Biosensor for dopamine based on stabilized lipid films with incorporated resorcin[4]arene receptor

This work reports a technique for the stabilization after storage in air of a lipid film with incorporated resorcin[4]arene receptor based biosensor for dopamine. Microporous filters composed of glass fibers (nominal pore sizes, 0.7 and 1.0 microm) were used as supports for the formation and stabilization of these devices and the lipid film is formed on the filter by polymerization prior its use. Methacrylic acid was the functional monomer, ethylene glycol dimethacrylate was the crosslinker and 2,2'-azobis-(2-methylpropionitrile) was the initiator. The stability of the lipid films by incorporation of a receptor for the preparation of stabilized lipid film biosensor is studied throughout this work. The response towards dopamine of the present stabilized for repetitive uses lipid membrane biosensor composed of dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidic acid was compared with planar freely suspended bilayer lipid membranes (BLMs). The stabilized lipid membranes provided similar artificial ion gating events as BLMs in the form of transient signals and can function for repetitive uses after storage in air. However, the response of the stabilized lipid films was slower than that of the freely suspended BLMs. This will allow the practical use of the techniques for chemical sensing based on lipid films and commercialization of these devices, because it is now possible to prepare stabilized lipid film based biosensors and store them in the air.     

Solid-supported biomimetic membranes with tailored lipopolymer tethers

Stable lipid membranes with controlled substrate-membrane spacing can be prepared using well-defined lipopolymers as a tether. Based on the living cationic ring-opening polymerization of 2-methyl- or 2-ethyl-2-oxazoline, lipopolymers can be synthesized bearing a lipid head group as well as a silanol reactive coupling end group. Using a “grafting onto” procedure these polymers can form dense, brush like monolayers, whose layered structures can be obtained by x-ray reflectivity measurements. By transfer of a pre-organized monolayer that is followed by vesicle fusion, stable polymer supported lipid membranes can be prepared. The substrate-membrane spacing can be controlled via the degree of polymerization, while the lateral diffusion of lipids within the membrane depends on the density of polymer tethers. Preliminary experiments implied that the membrane with long (N = 40) polymer tethers could reside trans-membrane receptors homogeneously, suggesting a large potential of this strategy.     

Anchor-lipid monolayers at the air-water interface; prearranging of model membrane systems

Model membrane systems are gaining more and more interest both for basic studies of membrane-related processes as well as for biotechnological applications. Several different model systems have been reported among which the tethered bilayer lipid membranes (tBLMs) form a very attractive and powerful architecture. In all the proposed architectures, a control of the lateral organization of the structures at a molecular level is of great importance for an optimized preparation. For tBLMs, a homogeneous and not too dense monolayer is required to allow for the functional incorporation of complex membrane proteins. We present here an alternative approach to the commonly used self-assembly preparation. Lipids are spread on the air-water interface of a Langmuir film balance and form a monomolecular film. This allows for a better control of the lateral pressure and distribution for subsequent transfer to solid substrates. In this paper, we describe the properties of the surface monolayer, in terms of surface pressure, structure of the lipid molecule, content of lipid mixtures, temperature, and relaxations features. It is shown that a complete mixing of anchor-lipids and free lipids can be achieved. Furthermore, an increase of the spacer lengths and a decrease of the temperature lead to more compact films. This approach is a first step toward the fully controlled assembly of a model membrane system.     

Calixarenes in a membrane environment: a monolayer study on the miscibility of three p-tert-butylcalix[4]arene beta-lactam derivatives with 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine

Literature data indicate that some calixarene derivatives with antimicrobial activities may be useful as drugs; one of the aspects of the biological activity of different classes of antibiotics concerns interactions with lipid membranes. Here, the possibility of incorporation and/or translocation of three amphiphilic p-tert-butylcalix[4]arene derivatives across membranes was studied using lipid monolayers. The derivatives used have 6-aminopenicillanic acid or benzylpenicillin moieties grafted in alternate positions at the calixarene lower rim; 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), a model bacterial membrane lipid, was used to prepare the monolayers. The miscibility of calixarene-antibiotic conjugates with lipid films was studied using surface pressure and surface potential measurements, as well as Brewster angle microscopy. The results obtained show that the miscibility is significantly different for the 6-aminopenicillanic acid and the two benzylpenicillin derivatives. Molecular modeling allowed the assessment of the lowest energy conformations of the calixarene derivatives and gave more insight into the interactions with the DMPE films.     

