AFM characterization of solid-supported lipid multilayers prepared by spin-coating

Lipids are the principal components of biologically relevant structures as cellular membranes. They have been the subject of many studies due to their biological relevance and their potential applications. Different techniques, such as Langmuir-Blodgett and vesicle-fusion deposition, are available to deposit ordered lipid films on etched surfaces. Recently, a new technique of lipid film deposition has been proposed in which stacks of a small and well-controlled number of bilayers are prepared on a suitable substrate using a spin-coater.We studied the morphological properties of multi-layers made of cationic and neutral lipids (DOTAP and DOPC) and mixtures of them using dynamic mode atomic force microscopy (AFM). After adapting and optimizing, the spin-coating technique to deposit lipids on a chemically etched Silicon (1,0,0) substrate, a morphological nanometer-scale characterization of the aforementioned samples has been provided. The AFM study showed that an initial layer of ordered vesicles is formed and, afterward, depending on details of the spin-coating preparation protocol and to the dimension of the silicon substrate, vesicle fusion and structural rearrangements of the lipid layers may occur.The present data disclose the possibility to control the lipid's structures by acting on spin-coating parameters with promising perspectives for novel applications of lipid films.     

Advances in the characterization of supported lipid films with the atomic force microscope

During the past decade, the atomic force microscope (AFM) has become a key technique in biochemistry and biophysics to characterize supported lipid films, as testified by the continuous growth in the number of papers published in the field. The unique capabilities of AFM are: (i) capacity to probe, in real time and in aqueous environment, the surface structure of lipid films; (ii) ability to directly measure physical properties at high spatial resolution; (iii) possibility to modify the film structure and biophysical processes in a controlled way. Such experiments, published up to June 2000, are the focus of the present review. First, we provide a general introduction on the preparation and characterization of supported lipid films as well as on the principles of AFM. The section 'Structural properties' focuses on the various applications of AFM for characterizing the structure of supported lipid films: visualization of molecular structure, formation of structural defects, effect of external agents, formation of supported films, organization of phase-separated films (coexistence region, mixed films) and, finally, the use of supported lipid bilayers for anchoring biomolecules such as DNA, enzymes and crystalline protein arrays. The section 'Physical properties' introduces the principles of force measurements by AFM, interpretation of these measurements and their recent application to supported lipid films and related structures. Finally, we highlight the major achievements brought by the technique and some of the current limitations.     

Archaeal tetraether bipolar lipids: Structures,functions and applications

Archaea have developed specific tools permitting life under harsh conditions and archaeal lipids are one of these tools. This microreview describes the particular features of tetraether-type archaeal lipids and their potential applications in biotechnology. Natural and synthetic tetraether lipid structures as well as their applications in drug/gene delivery, vaccines and proteoliposomes or as lipid films are reviewed.     

Following the formation of supported lipid bilayers on mica: a study combining AFM, QCM-D, and ellipsometry

Supported lipid bilayers (SLBs) are popular models of cell membranes with potential biotechnological applications and an understanding of the mechanisms of SLB formation is now emerging. Here we characterize, by combining atomic force microscopy, quartz crystal microbalance with dissipation monitoring, and ellipsometry, the formation of SLBs on mica from sonicated unilamellar vesicles using mixtures of zwitterionic, negatively and positively charged lipids. The results are compared with those we reported previously on silica. As on silica, electrostatic interactions were found to determine the pathway of lipid deposition. However, fundamental differences in the stability of surface-bound vesicles and the mobility of SLB patches were observed, and point out the determining role of the solid support in the SLB-formation process. The presence of calcium was found to have a much more pronounced influence on the lipid deposition process on mica than on silica. Our results indicate a specific calcium-mediated interaction between dioleoylphosphatidylserine molecules and mica. In addition, we show that the use of PLL-g-PEG modified tips considerably improves the AFM imaging of surface-bound vesicles and bilayer patches and evaluate the effects of the AFM tip on the apparent size and shape of these soft structures.     

