Supported Lipid Monolayers Research Articles

Probing Deuteration-Induced Phase Separation in Supported Lipid Monolayers using Hyperspectral TERS Imaging.

In this study, we investigate the impact of deuteration on the formation of phase-separated domains in supported lipid monolayers using hyperspectral Tip-Enhanced Raman Spectroscopy (TERS) imaging. The intricate organization of biological membranes plays a crucial role in cellular functions. Various factors that influence domain formation have been identified in previous studies such as lipid tail length and cholesterol concentration. Deuterium labeling of lipids has proven useful for probing cellular structures and dynamics, but its impact on lipid phase separation remains underexplored. By examining 1:1 mixed monolayers of dipalmitoylphosphatidylcholine (DPPC) and deuterated DPPC on Au(111) surfaces, we reveal partial segregation of domains rich in deuterated and nondeuterated lipids. This study addresses a gap in knowledge by examining the impact of deuteration on lipid tail behavior, offering new insights into how even subtle structural modifications can influence phase behavior. Furthermore, it demonstrates that TERS can be a powerful, nondestructive, and label-free nanoanalytical tool for analyzing lipid membranes and advance the field of membrane biophysics.

Nanoscale visualization of phase separation in binary supported lipid monolayer using tip-enhanced Raman spectroscopy.

Supported lipid membranes are an important model system to study the phase separation behavior at the nanoscale. However, the conventional nanoanalytical tools often fail to provide reliable chemical characterization of the phase separated domains in a non-destructive and label-free manner. This study demonstrates the application of scanning tunneling microscopy-based tip-enhanced Raman spectroscopy (TERS) to study the nanoscale phase separation in supported d62-DPPC : DOPC lipid monolayers. Hyperspectral TERS imaging successfully revealed a clear segregation of the d62-DPPC-rich and DOPC-rich domains. Interestingly, nanoscale deposits of d62-DPPC were observed inside the DOPC-rich domains and vice versa. High-resolution TERS imaging also revealed the presence of a 40-120 nm wide interfacial region between the d62-DPPC-rich and DOPC-rich domains signifying a smooth transition rather than a sharp boundary between them. The novel insights obtained in this study demonstrate the effectiveness of TERS in studying binary lipid monolayers at the nanoscale.

Real-time topography inspection of DPPC monolayers using a surface-plasmon resonance sensor

This paper presents an alternative optical characterization of biosensors based on supported lipid monolayers (SLMs). Developing these biosensors requires precise thickness characterization of the films to understand their structure and dynamics. This paper proposes an optical technique to measure the thickness, optical properties, and location of a dipalmitoyl-phosphatidylcholine (DPPC) SLM on top of a metallic thin film. DPPC SLMs are of interest for biosensing applications, such as detecting pulmonary-related infections like SARS-CoV-2, Avian Influenza, and the H1N1 influenza virus. The monolayer was fabricated using the Langmuir–Blodgett technique, and the experimental characterization consisted of measuring the surface-plasmon resonance angle in the Kretschmann configuration. This technique provides an alternative option for real-time visual inspection and determination of the location and shape of DPPC monolayers in large areas. Therefore, it offers a useful tool for further developing SLM-based biosensors.