Binding of a truncated form of lecithin:retinol acyltransferase and its N- and C-terminal peptides to lipid monolayers

Lecithin:retinol acyltransferase (LRAT) is a 230 amino acid membrane-associated protein which catalyzes the esterification of all-trans-retinol into all-trans-retinyl ester. A truncated form of LRAT (tLRAT), which contains the residues required for catalysis but which is lacking the N- and C-terminal hydrophobic segments, was produced to study its membrane binding properties. Measurements of the maximum insertion pressure of tLRAT, which is higher than the estimated lateral pressure of membranes, and the positive synergy factor a argue in favor of a strong binding of tLRAT to phospholipid monolayers. Moreover, the binding, secondary structure and orientation of the peptides corresponding to its N- and C-terminal hydrophobic segments of LRAT have been studied by circular dichroism and polarization-modulation infrared reflection absorption spectroscopy in monolayers. The results show that these peptides spontaneously bind to lipid monolayers and adopt an α-helical secondary structure. On the basis of these data, a new membrane topology model of LRAT is proposed where its N- and C-terminal segments allow to anchor this protein to the lipid bilayer.     

Ceramide-enriched microdomains in planar membranes

Ceramide has a large effect on the properties of biological membranes, increasing lipid order and promoting lateral phase separation, and plays an important role in cell signaling. This review provides an overview of recent studies of the effects of direct ceramide incorporation and enzymatic ceramide generation on planar supported membranes, including lipid monolayers and supported lipid bilayers. Recent studies have focused on understanding the nucleation, growth and morphology of ceramide gel domains, characterizing the properties of ceramide-rich membrane phases and investigating the effects of ceramide on phase-separated membranes with co-existing liquid-ordered and fluid phases, as models for cellular membranes.     

Critical exponents for line tension and dipole density difference from lipid monolayer domain boundary fluctuations

The potential physiological relevance of liquid-liquid phase separation in lipid membranes to the formation and stability of "lipid rafts" in cellular plasma membranes has prompted extensive investigation of the physical chemistry underlying these phenomena. In this contribution, the line tension (gamma) and dipole density differences ( micro) between demixed fluid phases of monolayers comprised of dimyristoylphosphatidylcholine (DMPC) and dihydrocholesterol (DChol) were investigated by measuring the two-dimensional thermal fluctuations of domain boundaries visualized by the inclusion of a fluorescent tracer lipid. These parameters are essential determinants of domain stability, and their quantification will yield an increased understanding of the physical processes responsible for aspects of lateral phase separation. Employing an extensive data set, the surface pressure dependence of gamma and mu was determined at three different monolayer compositions (30%, 35%, and 40% DChol). Both parameters were found to decrease with a power law dependence as the surface pressure approached the phase transition pressure (pi t), in agreement with previous measurements. Additionally, photobleaching effects and domain size influence were quantified and found to be small in our system. We suggest that the method of flicker spectroscopy can be helpful in identifying line-active compounds.     

Interaction of hexadecylbetainate chloride with biological relevant lipids

The present work investigates the interaction of hexadecylbetainate chloride (C(16)BC), a glycine betaine-based ester with palmitoyl-oleoyl-phosphatidylcholine (POPC), sphingomyelin (SM), and cholesterol (CHOL), three biological relevant lipids present in the outer leaflet of the mammalian plasma membrane. The binding affinity and the mixing behavior between the lipids and C(16)BC are discussed based on experimental (isothermal titration calorimetry (ITC) and Langmuir film balance) and molecular modeling studies. The results show that the interaction between C(16)BC and each lipid is thermodynamically favorable and does not affect the integrity of the lipid vesicles. The primary adsorption of C(16)BC into the lipid film is mainly governed by a hydrophobic effect. Once C(16)BC is inserted in the lipid film, the polar component of the interaction energy between C(16)BC and the lipid becomes predominant. Presence of CHOL increases the affinity of C(16)BC for membrane. This result can be explained by the optimal matching between C(16)BC and CHOL within the film rather by a change of membrane fluidity due to the presence of CHOL. The interaction between C(16)BC and SM is also favorable and gives rise to highly stable monolayers probably due to hydrogen bonds between their hydrophilic groups. The interaction of C(16)BC with POPC is less favorable but does not destabilize the mixed monolayer from a thermodynamic point of view. Interestingly, for all the monolayers investigated, the exclusion surface pressures are above the presumed lateral pressure of the plasma membranes suggesting that C(16)BC would be able to penetrate into mammalian plasma membranes in vivo. These results may serve as a useful basis in understanding the interaction of C(16)BC with real membranes.     

