Two-dimensional arrays of human C-reactive protein formed on a dense aqueoys subphase underneath a lipid layer

A recently developed protein spreading technique, using a subphase of higher density than that of the injected protein solution, was combined with the lipid monolayer approach for two-dimensional crystallization experiments of human C-reactive protein (hCRP) at the air-water interface. Densely packed two-dimensional arrays of hCRP adsorbed on a stearylamine/egg yolk phosphatidylcholine lipid layer have been obtained in the pH range from 5.4 to 5.8. Correct choices of the lipid mixture and of the pH of the subphase were important for the formation of large arrays. Image analysis of transmission electron micrographs showed that the densely packed arrays do not possess crystalline order. The orientational homogeneity of the protein molecules adsorbed on the lipid layer could be used, however, to facilitate single particle averaging techniques. At a resolution of about 1.5 nm a clear handedness of the five-fold symmetric molecule becomes visible, and an asymmetric mass distribution of each subunit is revealed.     

Nanomolar protein sensing with embedded receptor molecules

A new concept of protein sensing at the air-water interface is introduced, based on amphiphilic receptor molecules embedded in a lipid monolayer. The process begins with incorporation of a small amount (0.13 equiv) of one or two different calix[4]arenes, adorned with charged functional groups at their upper rims, into a stearic acid monolayer. These doped monolayers are subsequently shown to attract peptides and proteins from the aqueous subphase. Depending on the host structure, the monolayers can be made selective for basic or acidic proteins. A working model is proposed, which explains the large observed p/A shifts with reincorporation of excess receptor molecules into the lipid monolayer after complex formation with the oppositely charged protein. This requires a self-assembly of multiple calixarene units over the protein surface, which bind the protein in a cooperative fashion. Oppositely charged calixarene derivatives do not form molecular capsules inside the monolayer, but rather remain separate inside the lipid layer, adopting a perpendicular orientation. They combine their hydrogen bond donor and acceptor capacities, and thus markedly enhance the sensitivity of the sensor system toward proteins, pushing the detection limits to 10 pM concentrations. The response pattern obtained from various receptor units inside the monolayer toward the same protein creates a fingerprint for this protein, which can hence be selectively detected at nanomolar concentrations (pattern recognition).     

Adsorption of carboxylate-modified gold nanoparticles on an octadecylamine monolayer at the air/liquid interface

Gold nanoparticles (Au NPs) were prepared and surface-modified by mercaptosuccinic acid (MSA) to render a surface with carboxylic acid groups (MSA-Au). Octadecylamine (ODA) was used as a template monolayer to adsorb the Au NPs dispersed in the subphase. The effect of MSA concentration on the incorporation of Au NPs on the ODA monolayer and the relevant behavior of the mixed monolayer were studied using the pressure-area (pi-A) isotherm and transmission electron microscopy (TEM) observations. The experimental results showed that the adsorbed density of Au NPs is low without the surface modification by MSA. When MSA was added into the Au NP-containing subphase, the incorporation amount of Au NPs increased with increasing MSA concentration up to approximately 1 x 10-5 M for the particle density of 1.3 x 1011 particles/mL. With a further increase in the MSA concentration, the adsorbed particle density decreases due to competitive adsorption between the free MSA molecules and the MSA-Au NPs. It is inferred that free MSA molecules adsorb more easily than the MSA-Au NPs on the ODA monolayer. Therefore, an excess amount of MSA present in the subphase is detrimental to the incorporation of gold particles. The study on the monolayer behavior also shows that the pi-A isotherm of the ODA monolayer shifts right when small amounts of Au NPs or free MSA molecules are incorporated. However, when larger amounts of particles are adsorbed at the air/liquid interface, a left shift of the pi-A isotherm appears, probably due to the adsorption of ODA molecules onto the particle surface and the transferring of the particles from beneath the ODA monolayer to the air/water interface. According to the present method, it is possible to prepare uniform particulate films of controlled densities by controlling the particle concentration in the subphase, the MSA concentration, and the surface pressure of a mixed monolayer.     