Homogeneous supported monolayer from microbial glycolipid biosurfactant

The development of supported glycosylated lipid layers is an important trend in the field of glyconanomaterials for their interest in understanding sugar-sugar and protein–sugar interactions, these being at the core of cellular, bacterial or viral adhesion. The conventional self-assembled monolayer (SAM) approach generally requires a thiolated glycoconjugate and a gold substrate. In this work, we show how glycolipid amphiphiles of natural origin, commonly known as microbial biosurfactants, can be easily deposited onto a substrate. Spontaneously produced by microorganisms but lacking a thiol group, one can take advantage of their self-assembly properties to prepare homogeneous supported lipid monolayers (SLM). We then choose a saturated glucolipid, G-C18:0, which forms a colloidal lamellar phase under diluted conditions. The lamellae can then be deposited onto a substrate (silicon, gold) using a physical method (dip coating). Dip coating is preferred over more classical deposition methods (Langmuir-Blodgett-LB-, vesicle fusion or spin-coating) because of its versatility, compatibility with aqueous solutions and robust control of the thickness below 10 nm. Defect-free glycosylated SLM from a microbial biosurfactant are then easily developed. A combination of ellipsometry, fluorescence microscopy, atomic force microscopy and infrared nanospectroscopy (AFMIR) show that the glycosylated SLM are defect-free, have a thickness of 2.8 ± 1.0 nm and they are highly homogeneous at scales going from the nm to cm.

Nanoscale Chemical Imaging of Supported Lipid Monolayers using Tip‐Enhanced Raman Spectroscopy

AbstractVisualizing the molecular organization of lipid membranes is essential to comprehend their biological functions. However, current analytical techniques fail to provide a non‐destructive and label‐free characterization of lipid films under ambient conditions at nanometer length scales. In this work, we demonstrate the capability of tip‐enhanced Raman spectroscopy (TERS) to probe the molecular organization of supported DPPC monolayers on Au (111), prepared using the Langmuir–Blodgett (LB) technique. High‐quality TERS spectra were obtained, that permitted a direct correlation of the topography of the lipid monolayer with its TERS image for the first time. Furthermore, hyperspectral TERS imaging revealed the presence of nanometer‐sized holes within a continuous DPPC monolayer structure. This shows that a homogeneously transferred LB monolayer is heterogeneous at the nanoscale. Finally, the high sensitivity and spatial resolution down to 20 nm of TERS imaging enabled reproducible, hyperspectral visualization of molecular disorder in the DPPC monolayers, demonstrating that TERS is a promising nanoanalytical tool to investigate the molecular organization of lipid membranes.

Nanoscale Chemical Imaging of Supported Lipid Monolayers using Tip-Enhanced Raman Spectroscopy.

Visualizing the molecular organization of lipid membranes is essential to comprehend their biological functions. However, current analytical techniques fail to provide a non‐destructive and label‐free characterization of lipid films under ambient conditions at nanometer length scales. In this work, we demonstrate the capability of tip‐enhanced Raman spectroscopy (TERS) to probe the molecular organization of supported DPPC monolayers on Au (111), prepared using the Langmuir–Blodgett (LB) technique. High‐quality TERS spectra were obtained, that permitted a direct correlation of the topography of the lipid monolayer with its TERS image for the first time. Furthermore, hyperspectral TERS imaging revealed the presence of nanometer‐sized holes within a continuous DPPC monolayer structure. This shows that a homogeneously transferred LB monolayer is heterogeneous at the nanoscale. Finally, the high sensitivity and spatial resolution down to 20 nm of TERS imaging enabled reproducible, hyperspectral visualization of molecular disorder in the DPPC monolayers, demonstrating that TERS is a promising nanoanalytical tool to investigate the molecular organization of lipid membranes.

Discerning perturbed assembly of lipids in a model membrane in presence of violacein

Violacein is a naturally found pigment that is used by some gram negative bacteria to defend themselves from various gram positive bacteria. As a result, this molecule has caught attention for its potential biomedical applications and has already shown promising outcomes as an antiviral, an antibacterial, and an anti-tumor agent. Understanding the interaction of this molecule with a cellular membrane is an essential step to extend its use in the pharmaceutical paradigm. Here, the interaction of violacein with a lipid monolayer formed at the air–water interface is found to depend on electrostatic nature of lipids. In presence of violacein, the two dimensional (2D) pressure–area isotherms of lipids have exhibited changes in their phase transition pressure and in-plane elasticity. To gain insights into the out-of-plane structural organization of lipids in a membrane, X-ray reflectivity (XRR) study on a solid supported lipid monolayer on a hydrophilic substrate has been performed. It has revealed that the increase in membrane thickness is more pronounced in the zwitterionic and positively charged lipids compared to the negatively charged one. Further, the lipid molecules are observed to decrease their tilt angle made with the normal of lipid membrane along with an alteration in their in-plane ordering. This has been quantified by grazing incidence X-ray diffraction (GIXD) experiments on the multilayer membrane formed in an environment with controlled humidity. The structural reorganization of lipid molecules in presence of violacein can be utilized to provide a detailed mechanism of the interaction of this molecule with cellular membrane.

