Confocal Raman Microscopy Investigation of Phospholipid Monolayers Deposited on Nitrile-Modified Surfaces in Porous Silica Particles

Abstract

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 Å 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.

Inter-Leaflet Phospholipid Exchange Impacts the Ligand Density Available for Protein Binding at Supported Lipid Bilayers

“…This shift to lower frequency is consistent with a change in the interfacial environment from the highdielectric constant of an aqueous solution to a low-dielectric constant environment equivalent to a nitrile group in a hydrocarbon environment. 36,46 This result provides evidence that the surface nitrile groups are in contact with the acyl chains of the adsorbed lipid, 36 in agreement with the tailsdown monolayer structure proposed for lipid monolayers on the surfaces of nitrile-derivatized glass nanopores. 47,48 Further evidence for DPPC and POPC forming supported lipid monolayers on the nitrile-derivatized silica is provided by the Raman scattering intensities from accumulated phospholipids on bare versus nitrile-derivatized silica surfaces.…”

“…These quantitative results support the conclusion that POPC and DPPC deposited on the nitrile-terminated silica form phospholipid monolayers, where the nitrile frequency response is consistent with lipid acyl chains oriented toward the nitrile interface. 36,47 Quantitative Determination of Lipid Densities of Supported Bilayers and Monolayers. The high specific surface area of the porous silica particles allows a small (10 mg) sample of particles to contain sufficient carbon in a lipid bilayer or monolayer to be quantified by elemental carbon analysis.…”

“…From these results, the structure of these porous silica-supported lipid bilayers (headgroup spacing, lipid bilayer thickness, and melting phase transition) was found to be comparable to supported bilayers formed on planar surfaces. 35,36 Raman scattering from these bilayers has also been used to quantify the association response of a lectin protein, concanavalin A, with mannose-capped phospholipids incorporated into the porous silica-supported lipid bilayers. 6 This study demonstrated that confocal Raman microscopy of ligand-modified phospholipid bilayers in high-surface-area support particles can be an effective method for in situ, labelfree, and quantitative investigation of protein−ligand interactions at supported lipid bilayer−solution interfaces.…”

Using single-vesicle technologies to unravel the heterogeneity of extracellular vesicles

“…Therefore, RTM, with a typical processing capacity of 0.2 particles per min, is not considered a high-throughput methodology 71 . RTM can, however, be used to obtain interesting, unique information not only for EVs 57,60,61,[70][71][72][73][74][75][76][77] but also for many other bioparticles such as liposomes, lipid layers on synthetic nanoparticles and others [78][79][80][81][82][83] .…”

Confocal Raman Microscopy Enables Label-Free, Quantitative, and Structurally Informative Detection of DNA Hybridization at Porous Silica Surfaces

“…Raman spectra of the silica following functionalization are accompanied by the appearance of the 2580 cm –1 S–H stretching mode indicative of the presence of surface-immobilized thiol (Figure ). To quantify the surface coverage of the bound silane reagent, the total intensity of scattering from the C–H stretching modes of the immobilized silane was used for calibration through comparison with scattering from a commercial propyl nitrile-derivatized silica having a previously determined ligand surface density and specific surface area . Using this means of surface characterization, we estimate our thiol surface density to be 180 ± 20 nmol/m 2 or about 5% of a full monolayer.…”