Interaction between puroindolines and the major polar lipids of wheat seed endosperm at the air-water interface

To understand the role of the puroindolines (PIN-a and PIN-b) in the defense mechanism and stabilization of lipid films in the gas cell of bread dough, we have isolated the proteins and lipids from wheat seed endosperm and studied their interaction at the air/water interface using a Langmuir trough. The nature and shape of the pressure–area compression isotherms of the lipid monolayer in the presence of puroindolines in the subphase depended on the concentration of protein. A distinct phase separation occurred, when the concentration of protein in the subphase increased. The interfacial elasticity of the lipid monolayer in the presence of puroindolines in the subphase was higher than the pure lipid. Injection of protein beneath the preexisting lipid monolayer resulted in the increase of surface pressure due to the penetration of proteins. The extent of penetration depended on the nature of lipid head groups as well as on the initial surface pressure. The penetration of puroindolines to lipid monolayer was observed to be zero after crossing a critical initial surface pressure. The magnitude of the critical initial surface pressure for anionic lipids was significantly higher than the zwitterionic and nonionic lipids. The experimental results showed that both PIN-a and PIN-b had more affinity for anionic polar lipids than the neutral polar lipids and stabilized the lipid monolayer.     

Mixed DPPC/DPTAP monolayers at the air/water interface: influence of indolilo-3-acetic acid and selenate ions on the monolayer morphology

The interactions of mixed monolayers of two lipids, zwitterionic 1,2-dipalmitoyl-phosphatidylcholine (DPPC) and positively charged 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), with phytohormone indolilo-3-acetic acid (IAA) and selenate anions in the aqueous subphase were studied. For this purpose, isotherms of the surface pressure versus the mean molecular area were recorded. Domain formation was investigated by using Brewster angle microscopy (BAM). The method of grazing incidence X-ray diffraction (GIXD) was also applied for the characterization of the organization of lipid molecules in condensed monolayers. It was found that selenate ions contribute to monolayer condensation by neutralizing the positive net charge of mixed monolayers whereas IAA molecules penetrated the lipid monolayer, causing its expansion/fluidization. When both solutes were introduced into the subphase, a competition between them for interaction with the positively charged lipids in the monolayer was observed.     

Incorporation conditions guiding the aggregation of a glycosylphosphatidyl inositol (GPI)-anchored protein in Langmuir monolayers

This work investigates the process of incorporation of a glycosylphosphatidyl inositol (GPI)-anchored alkaline phosphatase into Langmuir monolayers of dimyristoyl phosphatidic acid (DMPA). Three different methods of protein incorporation were assayed. When the protein solution was injected below the air–water interface after formation of the lipid monolayer a micro-heterogeneous distribution of alkaline phosphatase throughout the interface was observed. Adsorption kinetics studied by fluorescence microscopy, associated with surface pressure measurements, led to the proposition of a model in which the protein penetration is modulated by the surface packing of the monolayer and intermolecular interactions occurring between the phospholipid and the protein. At initial surface pressures higher than 20 mN m⁻¹, the protein is quickly adsorbed on the interface and the lateral diffusion drives the alkyl chains to turn towards the air phase while the polypeptide moiety faces the aqueous subphase.     