Elasticity‐Driven Membrane Budding through Cholesterol Concentration on Supported Lipid Monolayer–Bilayer Junction

AbstractMembrane budding is an essential process during various biological activities, such as intercellular communication and transportation of life‐sustaining molecules and signals, which primarily occur with the aid of particular proteins. During such processes, the presence of cholesterol and its assembly into particular domains within the membranes have attracted considerable attention due to their unique characteristics in terms of the regulation of signal activities, membrane fluidity, and hormone production during metabolic pathways. However, despite these crucial roles, the precise mechanisms of their spatiotemporal localization toward specific areas and their physiological roles in membrane budding without the assistance of particular proteins remain unclear. Herein, a model membrane platform is reconstituted with lipid monolayer–bilayer junctions, which facilitate the selective concentration of cholesterol on the lipid monolayer regions. In vitro membrane budding is demonstrated by the remaining vesicles where the lipid monolayer membrane is ruptured. The physicochemical approach to the selective concentration of cholesterol and its consequent membrane vesiculation offer important clues to help understand the underlying mechanisms of cholesterol in the lipid‐driven membrane‐budding process.

Broadband vibrational sum-frequency generation spectrometer at 100 kHz in the 950-1750 cm-1 spectral range utilizing a LiGaS2 optical parametric amplifier.

We present a 100 kHz broadband vibrational sum-frequency generation (VSFG) spectrometer operating in the 5.7-10.5 µm (950-1750 cm-1) wavelength range. The mid-infrared beam of the system is obtained from a collinear, type-I LiGaS2-crystal-based optical parametric amplifier seeded by a supercontinuum and pumped directly by 180 fs, ~32 µJ, 1.03 µm pulses from an Yb:KGd(WO4)2 laser system. Up to 0.5 µJ mid-infrared pulses with durations below 100 fs were obtained after dispersion compensation utilizing bulk materials. We demonstrate the utility of the spectrometer by recording high-resolution, low-noise vibrational spectra of Langmuir-Blodgett supported lipid monolayers on CaF2. The presented VSFG spectrometer scheme offers superior signal-to-noise ratios and constitutes a high-efficiency, low-cost, easy-to-use alternative to traditional schemes relying on optical parametric amplification followed by difference frequency generation.

Ultrathin Supported Lipid Monolayer with Unprecedented Mechanical and Dielectric Properties

AbstractThe development of ultrathin dielectrics for low power electronics operations, flexible and printed electronics, and field‐effect‐transistor‐based sensors is still a challenge. Here, monolayers of engineered lipids supported on silicon are reported presenting exceptional mechanical and dielectric properties. The lipid monolayers are stabilized using a simple procedure based on a two‐stage reticulation process in both their aliphatic chains and their head‐group. With a thickness lower than 3 nm, such layers are demonstrated to offer exceptional mechanical and dielectric stability with unprecedented low leakage current and dielectric strength. Surprisingly, the mechanical and dielectric pressures required to rupture/breakdown the monolayers are shown to be similar. These results suggest the presence of a strong correlation between mechanical and dielectric properties, as well as between the mechanisms of rupture and breakdown.

Direct observation of nanometer-scale pores of melittin in supported lipid monolayers.