The action of indomethacin on neutral and positively charged monolayers

The penetration ability of indomethacin in neutral and positively charged monolayers has been studied. Neutral monolayers of cholesterol and dipalmitoyl phosphatidyl choline present a slight but significant increase of surface pressure. The presence of stearylamine in the films results in an increase of surface pressure due to an electrostatic effect between the carboxylic anion of indomethacin and the polar head group of the stearylamine. These values can afford a reference point to choose the best lipid composition of liposomes encapsulating indomethacin to avoid the drug causing leakage of liposomes.     

Interactions of a tetravalent branched peptide from VP3 capsid protein of hepatitis A virus with monolayers as biomembrane models

The interactions between a synthetic multiple antigenic peptide containing four units of a peptide corresponding to the sequence (110–121) of VP3 protein of the hepatitis A virus, termed MAP₄-VP3(110–121), and phospholipids as the main components of biological membranes have been studied in detail. Surface activity of the multiple antigenic peptide was determined as a function of its bulk concentration in an aqueous solution. Saturation was reached at 0.33 μM concentration. The ability of the peptide to insert into lipid monolayers of dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol and stearyl amine was determined. The peptide interacts preferably with the positive phospholipid according to its negative charge.     

Photopolymerization of mixed monolayers and black lipid membranes containing gramicidin A and diacetylenic phospholipids

We formed monolayers and black lipid membranes (BLMs) of photopolymerizable lipids mixed with the channel-forming protein gramicidin A to evaluate their miscibility and the potential for improved stability of the BLM scaffold through polymerization. Analyses of surface pressure vs area isotherms indicated that gramicidin A dispersed with three different synthetic, polymerizable, diacetylene-containing phospholipids, 1,2-di-10,12-tricosadiynoyl-sn-glycero-3-phosphocholine (DTPC), 1,2-di-10,12-tricosadiynoyl-sn-glycero-3-phosphoethanolamine (DTPE), and 1-palmitoyl-2,10,12-tricosadiynoyl-sn-glycero-3-phosphoethanolamine (PTPE) to form mixed monolayers at the air-water interface on a Langmuir-Blodgett (LB) trough. Conductance measurements across a diacetylenic lipid-containing BLM confirmed dispersion of the gramicidin channel with the lipid layer and demonstrated gramicidin ion-channel activity before and after UV exposure. Polymerization kinetics of the diacetylenic films were monitored by film pressure changes at constant LB trough area and by UV-vis absorption spectroscopy of polymerized monolayers deposited onto quartz. An initial increase in film pressure of both the pure diacetylene lipid monolayers and mixed films upon exposure to UV light indicated a change in the film structure. Over the time scale of the pressure increase, an absorbance peak indicative of polymerization evolved, suggesting that the structural change in the lipid monolayer was due to polymerization. Film pressure and absorbance kinetics also revealed degradation of the polymerized chains at long exposure times, indicating an optimum time of UV irradiation for maximized polymerization in the lipid layer. Accordingly, exposure of polymerizable lipid-containing black lipid membranes to short increments of UV light led to an increase in the bilayer lifetime.     

Interaction of rabbit C-reactive protein with phospholipid monolayers at air/water interface

C-reactive protein (CRP) is a major acute phase reactant in most mammalian species. The structure of CRP has been previously established by crystallography, and the significance of its interaction with lipid membranes is accepted in the literature. However, the nature of the interaction between CRP and phospholipids is not yet well understood. In this paper we use monolayer technique to study the characteristics of the interaction of rabbit C-reactive protein (rCRP) with the phospholipid membranes. The results show that rCRP is surface active and can spontaneously insert into the lipid monolayers. The critical pressure for rCRP inserting into the phospholipid monolayers is about 34.5 mN/m, which is not sensitive to the types of the lipid headgroups and the presence of calcium ions in the subphase. The findings of this paper may provide a clue to the further understanding of the mechanism of the interactions between rCRP and the biological membranes.     