Study of pH-sensitive copolymer/phospholipid complexes using the langmuir balance technique: effect of anchoring sequence and copolymer molecular weight

The behavior of three copolymers of N-isopropylacrylamide (NIPAM), methacrylic acid (MAA), and hydrophobic moiety was studied at phospholipid monolayer/subphase interfaces. The hydrophobic moieties, N-terminal dioctadecylamine (DODA) and random octadecylacrylate (ODA), were used as anchoring groups. The interactions between a 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC) monolayer and the copolymers were studied using the Langmuir balance technique. The effect of subphase pH, distribution of anchors along the copolymer chain, and copolymer molecular weight on the nature of the interactions between the copolymer chains and the DSPC monolayer were investigated. A first-order kinetics model was used to analyze the copolymers adsorption at the DSPC monolayer/subphase interface and allowed the interaction area between the copolymer chains and the DSPC monolayer, A(x), to be determined. The interaction area appears to depend on the subphase pH and the copolymer molecular weight. On decreasing pH, the interaction area of high molecular weight copolymers increases significantly; this is consistent with the copolymer chain phase transition from an extended coil to a collapsed globule while pH is lowered. In the latter conformation, strong hydrophobic attractive interactions between the copolymer chains and the hydrophobic part of the DSPC monolayer favor the copolymer intercalation, which could eventually provoke the phospholipidic layer destabilization or rupture.     

Effect of gramicidin on phospholipid-modified monolayers and on ion transfer at a liquid-liquid interface.

The interaction of hybrid lipid/gramicidin A (gA) monolayers with dextran sulfate (DS) and the effect of this interaction on ion transfer at a liquid-liquid interface is reported. The interfacial and physicochemical properties are studied with Langmuir-Blodgett (LB) and electrochemical techniques. The results obtained from compression isotherms demonstrate that the interactions between the different species in the hybrid monolayer vary according to the chemical nature of the lipid (hydrocarbon region and charge of the head group). Interfacial capacitance measured with AC voltammetry indicates that the DS chains form a rather flat and compact layer when adsorbed to either zwitterionic or negatively charged phospholipid monolayers, and that calcium, even at low concentrations, interacts with the monolayers. These results are successfully described by a model based on the solution of the Poisson-Boltzmann equation in the interfacial region. Ion transfer and interactions with the lipid/gA/DS-modified monolayers were also studied with electrochemical techniques. Admittance data show that although the studied ions are not using gA channels for the transfer through the lipid membranes, the incorporation of gA in the lipid domain and the adsorption of DS at the interface have a significant effect on ion transfer across the monolayers. This effect can be explained as a consequence of the modified surface charge and of the compactness of the lipid domain due to its interaction with gA and to calcium and DS adsorption at the interface. The ion-transfer rate, therefore, depends on the composition of the monolayer and the chemical nature of the ion.     

Epi-fluorescence microscopic characterization of potential-induced changes in a DOPC monolayer on a Hg drop

Characterization of the potential-induced changes of a lipid-coated Hg-0.1 M KCl interface through electrochemical techniques and newly developed in situ fluorescence microscopy is described. Fluorescence of a fluorophore-containing dioleoyl phosphatidylcholine (DOPC) layer deposited from the gas-solution interface was observed to be dependent upon the potential of the Hg surface. The largest changes occurred for potentials where the lipid layer was desorbed: the lipid moved away from the electrode surface, reducing the efficiency of metal-mediated quenching of the excited state resulting in an increase in fluorescence. Electric potential-induced changes in the morphology of the adsorbed or desorbed DOPC lipid monolayer were observed optically for the first time using this technique. The observed potential-dependent fluorescence was compared to previous studies on an octadecanol-coated Au(111) electrode. Fluorescence microscopy was also used to characterize the fusion of DOPC liposomes with a previously adsorbed DOPC layer. Large changes in fluorescence were observed for the DOPC layer after fusion with liposomes. The fusion was accomplished via potential-created defects in the adsorbed DOPC monolayer through which the liposomes interact. The integration of the liposomes into the adsorbed monolayer results in a hybrid layer in which some lipid exists further from the electrode surface, resulting in a large increase in fluorescence. Possibilities for the creation of a biomimetic adsorbed hybrid lipid layer on Hg are also discussed.     