Melittin is the most studied membrane-active peptide and archetype within a large and diverse group of pore formers. However, the molecular characteristics of melittin pores remain largely unknown. Herein, we show by atomic force microscopy (AFM) that lipid monolayers in the presence of melittin are decorated with numerous regularly shaped circular pores that can be distinguished from nonspecific monolayer defects. The specificity of these pores is reinforced through a statistical evaluation of depressions found in Langmuir-Blodgett monolayers in the presence and absence of melittin, which eventually allows characterization of the melittin-induced pores at a quantitative low-resolution level. We observed that the large majority of pores exhibit near-circular symmetry and a Gaussian distribution in size, with a mean diameter of ∼8.7 nm. A distinctive feature is a ring of material found around the pores, made by, on average, three positive peaks, with a height over the level of the lipidic background of ∼0.23 nm. This protruding rim is most likely due to the presence of melittin near the pore border. Although the current resolution of the AFM images in the {x, y} plane does not allow distinction of the specific organization of the peptide molecules, these results provide an unprecedented view of melittin pores formed in lipidic interfaces and open new perspectives for future structural investigations of these and other pore-forming peptides and proteins using supported monolayers.

High Precision, Electrochemical Detection of Reversible Binding of Recombinant Proteins on Wide Bandgap GaN Electrodes Functionalized with Biomembrane Models

We report a novel hybrid charge sensor realized by the deposition of phospholipid monolayers on highly doped n‐GaN electrodes. To detect the binding of recombinant proteins with histidine‐tags, lipid vesicles containing chelator lipids were deposited on GaN electrodes pre‐coated with octadecyltrimethoxysilane monolayers. Owing to its optical transparency, GaN allows the confirmation of the fluidity of supported membranes by fluorescence recovery after photo‐bleaching (FRAP). The electrolyte‐(organic) insulator‐semiconductor (EIS) setup enables one to transduce variations in the surface charge density ΔQ into a change in the interface capacitance ΔC p and, thus, the flat‐band potential ΔU FB. The obtained results demonstrate that the membrane‐based charge sensor can reach a high sensitivity to detect reversible changes in the surface charge density on the membranes by the formation of chelator complexes, docking of eGFP with histidine tags, and cancellation by EDTA. The achievable resolution of ΔQ ≥ 0.1 μC/cm2 is better than that obtained for membrane‐functionalized p‐GaAs, 0.9 μC/cm2, and for ITO coated with a polymer supported lipid monolayer, 2.2 μC/cm2. Moreover, we examined the potential application of optically active InGaN/GaN quantum dot structures, for the detection of changes in the surface potential from the photoluminescence signals measured at room temperature.

Biomimetic surface modification with bolaamphiphilic archaeal tetraether lipids via liposome spreading.

Through investigations of the self-assembly behavior of three different tetraether lipids, the authors successfully established a solid supported, biomimetic tetraether lipid membrane via liposome spreading. These bolaamphiphilic lipids are the main compound in membranes of archaea, extremophile microorganisms, which underwent an enormous adaptation to extreme conditions in their natural environment with regard to temperature, pH, and high salt concentrations. Starting from a mathematical point of view, the authors calculated hydrophilic-lipophilic balance values for each lipid and recognized a wide difference in self-assembly potentials relying on size and hydrophilic properties of the lipid head groups. These results were in good accordance with data generated by lipid experiments at the air-water interface applying a Langmuir-Blodgett film balance so that the self-assembly potential of two different tetraether lipids was found to be sufficient to form stable liposomes in aqueous media. Liposomes composed of the main phospholipid of the archaea strain Sulfolobus acidocaldarius fused covalently on silanized glass substrates and formed a monomolecular lipid layer with upright standing molecules at film consistent thicknesses of approximately 5 nm determined by ellipsometry and atomic force microscopy. This work can be considered as a basic strategy to find optimized lipid properties in terms of liposome formation and spreading in water, and it is the first report about archaeal liposome fusing on surfaces to establish a solid supported lipid monolayer.

