Enhanced activity of horseradish peroxidase in Langmuir-Blodgett films of phospholipids

The immobilization of enzymes in nanostructured films has potential applications, e.g. in biosensing, for which the activity may not only be preserved, but also enhanced if optimized conditions are identified. Optimization is not straightforward because several requirements must be fulfilled, including a suitable matrix and film-forming technique. In this study, we show that horseradish peroxidase (HRP) has its activity enhanced when immobilized in Langmuir-Blodgett (LB) films, in conjunction with dipalmitoylphosphaticlylglycerol (DPPG). Incorporation of HRP into a DPPG monolayer at the air-water interface was demonstrated with compression isotherms, and Polarization-Modulation Infrared Reflection Absorption Spectroscopy (PM-IRRAS). From the PM-IRRAS data, we inferred that HRP was not denatured when adsorbed on a pre-formed, low pressure DPPG monolayer. A change in orientation was induced by the phospholipid matrix, with the amide C=O and NH groups from HRP being oriented perpendicular to the surface, parallel to the DPPG acyl chains, i.e. the alpha-helix was inserted into the monolayer. The mixed DPPG-HRP monolayer could be transferred onto solid supports, to which HRP activity was ca. 23% higher than in solution. The control of molecular architecture and choice of a suitable phospholipid matrix allowed HRP-containing LB films to be used in sensing peroxide. (c) 2008 Elsevier B.V. All rights reserved.

Surface instability of confined elastic bilayers: Theory and simulations

The surface of a soft elastic film becomes unstable and forms a self-organized undulating pattern because of adhesive interactions when it comes in contact proximity with a rigid surface. For a single film, the pattern length scale lambda, which is governed by the minimization of the elastic stored energy, gives lambda similar to 3h, where h is the film thickness. Based on a linear stability analysis and simulations of adhesion and debonding, we consider the contact instability of an elastic bilayer, which provides greater flexibility in the morphological control of interfacial instability. Unlike the case of a single film, the morphology of the contact instability patterns, debonding distance, and debonding force in a bilayer can be controlled in a nonlinear way by varying the thicknesses and shear moduli of the films. Interestingly, the pattern wavelength in a bilayer can be greatly increased or decreased compared to a single film when the adhesive contact is formed by the stiffer or the softer of the two films, respectively. In particular, lambda as small as 0.5h can be obtained. This indicates a new strategy for pattern miniaturization in elastic contact lithography.

Exchange biased FeNi/FeMn bilayers with coercivity and switching field enhanced by FeMn surface oxidation

FeNi/FeMn bilayers were grown in a magnetic field and subjected to heat treatments at temperatures of 50 to 350 degrees C in vacuum or in a gas mixture containing oxygen. In the as-deposited state, the hysteresis loop of 30 nm FeNi layer was shifted. Low temperature annealing leads to a decrease of the exchange bias field. Heat treatments at higher temperatures in gas mixture result in partial oxidation of 20 nm thick FeMn layer leading to a nonlinear dependence of coercivity and a switching field of FeNi layer on annealing temperature. The maximum of coercivity and switching field were observed after annealing at 300 degrees C.

Surface and interface morphology of polystyrene/poly(methyl methacrylate) thin-film blends and bilayers

The surface and interface morphologies of polystyrene (PS)/poly(methyl methacrylate) (PMMA) thin-film blends and bilayers were investigated by means of atomic force microscopy (AFM) and X-ray photoelectron spectroscopy. Spin-coating a drop of a PS solution directly onto a PMMA bottom layer from a common solvent for both polymers yielded lateral domains that exhibited a well-defined topographical structure. Two common solvents were used in this study. The structure of the films changed progressively as the concentration of the PS solution was varied. The formation of the blend morphology could be explained by the difference in the solubility of the two polymers in the solvent and the dewetting of PS-rich domains from the PMMA-rich phase. Films of the PS/PMMA blend and bilayer were annealed at temperatures above their glass-transition temperatures for up to 70 h. All samples investigated with AFM were covered with PS droplets of various size distributions. Moreover, we investigated the evolution of the annealed PS/PMMA thin-film blend and bilayer and gave a proper explanation for the formation of a relatively complicated interface inside a larger PS droplet.