Configuration and photochemical reaction of a bolaamphiphilic diacid with a diazo resin in monolayers and Langmuir-Blodgett films

The monolayer formation of a bolaamphiphile, 1,18-octadecanedicarboxylic acid (ODA), on pure water and the subphase containing a positively charged photoactive 2-nitro-N-methyldiphenylamine-4-diazoniumformaldehyde resin (NDR) have been investigated by pi-A isotherms, pi-t curves, and Brewster angle microscopy (BAM) measurements. It has been revealed that although an unstable monolayer was formed by ODA alone, a stable complex monolayer between ODA and NDR could be formed at the interface through electrostatic adsorption and hydrogen bonding. It has been shown that the ODA formed a U-shaped monolayer at a lower pressure and was converted to a stretched configuration upon compression to a higher surface pressure on the subphase containing NDR. Under UV irradiation at the interface photoreaction can occur in the complex monolayer, which causes shrinkage of the monolayer. Photochemical reactions can also occur in deposited Langmuir-Blodgett films. In reactions occurring at the air/water interface, the two ends of ODA can react with NDR to form an ester containing aromatic rings. This makes the compound more hydrophobic and can easily be stretched without any phase transition upon compression. When the film with U-shaped configuration was deposited onto solid substrates, the configuration could be kept even upon photoirradiation.     

Attachment of 80S ribosome to lipid monolayers at air/water interface: An electron microscopy study

The adsorption of 80S ribosome from rat liver to the surface of lipid monolayers at the air/water interface was examined by electron microscopy (EM) using a negative staining method. The results showed that, a large number of 80S ribosomes can be adsorbed to the lipid monolayers containing positively charged octadecylamine (SA), whereas the adsorption of ribosomes to the surface of neutral or negatively charged lipid monolayers was negligible. There existed a proper ratio of SA to complemented neutral lipids which facilitated the maximum binding of ribosomes. Increasing the subphase pH value will enhance the adsorption of ribosome, but when raising the subphase concentrations of K⁺, Mg²⁺ and glycerol, the adsorption of ribosomes can be weakened, suggesting that the driving forces of the adsorption mainly come from the electrostatic interactions between the ribosome and the lipids. The important characteristics of such interactions between the 80S rat liver ribosomes and the lipid membranes, as revealed by this new technology, which may help in the further understanding of the protein biosynthesis is discussed.     

Formation of langmuir-blodgett films containing two-dimensional monoatomic arrays of rare-earth cations

The work is focused on the preparation and characterisation of rare-earth containing stearic acid Langmuir-Blodgett (LB) films. Stearic acid monolayer behavior on the rare-earth containing aqueous subphase have been analyzed by thermodynamic measurements. The compression isotherm shape changed substantially under varying the anion type at the constant rare-earth cation content in aqueous subphase. The structure of formed multilayer rare-earth containing LB films was studied using small angle X-rays diffraction. The considerable structural defects in multilayer films were found when rare-earth chloride solution was exploited as aqueous subphase, caused presumably by electrostatic interactions at the monolayer surface under high binding of rare-earth cations. The use of monodentate complexones like acetic acid was proposed to form bulk phase quasi-neutral rare-earth complexes. The electroneutral ligand exchange reactions of such complexes with stearic acid monolayer allowed to neutralise monolayer surface under rare-earth cations binding and to form condenced monolayer below the triple-point temperature. The compression of such monolayer to the state of minimal compressibility and subsequent monolayer deposition resulted in the formation of high ordered LB films with high content of adsorbed multivalent rare-earth cations arranged in two-dimensional monoatomic arrays.     