Ca2 + induces PI(4,5)P2 clusters on lipid bilayers at physiological PI(4,5)P2 and Ca2 + concentrations

Calcium has been shown to induce clustering of PI(4,5)P2 at high and non-physiological concentrations of both the divalent ion and the phosphatidylinositol, or on supported lipid monolayers. In lipid bilayers at physiological conditions, clusters are not detected through microscopic techniques. Here, we aimed to determine through spectroscopic methodologies if calcium plays a role in PI(4,5)P2 lateral distribution on lipid bilayers under physiological conditions. Using several different approaches which included information on fluorescence quantum yield, polarization, spectra and diffusion properties of a fluorescent derivative of PI(4,5)P2 (TopFluor(TF)-PI(4,5)P2), we show that Ca2+ promotes PI(4,5)P2 clustering in lipid bilayers at physiological concentrations of both Ca2+ and PI(4,5)P2. Fluorescence depolarization data of TF-PI(4,5)P2 in the presence of calcium suggests that under physiological concentrations of PI(4,5)P2 and calcium, the average cluster size comprises ~15 PI(4,5)P2 molecules. The presence of Ca2+-induced PI(4,5)P2 clusters is supported by FCS data. Additionally, calcium mediated PI(4,5)P2 clustering was more pronounced in liquid ordered (lo) membranes, and the PI(4,5)P2-Ca2+ clusters presented an increased affinity for lo domains. In this way, PI(4,5)P2 could function as a lipid calcium sensor and the increased efficiency of calcium-mediated PI(4,5)P2 clustering on lo domains might provide targeted nucleation sites for PI(4,5)P2 clusters upon calcium stimulus.

Mechanistic Insight into Patterned Supported Lipid Bilayer Self-Assembly

Patterned supported lipid bilayers (SLBs) provide a model system for studying fluid lipid bilayers and transmembrane proteins in an array format. SLB arrays self-assemble on patterned self-assembled monolayers (SAMs) consisting of hexadecanethiol and glycol-terminated regions. While the mechanism of SLB formation on glass has been studied extensively, the formation of SLBs on other substrates is not necessarily well understood. Moreover, SLB arrays on patterned SAMs represent an intriguing system, since lipid vesicles do not adhere to glycol-terminated monolayers. Here, we utilize surface plasmon resonance imaging (SPRi) and kinetic analysis to examine the mechanism of SLB formation on the glycol-terminated regions of patterned SAMs and supported lipid monolayer (SLM) formation on alkyl-terminated regions of patterned SAMs. We determine that vesicles rupture to form a patterned SLB through a two-step mechanism that is dependent upon vesicle attachment at the interface of the two regions of the patterned monolayer.

Supported Lipid Monolayer with Improved Nanomechanical Stability: Effect of Polymerization

We study the effect of polymerization on the nanomechanical stability of supported lipid monolayers consisting of 1,2-di-(10Z,12Z-tricosadiynoyl)-sn-glycero-3-phosphocholine by means of force mapping using an atomic force microscope. For both nonpolymerized and polymerized lipid monolayers, we investigate the break-through forces required to rupture the monolayers for a whole range of loading velocities. We show that the average break-through force exerted by the tip and required to penetrate the monolayer has a logarithmic dependence on the loading rate. Both Young moduli and intrinsic Gibbs energies have been determined for the nonpolymerized and polymerized lipid monolayers, and we show a drastic effect of polymerization on the nanomechanical stability of the monolayer with an increase by a factor of ∼100 for the young modulus and ∼3 for the intrinsic Gibbs activation energy.