Effects of the cationic antimicrobial peptide eumenitin from the venom of solitary wasp Eumenes rubronotatus in planar lipid bilayers: Surface charge and pore formation activity

Eumenitin, a novel cationic antimicrobial peptide from the venom of solitary wasp Eumenes rubronotatus, was characterized by its effects on black lipid membranes of negatively charged (azolectin) and zwitterionic (1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC) or DPhPC-cholesterol) phospholipids: surface potential changes, single-channel activity, ion selectivity, and pore size were studied. We found that eumenitin binds preferentially to charged lipid membranes as compared with zwitterionic ones. Eumenitin is able to form pores in azolectin (G(1) = 118.00 +/- 3.67 pS or G(2) = 160.00 +/- 7.07 pS) and DPhPC membranes (G = 61.13 +/- 7.57 pS). Moreover, cholesterol addition to zwitterionic DPhPC membranes inhibits pore formation activity but does not interfere with the binding of peptide. Open pores presented higher cation (K (+)) over anion (Cl-) selectivity. The pore diameter was estimated at between 8.5and 9.8 angstrom in azolectin membranes and about 4.3 angstrom in DPhPC membranes. The results are discussed based on the toroidal pore model for membrane pore-forming activity and ion selectivity. (c) 2007 Elsevier Ltd. All rights reserved.

Natural articular joints : model of lamellar-rollerbearing lubrication and the nature of the cartilage surface

The influence of pH on interfacial energy and wettability distributed over the phospholipid bilayer surface were studied, and the importance of cartilage hydrophobicity (wettability) on the coefficient of friction (f) was established. It is argued that the wettability of cartilage signifi antly depends on the number of phospholipid bilayers acting as solid lubricant; the hypothesis was proven by conducting friction tests with normal and lipid- depleted cartilage samples. A lamellar-roller-bearing lubrication model was devised involving two mechanisms: (i) lamellar frictionless movement of bilayers, and (ii) roller-bearing lubrication mode through structured synovial fluid, which operates when lamellar spheres, liposomes and macromolecules act like a roller-bearing situated between two cartilage surfaces in effective biological lubrication.

Binding of small mono- and oligomeric integrin ligands to membrane-embedded integrins monitored by surface plasmon-enhanced fluorescence spectroscopy

We recently developed a binding assay format by incorporating native transmembrane receptors into artificial phospholipid bilayers on biosensor devices for surface plasmon resonance spectroscopy. By extending the method to surface plasmon-enhanced fluorescence spectroscopy (SPFS), sensitive recording of the association of even very small ligands is enabled. Herewith, we monitored binding of synthetic mono- and oligomeric RGD-based peptides and peptidomimetics to integrins alphavbeta3 and alphavbeta5, after having confirmed correct orientation and functionality of membrane-embedded integrins. We evaluated integrin binding of RGD multimers linked together via aminohexanoic acid (Ahx) spacers and showed that the dimer revealed higher binding activity than the tetramer, followed by the RGD monomers. The peptidomimetic was also found to be highly active with a slightly higher selectivity toward alphavbeta3. The different compounds were also evaluated in in vitro cell adhesion tests for their capacity to interfere with alphavbeta3-mediated cell attachment to vitronectin. We hereby demonstrated that the different RGD monomers were similarly effective; the RGD dimer and tetramer showed comparable IC50 values, which were, however, significantly higher than those of the monomers. Best cell detachment from vitronectin was achieved by the peptidomimetic. The novel SPFS-binding assay platform proves to be a suitable, reliable, and sensitive method to monitor the binding capacity of small ligands to native transmembrane receptors, here demonstrated for integrins.

Interaction between alpha-actinin and negatively charged lipids membrane investigated by surface plasmon resonance and electrochemical methods

alpha-Actinin has been shown to be capable of interacting with some special membrane phospholipids directly, which is important for its function. In this study, hybrid bilayer membranes composed of negatively charged lipids are constructed on the surface plasmon resonance gold substrate and on the gold electrode, respectively, and the interaction between alpha-actinin and negatively charged lipids membrane is investigated by surface plasmon resonance, cyclic voltammetry and electrochemical impedance spectroscopy methods. alpha-Actinin is proved to be able to interact with the negatively charged lipids membrane directly. It can also insert at least partly into the membrane or lead to some defect or lesion in the membrane, which increase the permeability of the membrane. This study would bring some insight on the interaction between the alpha-actinin and the cell membranes in vivo.

