the ability of the bilayer to accommodate the peptide at high concentrations within the bilayer. Raman spectra show that GLP-1 association with the supported bilayer is accompanied by structural changes consistent with the intercalation of the peptide into the bilayer, where the observed increase in acyl-chain order would increase the lipid density and provide free volume needed to accommodate the peptide. These results were compared with previous measurements of the association of fluorescently labeled GLP-1 with a planar-supported bilayer; the unlabeled peptide exhibits a 3-fold greater affinity for the lipid bilayer on the porous silica support, suggesting that the fluorescent label alters the GLP-1 lipid bilayer association.

Phospholipid bilayers deposited on a variety of surfaces provide models for investigation of the lipid membrane structure and supports for biocompatible sensors. Hybrid-supported phospholipid bilayers (HSLBs) are stable membrane models for these investigations, typically prepared by self-assembly of a lipid monolayer over an n-alkane-modified surface. HSLBs have been prepared on nalkyl chain-modified silica and used for lipophilicity-based chromatographic separations. The structure of these hybrid bilayers differs from vesicle membranes where the lipid head group spacing is greater due to interdigitation of the lipid acyl chains with the underlying n-alkyl chains bound to the silica surface. This interdigitated structure exhibits a broader melting transition at a higher temperature due to strong interactions between the lipid acyl chains and the immobile n-alkyl chains bound to silica. In the present work, we seek to reduce the interactions between a lipid monolayer and its supporting substrate by self-assembly of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) on porous silica functionalized with nitrile-terminated surface ligands. The frequency of Raman scattering of the surface -C N stretching mode at the lipid-nitrile interface is consistent with an n-alkane-like environment and insensitive to lipid head group charge, indicating that the lipid acyl chains are in contact with the surface nitrile groups. The head group area of this lipid monolayer was determined from the within-particle phospholipid concentration and silica specific surface area and found to be 54 +/- 2 A(2), equivalent to the head group area of a DMPC vesicle bilayer. The structure of these nitrile-supported phospholipid monolayers was characterized below and above their melting transition by confocal Raman microscopy and found to be nearly identical to DMPC vesicle bilayers. Their narrow gel-to-fluid-phase melting transition is equivalent to dispersed DMPC vesicles, suggesting that the acyl chain structure on the nitrile support mimics the outer leaflet structure of a vesicle membrane.

Hybrid-supported phospholipid bilayers are a model structure utilized for measurement of molecular interactions that typically occur at cell membranes. These membrane models are prepared by adsorption of a lipid monolayer onto a stable n-alkyl chain layer that is covalently bound to a support surface. Hybrid bilayers have been adapted to chromatographic retention measurements of lipophilicity through the assembly of a phospholipid monolayer onto n-alkane-modified silica surfaces in reversed-phase chromatographic particles. Recent Raman microscopy studies of these particles have shown that the acyl chains of the phospholipid interact with the C18-alkyl chains immobilized on the silica surface, where both lipid and C18 alkyl chains become ordered because of chain interdigitation. Confocal Raman microscopy has also been used to investigate the association of small molecules with hybrid-lipid bilayers in C18 chromatographic silica particles; the partitioning of model solutes compares favorably to that in lipid vesicle membranes with similar changes in acyl-chain structure (disordering) with solute partitioning. The present study seeks information about how these membrane-mimetic bilayers assemble onto the C18-derivatized silica surfaces of reversed-phase chromatographic silica particles. Confocal Raman microscopy is capable of interrogating the time-dependent internal composition and structure within individual silica particles. The Raman scattering data can be resolved into component Raman spectra and corresponding composition vectors that describe the time-dependent changes in intensity of the component spectra. This analysis provides insight into how the structures of both the lipid and C18 alkyl chains of hybrid lipid bilayers evolve during deposition and organization on the internal surfaces of reversed-phase chromatographic silica particles.

