We have studied the spreading of phospholipid vesicles on photochemically patterned n-octadecylsiloxane monolayers using epifluorescence and imaging ellipsometry measurements. Self-assembled monolayers of n-octadecylsiloxanes were patterned using short-wavelength ultraviolet radiation and a photomask to produce periodic arrays of patterned hydrophilic domains separated from hydrophobic surroundings. Exposing these patterned surfaces to a solution of small unilamellar vesicles of phospholipids and their mixtures resulted in a complex lipid layer morphology epitaxially reflecting the underlying pattern of hydrophilicity. The hydrophilic square regions of the photopatterned OTS monolayer reflected lipid bilayer formation, and the hydrophobic OTS residues supported lipid monolayers. We further observed the existence of a boundary region composed of a nonfluid lipid phase and a lipid-free moat at the interface between the lipid monolayer and bilayer morphologies spontaneously corralling the fluid bilayers. The outer-edge of the boundary region was found to be accessible for subsequent adsorption by proteins (e.g., streptavidin and BSA), but the inner-edge closer to the bilayer remained resistant to adsorption by protein or vesicles. Mechanistic implications of our results in terms of the effects of substrate topochemical character are discussed. Furthermore, our results provide a basis for the construction of complex biomembrane models, which exhibit fluidity barriers and differentiate membrane properties based on correspondence between lipid leaflets. We also envisage the use of this construct where two-dimensionally fluid, low-defect lipid layers serve as sacrificial resists for the deposition of protein and other material patterns.
Subnanometer-scale vertical z-resolution coupled with large lateral area imaging, label-free, noncontact, and in situ advantages make the technique of optical imaging ellipsometry (IE) highly suitable for quantitative characterization of lipid bilayers supported on oxide substrates and submerged in aqueous phases. This article demonstrates the versatility of IE in quantitative characterization of structural and functional properties of supported phospholipid membranes using previously well-characterized examples. These include 1), a single-step determination of bilayer thickness to 0.2 nm accuracy and large-area lateral uniformity using photochemically patterned single 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers; 2), hydration-induced spreading kinetics of single-fluid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers to illustrate the in situ capability and image acquisition speed; 3), a large-area morphological characterization of phase-separating binary mixtures of 1,2-dilauroyl-sn-glycero-3-phosphocholine and galactosylceramide; and 4), binding of cholera-toxin B subunits to GM1-incorporating bilayers. Additional insights derived from these ellipsometric measurements are also discussed for each of these applications. Agreement with previous studies confirms that IE provides a simple and convenient tool for a routine, quantitative characterization of these membrane properties. Our results also suggest that IE complements more widely used fluorescence and scanning probe microscopies by combining large-area measurements with high vertical resolution without the use of labeled lipids.
We report the development of a microdevice for detecting local interferon gamma (IFN-γ) release from primary human leukocytes in real-time. Our microdevice makes use of miniature aptamer-modified electrodes integrated with microfluidics to monitor cellular production of IFN-γ. The aptamer species consists of a DNA hairpin molecule with thiol groups on the 3′-end for self-assembly onto Au electrodes. A redox reporter is covalently attached at the 5′-end for electrochemical sensing. This aptasensor has excellent sensitivity for IFN-γ (<60 pM detection limit) and responds to the target analyte in real-time without additional washing or labeling steps. Aptamer-functionalized electrode arrays are fabricated on glass slides containing poly(ethylene glycol) (PEG) hydrogel patterns designed to expose glass regions adjacent to electrodes while protecting the remainder of the surface from non-specific adsorption. The micropatterned substrates are integrated with PDMS microfluidic channels and incubated with T-cell-specific antibodies (Ab) (anti-CD4). Upon injection of blood, leukocytes are bound to Ab-modified glass regions in proximity to aptasensors. Cytokine release from captured cells is triggered by mitogenic activation and detected at the aptamer-modified electrodes using square wave voltammetry (SWV). The IFN-γ signal is monitored in real-time with signal appearing as early as 15 min post-stimulation from as few as 90 T-cells. The observed IFN-γ release profiles are used to calculate an initial IFN-γ production rate of 0.0079 pg cell−1 h−1 upon activation. The work described here represents an important step towards development of aptasensors for immune cell analysis and blood-based diagnostics.
The asymmetric distribution of charged molecules between the leaflets of solid-substrate-supported phospholipid bilayers is studied using imaging ellipsometry, fluorescence microscopy, and numerical solutions of the Poisson-Boltzmann equation. Experiments are facilitated by the use of patterned substrates that allow for side-by-side comparison of lipid monolayers and supported bilayers. On silica surfaces, negatively charged lipid components are shown to be enriched in the outer leaflet of a supported bilayer system at modest salt concentrations. The approaches developed provide a general means for determining asymmetries of charged components in supported lipid bilayers.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.