Single molecule probes of membrane structure: orientation of BODIPY probes in DPPC as a function of probe structure

Single molecule fluorescence measurements have recently been used to probe the orientation of fluorescent lipid analogs doped into lipid films at trace levels. Using defocused polarized total internal reflection fluorescence microscopy (PTIRF-M), these studies have shown that fluorophore orientation responds to changes in membrane surface pressure and composition, providing a molecular level marker of membrane structure. Here we extend those studies by characterizing the single molecule orientations of six related BODIPY probes doped into monolayers of DPPC. Langmuir-Blodgett monolayers transferred at various surface pressures are used to compare the response from fluorescent lipid analogs in which the location of the BODIPY probe is varied along the length of the acyl chain. For each BODIPY probe location along the chain, comparisons are made between analogs containing phosphocholine and smaller fatty acid headgroups. Together these studies show a general propensity of the BODIPY analogs to insert into membranes with the BODIPY probe aligned along the acyl chains or looped back to interact with the headgroups. For all BODIPY probes studied, a bimodal orientation distribution is observed which is sensitive to surface pressure, with the population of BODIPY probes aligned along the acyl chains increasing with elevated surface pressure. Trends in the single molecule orientations for the six analogs reveal a configuration where optimal placement of the BODIPY probe within the acyl chain maximizes its sensitivity to the surrounding membrane structure. These results are discussed in terms of balancing the effects of headgroup association with acyl chain length in designing the optimal placement of the BODIPY probe.     

Monolayer Characteristics of Bacteriocin AS-48, pH Effect and Interactions with Dipalmitoyl Phosphatidic Acid at the Air-Water Interface

Bacteriocin AS-48 produced by Enterococcus faecalis S-48 is a ribosomally synthesized cyclic peptide (7.4 kDa) of broad inhibitory spectrum against Gram-positive and Gram-negative bacteria. Simple monolayers of AS-48 and of dipalmitoyl phosphatidic acid (DPPA) at the air-water interface are studied. The AS-48 interfacial behavior in the function of pH explains the biological activity of the peptide. The lipid monolayers show the characteristic behavior of phosphatidic acid at the mentioned interface. The interactions between AS-48 and DPPA, a majority lipid of the bacterial cell membrane, are quantitatively investigated. The results indicate that only when the lipid molecules are charged enough (pH 10.5) is an attractive interaction between AS-48 and DPPA observed, although under these experimental conditions the results seem to indicate that a deformation of the peptide helical structure could take place. Copyright 2001 Academic Press.     

Compressibility study of quaternary phospholipid blend monolayers

The mechanical properties of liposome membranes are strongly dependent on type and ratio of lipid compounds, which can have important role in drug targeting and release processes when liposome is used as drug carrier. In this work we have used Brewster's angle microscopy to monitor the lateral compression process of lipid monolayers containing as helper lipids either distearoyl phosphatidylethanolamine (DSPE) or dioleoyl phophatidylethanolamine (DOPE) molecules on the Langmuir trough. The compressibility coefficient was determined for lipid blend monolayers containing the helper lipids above, cholesterol, distearoyl phosphatidylcholine (DSPC) and pegylated-DSPE at room temperature. Two variables, the cholesterol fraction and the ratio ρ between the helper lipid (either DSPE or DOPE) and the reference lipid DSPC, were studied by multivariate analysis to evaluate their impact on the compressibility coefficient of the monolayers. The cholesterol level was found to be the most significant variable for DSPE blends while the ratio ρ was the most significant one for DOPE blend monolayers. It was also found that these two variables can exhibit positive interaction and the same compressibility value can be obtained with different blend compositions.     