Optimized Electroless Silver Coating for Optical and Plasmonic Applications

Electroless metal deposition is a simple and convenient technique to fabricate metallic films and to provide isotropic metal functionalization of 3D structures with complex geometries. In this work, we describe the synthesis of silver coatings by means of a modified Tollens reaction and their use as optical coating. The chemical composition of the metallization bath is here addressed to optimize the metal coating deposition. The synthesis parameters have been tailored in order to deposit very smooth films which were characterized by scanning electron microscopy, atomic force microscopy, and optical spectroscopy. 2D diffraction gratings and sinusoidal plasmonic gratings were produced with the proposed method. Optical characterization confirmed the plasmonic activities of the resultant structures, proving the efficiency of the described method for optical applications. Thermal annealing was found to improve the surface roughness of the coating and therefore the optical properties of the plasmonic gratings.     

Biophysical investigations of the structure and function of the tear fluid lipid layer and the effect of ectoine. Part A: Natural meibomian lipid films

The tear fluid lipid layer is the outermost part of the tear film on the ocular surface which protects the eye from inflammations and injuries. We investigated the influence of ectoine on the structural organization of natural meibomian lipid films using surface activity analysis and topographical studies. These films exhibit a continuous pressure–area isotherm without any phase transition. With the addition of ectoine, the isotherm is expanded towards higher area per molecule values suggesting an increased area occupied by the interfacial lipid molecules. The AFM topology scans of natural meibomian lipid films reveal a presence of fiber-like structures. The addition of ectoine causes an appearance of droplet-like structures which are hypothesized to be tri-acyl-glycerols and other hydrophobic components excluded from the lipid film. Further the material properties of the droplet-like structure with respect to the surrounding were determined by using the quantitative imaging mode of the AFM technique. The droplet-like structures were found to be comparatively softer than the surrounding. Based on the observations a preliminary hypothesis is proposed explaining the mechanism of action of ectoine leading to the fluidization of meibomian lipid films. This suggests the possibility of ectoine as a treatment for the dry eye syndrome.     

Atomic force microscopy of supported planar membrane bilayers.

Membrane bilayers of dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylethanolamine (DPPE) adsorbed to a freshly cleaved mica substrate have been imaged by Atomic Force Microscopy (AFM). The membranes were mounted for imaging by two methods: (a) by dialysis of a detergent solution of the lipid in the presence of the substrate material, and (b) by adsorption of lipid vesicles onto the substrate surface from a vesicle suspension. The images were taken in air, and show lipid bilayers adhering to the surface either in isolated patches or in continuous sheets, depending on the deposition conditions. Epifluorescence light-microscopy shows that the lipid is distributed on the substrate surfaces as seen in the AFM images. In some instances, when DPPE was used, whole, unfused vesicles, which were bound to the substrate, could be imaged by the AFM. Such membranes should be capable of acting as natural anchors for imaging membrane proteins by AFM.     

In-Plane and Out-of-Plane Plasmons in Random Silver Nanoisland Films

Effective permittivity of closely spaced random nanoparticles supported by a substrate has been calculated using a modified Yamaguchi’s model (MYM) which involves the exact expression of a local field outside a metal nanoparticle (NP) along with the effective-medium approach. Pulsed laser deposition has been used to deposit silver nanoisland films on SiO₂ substrates. In-plane and out-of-plane plasmonic responses have been calculated using MYM for various filling fractions and the results are compared with those obtained from spectroscopic ellipsometry. Distinct features of out-of-plane and in-plane plasmons are observed with an spectroscopic ellipsometer and their behavior is supported by the present theoretical investigation. The comparison of the effective dielectric constants of the films obtained from ellipsometry data with those calculated using MYM shows uniaxial optical anisotropy in our case. The calculated morphological parameters (filling fraction, aspect ratio, and average particle size) using MYM are also found to be consistent with those obtained from FESEM images.     