A common approach to exploring the structure and dynamics of biological membranes is through the deposition of model lipid bilayers on planar supports by Langmuir-trough or vesicle-fusion methods. Planar-supported lipid bilayers have been shown to exhibit structure and properties similar to those of lipid-vesicle membranes and are suitable for biosensing applications. Investigations using these planar-membrane models are limited to high-sensitivity methods capable of detecting a small population of molecules at the interface between a planar support and aqueous solution. In this work, we present evidence that supported-lipid bilayers can be deposited by vesicle fusion onto the interior surfaces throughout the wide-pore network of chromatographic silica particles. The thickness of a 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) film and headgroup spacing are consistent with a single bilayer of DMPC deposited onto the pore surfaces. The high specific surface area of these materials generates phospholipid concentrations easily detected by confocal-Raman microscopy within an individual particle, which allows the structure of these supported bilayers to be investigated. Raman spectra of porous-silica-supported DMPC bilayers are equivalent to spectra of DMPC vesicle membranes, both above and below their melting phase transitions, suggesting comparable phospholipid organization and bilayer structure. These porous-silica-supported model membranes could share benefits that planar-supported lipid bilayers bring to biosensing applications, but in a material that overcomes the limited surface area of a planar support. To test this concept, the potential of these porous-silica-supported lipid bilayers as high-surface-area platforms for label-free Raman-scattering-based protein biosensing is demonstrated with detection of concanavalin A selectively binding to a lipid-immobilized mannose target.

The phospholipid-water partition coefficient is a commonly measured parameter that correlates with drug efficacy, small-molecule toxicity, and accumulation of molecules in biological systems in the environment. Despite the utility of this parameter, methods for measuring phospholipid-water partition coefficients are limited. This is due to the difficulty of making quantitative measurements in vesicle membranes or supported phospholipid bilayers, both of which are small-volume phases that challenge the sensitivity of many analytical techniques. In this work, we employ in situ confocal Raman microscopy to probe the partitioning of a model membrane-active compound, 2-(4-isobutylphenyl) propionic acid or ibuprofen, into both hybrid- and supported-phospholipid bilayers deposited on the pore walls of individual chromatographic particles. The large surface-area-to-volume ratio of chromatographic silica allows interrogation of a significant lipid bilayer area within a very small volume. The local phospholipid concentration within a confocal probe volume inside the particle can be as high as 0.5 M, which overcomes the sensitivity limitations of making measurements in the limited membrane areas of single vesicles or planar supported bilayers. Quantitative determination of ibuprofen partitioning is achieved by using the phospholipid acyl-chains of the within-particle bilayer as an internal standard. This approach is tested for measurements of pH-dependent partitioning of ibuprofen into both hybrid-lipid and supported-lipid bilayers within silica particles, and the results are compared with octanol-water partitioning and with partitioning into individual optically trapped phospholipid vesicle membranes. Additionally, the impact of ibuprofen partitioning on bilayer structure is evaluated for both within-particle model membranes and compared with the structural impacts of partitioning into vesicle lipid bilayers.

Interactions of lectins, proteins that selectively bind carbohydrates, play an important role in many biological processes including cell adhesion, immune response, and cell signaling. Given the range of lectin functions and their potential for application in disease detection, there is a need for methods to investigate lectin-carbohydrate interactions that are rapid, structurally specific, and sensitive to binding from low-concentration samples. In this work, we describe the preparation and application of supported phospholipid bilayers deposited in wide-pore chromatographic silica particles for confocal Raman-microscopy-based detection of specific binding of concanavalin-A to mannose-functionalized phospholipids. The high surface area of porous-silica supports provides an ample concentration of phospholipid and protein for rapid, label-free detection of lectin binding to be carried out in an individual lipid-bilayer-functionalized particle. The Raman spectrum provides structural information on the bound protein as well as the phospholipid bilayer. Using scattering from the supported-lipid bilayer as an internal standard, Raman scattering from accumulated protein can be interpreted quantitatively to determine its absolute surface coverage on the lipid bilayer. At low glycolipid fraction (<1 mol %) in the prepared bilayer, the surface coverage by protein increases linearly with mannose-lipid densities, where the lectin population corresponds to ∼96% occupancy of the mannose ligands. At increasing glycolipid site densities in excess of 1 mol %, the surface-associated protein population saturates at a coverage that is equivalent to a full monolayer of mannose-bound lectin proteins. The results suggest that Raman microscopy of supported phospholipid bilayers in high-surface-area support particles is a promising approach for in situ, label-free, and quantitative investigation of bilayer-localized protein-ligand interactions.