Chemical modification of the pendant structure of wholly aromatic polyamides: Toward functional high‐performance materials with tuned chromogenic and fluorogenic behavior

This work describes the preparation of functional aromatic polyamides with pendant fluorescent chemical structures. The preparation of a parent copolyamide with a lateral fluorene moiety anchored to the main chain through a urea group is described, along with the chemical modification of the fluorene moieties to render six copolymers with different fluorescent behaviors. The easy and clean chemical modification of the polyamide structure permits the preparation of high‐performance materials with “a la carte” fluorescence properties. The characteristics of these materials make them useful for cutting‐edge technologies associated with the fluorescence of the pendant fluorene moiety and with the host behavior of the urea motif, that is, fluorescent sensing of analytes or hybrid luminescent converter—light‐emitting diode systems. The chemical modification of the polymer structure was carried out with chemicals and conditions optimized for polyamide models. The influence of the chemical structure of the pendant fluorene core has also been addressed in terms of thermal properties, solubility, water uptake, and so forth. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 3823–3833, 2010     

Study of the interaction of beta-cyclodextrin with phospholipid monolayers by surface pressure measurements and fluorescence microscopy

The interaction of beta-cyclodextrin (beta-CD) with different lipids has been studied, using Langmuir monolayers kept at constant surface pressure or constant spreading surface. Results show that beta-CD, injected beneath the monolayer, is able to desorb unsaturated palmitoyloleoylphosphatidylcholine (POPC) and sphingomyelin (SM) under specific experimental conditions. In this last case, SM monolayers, labeled with the fluorescent NBD-PC probe, were also observed by fluorescence microscopy, before and after beta-CD injection. Images show that SM monolayers are more homogeneous after beta-CD injection, because of the lipid desorption. At last, it seems that lipid desorption occurs only in a restricted surface pressure range, depending on the lipid.     

Proton diffusion at phospholipid assemblies

A new scanning electrochemical microscopy proton feedback method has been developed for investigating lateral proton diffusion at phospholipid assemblies: specifically Langmuir monolayers at the water/air interface. In this approach, a base is electrogenerated by the reduction of a weak acid (producing hydrogen) at a "submarine" ultramicroelectrode (UME) placed in the aqueous subphase of a Langmuir trough close to a monolayer. The electrogenerated base diffuses to and titrates monolayer-bound protons and is converted back to its initial form, so enhancing the current response at the UME. Local deprotonation of the monolayer creates a concentration gradient for lateral proton diffusion. A numerical model has been developed, taking into account the potential-dependent association/dissociation constant of the interfacial acid groups. A comparison is made of monolayers comprising either acidic DL-alpha-phosphatidyl-L-serine, dipalmitoyl (DPPS) or zwitterionic L-alpha-phosphatidylcholine, dipalmitoyl (DPPC) monolayers at a range of surface pressures. It is demonstrated that lateral proton fluxes at DPPS are significant, but the lateral proton diffusion coefficient is lower than in bulk solution. In contrast, lateral proton diffusion cannot be detected at DPPC, suggesting that the acid/base character of the phospholipid is important in determining the magnitude of interfacial proton fluxes.     

Semiempirical quantum mechanical calculations of dipolar interaction between dipyridamole and dipalmitoyl phosphatidyl choline in Langmuir monolayers

Recent studies have shown that dipalmitoyl phosphatidyl choline (DPPC) monolayers respond cooperatively to the presence of dipyridamole (DIP) guest molecules even at small concentrations, which is a signature of molecular recognition. Using semiempirical quantum mechanical calculations for the DIP-DPPC system, we show that the incorporation of DIP causes large changes in the vertical dipole moment of the DIP-DPPC system, which can explain why measurable changes in surface potential are observed experimentally even at very low DIP concentrations. The calculations are also consistent with the anomalous concentration dependence of the surface pressure and surface potential isotherms for DIP-DPPC monolayers. Rather than saturation or a continuous increase in the effects caused by the incorporation of increasing amounts of DIP, the experimentally observed inversion in the behavior of the surface potential as the DIP concentration reaches 0.5 mol % would be caused by a change in DIP conformation, from a vertical arrangement for the DIP rings to a horizontal or intermediate arrangement. The strong dipolar interactions indicated in the calculations may also be the origin of the drastic changes in monolayer morphology seen in fluorescence microscopy images, with triskellion-shaped domains being formed for condensed DIP-DPPC monolayers.