Morphological and optical characterization of polyelectrolyte multilayers incorporating nanocrystalline cellulose

Aqueous layer-by-layer (LbL) processing was used to create polyelectrolyte multilayer (PEM) nanocomposites containing cellulose nanocrystals and poly(allylamine hydrochloride). Solution-dipping and spin-coating assembly methods gave smooth, stable, thin films. Morphology was studied by atomic force microscopy (AFM) and scanning electron microscopy (SEM), and film growth was characterized by X-ray photoelectron spectroscopy (XPS), ellipsometry, and optical reflectometry. Relatively few deposition cycles were needed to give full surface coverage, with film thicknesses ranging from 10 to 500 nm. Films prepared by spin-coating were substantially thicker than solution-dipped films and displayed radial orientation of the rod-shaped cellulose nanocrystals. The relationship between film color and thickness is discussed according to the principles of thin film interference and indicates that the iridescent properties of the films can be easily tailored in this system.     

Superhydrophobic surface based on a coral-like hierarchical structure of ZnO

Background

Fabrication of superhydrophobic surfaces has attracted much interest in the past decade. The fabrication methods that have been studied are chemical vapour deposition, the sol-gel method, etching technique, electrochemical deposition, the layer-by-layer deposition, and so on. Simple and inexpensive methods for manufacturing environmentally stable superhydrophobic surfaces have also been proposed lately. However, work referring to the influence of special structures on the wettability, such as hierarchical ZnO nanostructures, is rare.

Methodology

This study presents a simple and reproducible method to fabricate a superhydrophobic surface with micro-scale roughness based on zinc oxide (ZnO) hierarchical structure, which is grown by the hydrothermal method with an alkaline aqueous solution. Coral-like structures of ZnO were fabricated on a glass substrate with a micro-scale roughness, while the antennas of the coral formed the nano-scale roughness. The fresh ZnO films exhibited excellent superhydrophilicity (the apparent contact angle for water droplet was about 0°), while the ability to be wet could be changed to superhydrophobicity after spin-coating Teflon (the apparent contact angle greater than 168°). The procedure reported here can be applied to substrates consisting of other materials and having various shapes.

Results

The new process is convenient and environmentally friendly compared to conventional methods. Furthermore, the hierarchical structure generates the extraordinary solid/gas/liquid three-phase contact interface, which is the essential characteristic for a superhydrophobic surface.     

Metallosomes.

Structures and ordered arrays containing organometallic particles have potential application in nanofabrication, smaller computer components, optical devices, sensors, and membrane probes and as detection agents. Here, we describe construction of gold clusters covalently attached to lipids and their use in forming typical lipid structures: micelles, liposomes ("metallosomes"), and sheets on an air-water interface. Two sizes of gold clusters were used, undecagold, with an 11-gold atom core 0.8 nm in diameter, and the larger Nanogold, with a 1.4-nm gold core. The morphology of the structures formed was determined by electron microscopy at a resolution at which single gold-lipid molecules were visualized. Further modification by additional catalytic metal deposition enhanced detectability. The approach is flexible and permits a wide variety of metal particle structures to be created using known lipid structures as templates. Additionally, these gold-lipids may serve as useful membrane labels.     

Defects in ordered aggregates of cardiolipin visualized by atomic force microscopy

The formation and the nature of defects in ordered aggregates of cardiolipin (tetra acyl diphosphatidylglycerol) supported on solid substrates have been investigated by atomic force microscopy (AFM). The experiments were performed on two model systems, i.e. three-dimensional liquid crystals dispersed in water and partially de-hydrated on a hydrophilic surface, and two-dimensional films of molecules self-assembled onto an isotropic hydrophobic surface. Defects were induced both by varying the preparation temperature and by treatment with specific chemicals known to modify the order parameters in natural and artificial membranes, specifically: 2,4-dinitro-phenol (DNP) and pentachloro-phenol (PCP). The effect of lipid oxidation on the nanocrystalline order was also investigated. The images obtained by AFM allow to characterize the type of defects and their local density at nanoscale level. They also provide additional information to differentiate the specific role of acyl chains and polar heads in the process of lipid self-organization.     