Protein-lipid interactions at the air/water interface

Surface pressure measurements and external reflection FTIR spectroscopy have been used to probe protein-lipid interactions at the air/water interface. Spread monomolecular layers of stearic acid and phosphocholine were prepared and held at different compressed phase states prior to the introduction of protein to the buffered subphase. Contrasting interfacial behaviour of the proteins, albumin and lysozyme, was observed and revealed the role of both electrostatic and hydrophobic interactions in protein adsorption. The rate of adsorption of lysozyme to the air/water interface increased dramatically in the presence of stearic acid, due to strong electrostatic interactions between the negatively charged stearic acid head group and lysozyme, whose net charge at pH 7 is positive. Introduction of albumin to the subphase resulted in solubilisation of the stearic acid via the formation of an albumin-stearic acid complex and subsequent adsorption of albumin. This observation held for both human and bovine serum albumin. Protein adsorption to a PC layer held at low surface pressure revealed adsorption rates similar to adsorption to the bare air/water interface and suggested very little interaction between the protein and the lipid. For PC layers in their compressed phase state some adsorption of protein occurred after long adsorption times. Structural changes of both lysozyme and albumin were observed during adsorption, but these were dramatically reduced in the presence of a lipid layer compared to that of adsorption to the pure air/water interface.     

Effect of compression of a stearic acid monolayer on interfacial binding of copper ions and cluster formation

The effect of compression of a stearic acid Langmuir monolayer on the interaction of copper ions with the monolayer and on the formation of interface clusters has been studied as functions of pH and ionic content of the water subphase. Copper binding was estimated by direct electron paramagnetic resonance measurements of ion concentrations in the water subphase. A scanning tunnelling microscopy study of monolayer Langmuir-Blodgett films, deposited on graphite substrates, gave evidence for the formation of nanosized clusters on the monolayer surface. The data obtained showed that the interaction of the monolayer with copper ions and the accompaning cluster formation processes were determined by the arrangement, order and mobility of the stearic acid molecules in the monolayer and the electrostatics at the interface.     

Mixed Langmuir and Langmuir-Blodgett films of a proton sponge and a fatty acid: influence of the subphase nature on the interactions between the two components

The H+ acceptor activity of a proton sponge, namely, diphenyl bis(octadecylamino)phosphonium bromide, has been studied at the air-liquid interface using several subphases. Mixed Langmuir and Langmuir-Blodgett (LB) films containing the proton sponge and a fatty acid (behenic acid) in the whole composition range have been prepared. Surface pressure versus area per molecule isotherms were recorded and excess Gibbs energies of mixing calculated. The existence of strong interactions between the proton sponge and the fatty acid is observed when the subphase is either pure water or a NaOH aqueous solution. A stoichiometric 1:1 reaction between both molecules takes place at the air-water interface. This reaction has an efficiency close to 100% at high surface pressures, provided the majority anion present in the subphase is OH-. However, when the majority anion is another one, this complex is hardly formed. From the experimental results, we conclude that the acid-base reaction is highly dependent on the protonation state of the proton sponge at the air-liquid interface that is a function of the present counterion in the subphase. The floating films were also transferred onto solid substrates and characterized by means of IR spectroscopy, atomic force microscopy (AFM), and X-ray diffraction to investigate in more detail the complex formation. The interactions between the complex (when formed) and the excess component have been studied in terms of the subphase nature. It was found that the complex is immiscible with the proton sponge, yielding films made of different domains. Nevertheless, the complex is miscible with the fatty acid when the subphase used is an alkaline solution, presumably due to electrostatic interactions between the carboxylate group of the acid and the complex.     

Organization of complex monolayers by matrix controlled adsorption

Complex monolayers can be organized at the air | water interface by adsorption of a water-soluble, negatively charged porphyrin dye from the aqueous subphase to a matrix monolayer containing positively charged head groups at the surface of the solution. The organization of the complex monolayer depends on the composition of the matrix monolayer which controls the molecular interactions with the dissolved porphyrin. Surface pressure versus area isotherms in combination with measurement of the enhanced light reflection from the interface in the presence of the porphyrin provides information on the packing and orientation of the chromophores. In an optimal situation, the surface density of the positively charged head groups matches the surface density of the negative charges in the densely packed porphyrins attached to the matrix monolayer, and neutral molecules in the monolayer occupy the free space to provide a dense packing of the matrix.     