A comparison of detergent action on supported lipid monolayers and bilayers

Using spatially patterned supported lipid mono- and bilayers, we compare the effect of transleaflet dynamics on membrane solubilization by a common, non-ionic detergent in single samples. We find that at concentrations surrounding CMC, complete bilayers undergo 5–8% lateral expansion followed by rapid dissolution. In contrast, single supported monolayers remain remarkably resistant to solubilization, suggesting the central role of detergent or lipid flip-flop in driving membrane solubilization. In addition to the previously well-appreciated mode of detergent-resistance by tight lateral packing of saturated and cholesterol-rich lipids (e.g., rafts) in membrane bilayers, our results suggest that hindrance to interleaflet dynamics, such as by strong interaction with the cytoskeleton, provides an alternative mechanism by which membranes resist detergent solubilisation. Furthermore, we show that this differential resistance can be exploited to design spatial compositional patterns of lipid bilayers and monolayers.

Multi-Modal Imaging of Supported Membranes using TOF-SIMS, AFM and Fluorescence Microscopy

Multi-modal imaging methods that combine multiple contrast mechanisms have been widely applied to study the localization and cooperative rearrangement of membrane lipids and proteins in supported lipid membranes, as a model for the heterogeneity of natural cell membranes. We use a combination of fluorescence spectroscopy and microscopy, atomic force microscopy (AFM) and chemical mapping using time-of-flight secondary ion mass spectrometry (TOF-SIMS) to probe the organization of lipid domains in supported lipid monolayers and bilayers and to examine the distribution of lipids and proteins in these domains. The application of these methods will be illustrated with studies of the membrane reorganization promoted by both direct and enzymatic incorporation of ceramide in supported membranes. The enzymatic incorporation of ceramide by sphingomyelin hydrolysis is believed to cause coalescence of small ordered membrane domains to give larger signaling platforms, thus providing a mechanism to aggregate membrane receptors and enhance signaling efficiency. We have examined the consequences of ceramide incorporation in supported membranes prepared from ternary lipid mixtures that have coexisting fluid and liquid-ordered phases. The direct incorporation of ceramide leads to the formation of a new ceramide-rich ordered phase that is localized in small sub-domains within the original liquid-ordered domains. Chemical mapping using TOF-SIMS provides unequivocal identification of the ceramide-enriched phase. Enzymatic generation of ceramide also produces ceramide-enriched islands, but leads to a larger scale membrane reorganization that includes clustering of individual domains, formation of areas of fluid phase that are devoid of domains and formation of membrane defects. A combination of correlated AFM and fluorescence imaging and the use of a custom-designed cholesterol probe provide further information on the complex enzyme-mediated bilayer restructuring.

Multi-Technique Surface Analytical Characterization of Dessication-Resistant Supported Lipid Monolayers

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.

Thermodynamic and structural studies of mixed monolayers: Mutual mixing of DPPC and DPPG with DoTAP at the air–water interface

Phospholipid monomolecular films at the air–water interface are useful model membranes to understand miscibility among various components. Surface pressure ( π)–area ( A) isotherms of pure and mixed monolayers of dioleoyltrimethylammonium propane (DoTAP)–dipalmitoylphosphatidylcholine (DPPC) and DoTAP–dipalmitoyphosphatidylglycerol (DPPG) were constructed using a surface balance. DPPC and DPPG produced isotherms as expected and reported earlier. DoTAP, an unsaturated lipid, demonstrated a continuous π– A isotherm. Associative interactions were identified in DPPC–DoTAP mixtures compared to the pure components, while DPPG–DoTAP mixtures showed repulsive interaction up to an equimolar ratio. Compression moduli of the monolayers revealed that DPPC–DoTAP mixtures had increasing stability with increasing surface pressure, but addition of DoTAP to DPPG showed instability at low and intermediate concentrations. In both cases increased stability was returned at higher X DoTAP values and surface pressures. Lipid monolayer film thickness values, determined on a gold coated glass substrate by surface plasmon resonance spectroscopy (SPR), indicated a systematic change in height profile for DPPC–DoTAP mixtures with increasing X DoTAP. However, DPPG–DoTAP mixed monolayer systems demonstrated a biphasic response. The SPR data were in excellent agreement with our interpretation of the structure of solid supported lipid monolayers.