Pectin-Lipid Self-Assembly: Influence on the Formation of Polyhydroxy Fatty Acids Nanoparticles

Nanoparticles, named cutinsomes, have been prepared from aleuritic (9,10,16-trihidroxipalmitic) acid and tomato fruit cutin monomers (a mixture of mainly 9(10),16-dihydroxypalmitic acid (85%, w/w) and 16-hydroxyhexadecanoic acid (7.5%, w/w)) with pectin in aqueous solution. The process of formation of the nanoparticles of aleuritic acid plus pectin has been monitored by UV-Vis spectrophotometry, while their chemical and morphological characterization was analyzed by ATR-FTIR, TEM, and non-contact AFM. The structure of these nanoparticles can be described as a lipid core with a pectin shell. Pectin facilitated the formation of nanoparticles, by inducing their aggregation in branched chains and favoring the condensation between lipid monomers. Also, pectin determined the self-assembly of cutinsomes on highly ordered pyrolytic graphite (HOPG) surfaces, causing their opening and forming interconnected structures. In the case of cutin monomers, the nanoparticles are fused, and the condensation of the hydroxy fatty acids is strongly affected by the presence of the polysaccharide. The interaction of pectin with polyhydroxylated fatty acids could be related to an initial step in the formation of the plant biopolyester cutin.     

Structures of archaebacterial membrane lipids

Structural data on archaebacterial lipids is presented with emphasis on the ether lipids of the methanogens. These ether lipids normally account for 80–95% of the membrane lipids with the remaining 5–20% of neutral squalenes and other isoprenoids. Genus-specific combinations of various lipid core structures found in methanogens include diether-tetraether, diether-hydroxydiether, or diether-macrocyclic diether-tetraether lipid moieties. Some species have only the standard diether core lipid, but none are known with predominantly tetraether lipids as found in certain sulfur-dependent archaebacteria. The relative proportions of these lipid cores are known to vary in relation to growth conditions inMethanococcus jannaschii andMethanobacterium thermoautotrophicum. Polar headgroups in glycosidic or phosphodiester linkage to thesn-1 orsn-1 carbons of glycerol consist of polyols, carbohydrates, and amino compounds. The available structural data indicate a close similarity among the polar lipids synthesized within the species of the same genus. Detection of lipid molecular ions by mass spectrometry of total polar lipid extracts is a promising technique to provide valuable comparative data. Since these lipid structures are stable within the extreme environments that many archaebacteria inhabit, there may be specific applications for their use in biotechnology.     

金属离子对模拟脂筏结构和稳定性影响的LB膜和原子力显微镜研究

目的在于研究钠、钾、钙、镁这几种金属离子对模拟脂筏表面形貌结构和稳定性的影响。钠、钾、钙、镁离子在生物体中普遍存在,在生物体的各种生理活动中起着重要的作用。用LB膜技术在一定温度下测量脂筏成分在不同亚相上的表面压与平均分子面积（π-A）等温线,分析其热力学性质;并且用原子力显微镜（AFM）对其表面形貌进行观察。实验表明金属离子对脂筏的形成、尺度和表面形貌都有影响。     

Impact of surface electric properties of carbon-based thin films on platelets activation for nano-medical and nano-sensing applications