Porous silica is used as a support in a variety of separation processes, including chromatographic separation and solid-phase extraction. The resolution and efficiency of these applications is significantly impacted by the kinetics of partitioning and molecular transport into the interior of the porous particles. Molecular transport in porous silica has been explored previously by measuring chromatographic elution profiles, but such measurements are limited to relatively low retention conditions, where within-particle molecular transport must be inferred from elution profiles of solutes emerging from a packed column. In this work, a measurement of within-particle molecular transport is carried out using confocal Raman microscopy to probe the time-dependent accumulation of pyrene from an aqueous mobile phase into the center of individual C-18-chromatographic particles. The measured time constants for pyrene accumulation were much slower than diffusion-limited transport of solute in solution to the particle surface. Furthermore, the accumulation into the center of the particle did not show a time-lag characteristic of slow-transport into the particle interior. The exponential rise of pyrene concentration is, however, consistent with first-order Langmuir adsorption kinetics at low surface coverages. The linear dependence of the time-constant on particle radius indicates an adsorption barrier near the outer boundary of the particle, where the accumulation rate depends on flux across the boundary (proportional to the particle area) to satisfy the within-particle capacity at equilibrium (proportional to the particle volume). The pyrene accumulation kinetics into the porous particle, expressed as a heterogeneous rate constant, were nearly 50-times faster than the pyrene adsorption rate at a planar C-18-functionalized silica surface, which demonstrates the impact of multiple surface encounters within the porous structure leading to much greater capture efficiency compared to a planar surface. Monte Carlo simulations of within-particle pyrene diffusion, with the adsorption efficiency estimated from the planar-surface adsorption rate, predict a diffusion-to-capture distance within the porous particle that is within 40% of that observed in the radial dependence of the pyrene within-particle accumulation results.

Permeabilization of the outer mitochondrial membrane is an integral step in apoptosis. The resulting release of pro-apoptotic signaling proteins leads to cell destruction through activation of the cysteine-aspartic protease (caspase) cascade. However, the mechanism of outer mitochondrial membrane (OMM) permeabilization remains unclear. It was recently shown that cytochrome c can induce pore formation in cardiolipin-containing phospholipid membranes, leading to large dextran and protein permeability. In this work, the interaction of cytochrome c with cardiolipin-containing phospholipid vesicles, serving as models of the OMM, is investigated to probe cytochrome c-induced permeability. Lipid vesicles having either a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or mixed-DPPC/cardiolipin membrane and containing a membrane-impermeable Raman tracer 3-nitrobenzenesulfonate (3-NBS) were optically trapped, translated into a solution containing cytochrome c, and monitored for 3-NBS leakage. Cytochrome-correlated leakage was observed only in cardiolipin-containing vesicles. Structural changes observed in the Raman spectra during permeabilization indicated acyl chain disordering along with decreased intensity of the cardiolipin cis-double-bond stretching modes. When the vesicle-associated cytochrome c Raman spectrum is compared with a spectrum in buffer, heme-resonance bands are absent, indicating loss of Met-80 coordination. To verify selective interactions of cytochrome c with cardiolipin, these experiments were repeated where the DPPC acyl chains were deuterated (D62-DPPC), allowing spectral resolution of the DPPC acyl chain response from that of cardiolipin. Interestingly, D62-DPPC acyl chains were unaffected by cytochrome c accumulation, while cardiolipin showed major changes in acyl chain structure. These results suggest that cytochrome-induced permeabilization proceeds through selective interaction of cytochrome c with cardiolipin, resulting in protein unfolding, where the unfolded form interacts with cardiolipin acyl chains within the bilayer to induce permeability.

CO2 injected into depleted oil or gas reservoirs for long-term storage has the potential to mobilize organic compounds and distribute them between sediments and reservoir brines. Understanding this process is important when considering health and environmental risks, but little quantitative data currently exists on the partitioning of organics between supercritical CO2 and water. In this work, a high-pressure, in situ measurement capability was developed to assess the distribution of organics between CO2 and water at conditions relevant to deep underground storage of CO2. The apparatus consists of a titanium reactor with quartz windows, near-infrared and UV spectroscopic detectors, and switching valves that facilitate quantitative injection of organic reagents into the pressurized reactor. To demonstrate the utility of the system, partitioning coefficients were determined for benzene in water/supercritical CO2 over the range 35-65 °C and approximately 25-150 bar. Density changes in the CO2 phase with increasing pressure were shown to have dramatic impacts on benzene's partitioning behavior. Our partitioning coefficients were approximately 5-15 times lower than values previously determined by ex situ techniques that are prone to sampling losses. The in situ methodology reported here could be applied to quantify the distribution behavior of a wide range of organic compounds that may be present in geologic CO2 storage scenarios.