硬脂酸单分子膜上电沉积Ag膜的研究

通过电沉积方法,以气/液界面上形成的硬脂酸单分子膜为模板诱导沉积金属银膜.考察了镀液pH值、单分子膜表面压及沉积电位对银膜形貌及结构的影响.实验发现,酸性镀液的气/液界面上形成的单分子膜不能诱导沉积银,而在中性和碱性镀液的气/液界面上可以诱导银膜的生长.当单分子膜处于液态或固态时,气/液界面有银膜形成;液态单分子膜上的银膜生长速度较快,且银膜的结构一致.随着电极电位的升高,银膜沉积的速度加快,呈环状向外生长的圆形银膜逐渐变得不规则.将不同实验条件下的银膜转移出来,采用扫描电镜(SEM)、透射电镜(TEM)对银膜的结构与形貌进行了表征.研究表明,银首先在单分子膜上异相成核,由八面体构型逐渐发展成星型,最终在气/液界面形成具有松枝状微观结构的光亮银膜.     

Langmuir-Blodgett films of a Mo-blue nanoring [Mo(142)O(429)H(10)(H(2)O)(49)(CH(3)CO(2))(5)(CH(3)CH(2)CO(2))](30)(-) (Mo(142)) by the semiamphiphilic method

Langmuir monolayers and LB films of the ring-shaped mixed-valence polyoxomolybdate [Mo142O429H10(H2O)49(CH3CO2)5(CH3CH2CO2)](30-) (Mo142) dissolved in the aqueous subphase have been successfully fabricated by using the adsorption properties of a DODA monolayer. Infrared and ultraviolet-visible spectroscopy of the LB films indicates that Mo142 and DODA molecules are incorporated within these LB films. X-ray reflectivity experiments indicate that the LB films exhibit a well-defined lamellar structure formed by bilayers of DODA molecules alternating with monolayers of Mo142. Using behenic acid-modified hydrophobic quartz substrate is critical for the formation of the well-defined lamellar structure. From the values of the periodicity obtained by these experiments it is clear that the Mo142 clusters lie flat along the charged organic layers. AFM images also showed the flat and homogeneous films on the quartz substrates treated with behenic acid. Cyclic voltammograms of Mo142-LB films deposited on ITO substrates showed quasi-reversible oxidation/reduction waves with positive shift of the potential compared to the case of solution.     

Compression induced helical nanotubes in a spreading film of a bolaamphiphile at the air/water interface

Inspired by the elegant helical structures endowed by mother nature, we designed an L-glutamic acid terminated bolaamphiphile and obtained helical nanotubes through the manipulation on the two-dimensional Langmuir films at the air/water interface. It has been found that on the subphase with a pH value lower than 3, stable monolayers with plateau regions were obtained for the bolaamphiphile. Although a flat and uniform morphology was observed for the film deposited at a surface pressure below the plateau region, helical nanotube structures were obtained when the film was compressed over the plateau region. It was suggested that the compression of the monolayer at the air/water interface caused the one end of the bolaamphiphile to leave from the water surface and form an intermediate monolayer in which one end group attached on the water surface and the other extruded in the air. Such an intermediate monolayer subsequently rolled into a helical structure due to the chiral nature of the L-glutamic acid headgroup.     

Bilayer and trilayer crystalline formation by collapsing behenic acid monolayers at gas/aqueous interfaces

X-ray reflectivities and grazing incidence X-ray diffractions of behenic acid (BA) monolayers compressed to the collapse region reveal that the resulting structures are reproducible and exhibit a high degree of order. The structures of the collapsed monolayers depend on the subphase solution. On pure water, the collapsed monolayer forms a stable crystalline trilayer structure. For monolayers spread on Ca2+ solutions, we find that an inverted bilayer structure is formed; that is, stretched BA-Ca-BA (calcium dibehenate, with calcium ions bridging the polar headgroups) forms a monolayer with the hydrophobic tails in contact with the water surface.