Electric surface properties of biomaterials, playing key role to various biointerfacial interactions, were related to hemocompatibility and biosensing phenomena. In this study, the examination of surface electric properties of amorphous hydrogenated carbon thin films (a-C:H) was carried out by means of electrostatic force microscope (EFM) and observation of differences in spatial charge distribution on the surface of the examined films during platelets adhesion was made. The thrombogenic potential of a-C:H thin films developed by magnetron sputtering with ~42% sp³ content and hydrogen partial pressure during deposition was evaluated, by in situ observation with atomic force microscope (AFM) of platelets’ activation and their subsequent adhesion. Platelet-rich plasma drawn from healthy donors was used and semi-contact mode of AFM was applied. Platelets behavior and their correlation with the electric surface properties of the examined a-C:H films by EFM was made for hemocompatibility enhancement and sensing platelets that are less electrical negatively charged and with higher tendency to aggregate and form thrombus. The results are discussed in view of the effect of different deposition conditions of hydrogenated carbon films on their structural and morphological characteristics, surface roughness and electrical properties attributing to different hemocompatibility and sensing aspects.     

Infrared reflection-absorption spectroscopy: Principles and applications to lipid-protein interaction in Langmuir films

Infrared reflection-absorption spectroscopy (IRRAS) of lipid/protein monolayer films in situ at the air/water interface provides unique molecular structure and orientation information from the film constituents. The technique is thus well suited for studies of lipid/protein interaction in a physiologically relevant environment. Initially, the nature of the IRRAS experiment is described and the molecular structure information that may be obtained is recapitulated. Subsequently, several types of applications, including the determination of lipid chain conformation and tilt as well as elucidation of protein secondary structure are reviewed. The current article attempts to provide the reader with an understanding of the current capabilities of IRRAS instrumentation and the type of results that have been achieved to date from IRRAS studies of lipids, proteins, and lipid/protein films of progressively increasing complexity. Finally, possible extensions of the technology are briefly considered.     

Ligand-receptor interactions and membrane structure investigated by AFM and time-resolved fluorescence microscopy

The atomic force microscope (AFM) and the associated dynamic force spectroscopy technique have been exploited to quantitatively assess the interaction between proteins and their binding to specific ligands and membrane surfaces. In particular, we have studied the specific interaction between lung surfactant protein D and various carbohydrates. In addition, we have used scanning AFM and time-resolved fluorescence microscopy to image the lateral structure of different lipid bilayers and their morphological changes as a function of time. The various systems studied illustrate the potential of modern AFM techniques for application to biomedical research, specifically within immunology and liposome-based drug delivery.     

Biomimetic Synthesis of Nanosized Silica Structures on a Substrate with Silicatein

Using atomic force microscopy (AFM), the formation of nanosized silica structures on a substrate, catalyzed by the recombinant silicatein LoSilA1 from the marine sponge Latrunculia oparinae, was studied. It has been shown that at room temperature under neutral conditions, recombinant silicatein immobilized on a mica substrate causes the rapid polycondensation of tetrakis(2-hydroxyethyl) orthosilicate to form spherical particles. Thus, immobilized silicatein may acts as a catalyst in the preparation of ordered silica structures on various surfaces.     

Imaging biomolecule arrays by atomic force microscopy.

We describe here a method for constructing ordered molecular arrays and for detecting binding of biomolecules to these arrays using atomic force microscopy (AFM). These arrays simplify the discrimination of surface-bound biomolecules through the spatial control of ligand presentation. First, photolithography is used to spatially direct the synthesis of a matrix of biological ligands. A high-affinity binding partner is then applied to the matrix, which binds at locations defined by the ligand array. AFM is then used to detect the presence and organization of the high-affinity binding partner. Streptavidin-biotin arrays of 100 x 100 microns and 8 x 8 microns elements were fabricated by this method. Contact and noncontact AFM images reveal a dense lawn of streptavidin specific to the regions of biotin derivatization. These protein regions are characterized by a height profile of approximately 40 A over the base substrate with a 350-nm edge corresponding to the diffraction zone of the photolithography. High resolution scans reveal a granular topography dominated by 300 A diameter features. The ligand-bound protein can then be etched from the substrate using the AFM tip, leaving an 8 A shelf that probably corresponds to the underlying biotin layer.