Interdigited phospholipid/alkanethiol bilayers assembled on APTMS-supported gold colloid electrodes

The preparative procedure of a kind of phospholipid/alkanethiol bilayers on a planar macroelectrode was copied to the as-prepared gold colloid electrodes. The electrochemical and spectral results show that the bilayers on colloid electrodes are interdigited, which are different from their 2-D counterparts on a planar macroelectrode.

The interaction of recombinant Pseudomonas aeruginosa exotoxin A with DNA and mimic biomembrane investigated by surface plasmon resonance and electrochemical methods

Based on the multidomain structure of Pseudomonas aeruginosa exotoxin A, a fusion protein termed rPEA has been constructed, which is expected to serve as a gene carrier in vitro. The expression and purification of rPEA are described. The basal properties of rPEA as a gene carrier are evaluated by investigating its interaction with plasmid DNA and mimic biomembrane by surface plasmon resonance (SPR) and electrochemical methods. rPEA is proved to be able to bind with plasmid DNA with high affinity. It can also interact with lipid membrane and increase permeability of the membrane, so the probe molecules can easily reach the gold surface and exhibit the electrochemical response.

Self-assembled monolayer growth of phospholipids on hydrophobic surface toward mimetic biomembranes: Scanning probe microscopy study

Atomic force microscopy (AFM) and lateral force microscopy (LFM) were used simultaneously to analyze a model membrane bilayer structure consisting of a phospholipid outer monolayer deposited onto organosilane-derivatized mica surfaces, which were constructed by using painting and self-assembly methods. The phospholipid used as outer monolayer was dimyristoylphosphatidylcholine (DMPC). The hydrocarbon-covered substrate that formed the inner half bilayer was composed of a self-assembly monolayer (SAM) of octadecyltrichloroorganosilane (OTS) on mica. SAMs of DMPC were formed by exposing hydrophobic mica to a solution of DMPC in decane/isobutanol and subsequently immersing into pure water. AFM images of samples immersed in solution for varying exposure times showed that before forming a complete monolayer the molecules aggregated into dense islands (2.2-2.6 nm high) on the surface. The islands had a compact and rounded morphology. LFM, coupled with topographic data obtained with the atomic force mode, had made possible the distinction between DMPC and OTS. The rate constant of DMPC growth was calculated. This is the first systematic study of the SAM formation of DMPC by AFM and LFM imaging. It reveals more direct information about the film morphology than previous studies with conventional surface analytical techniques such as infrared spectroscopy, X-ray, or fluorescence microscopy.

Interaction of scopolamine and cholesterol with sphingomyelin bilayers by FT-Raman spectroscopy

The interaction of scopolamine and cholesterol with sphingomyelin bilayers has been investigated by FT-Raman spectroscopy in head-group region (600-1000 cm(-1)), the C-C stretching (1000-1200 cm(-1)), CH2 deformation (1400-1500 cm(-1)) and the C-H stretching (2800-3000 cm(-1)) mode regions. The results indicate that scopolamine and cholesterol do not change the conformation of O-C-C-N+ backbone in the choline group of sphingomyelin bilayers, the polar headgroup is still extending parallel to the bilayer surface and O-C-C-N+ group is still in its gauche conformer. Scopolamine and cholesterol lower the order of the interface, the interchain, CH2 crystal lattices and the lateral chain-chain packing, and increase their fluidity.

Paint-freeze method to form self-assembled alkanethiol/phospholipid bilayers on gold

A paint-freeze method for preparing self-assembled alkanethiol/phospholipid bilayers on a gold surface has been described (by cyclic voltammetry, a.c impedance, polarized FTIR-ATR) to be well-ordered and packed, stable, solvent-free bilayers. The lipid order parameter was 0.67, calculated from the dichroic ratio, consistent with a well-ordered lipid film in which the methylene groups have segmental flexibility and are disordered to a degree which is typical for a lipid bilayer in the liquid-crystalline phase. Such a supported membrane provides a useful way for studies in biophysics, physiology and electrochemistry.

K+ sensors based on supported alkanethiol/phospholipid bilayers

A novel kind of K+ sensor with valinomycin-incorporated bilayers supported on a gold electrode consisting of self-assembled alkanethiol monolayers (SAMs) and a lipid monolayer has been fabricated successfully. The lipid monolayer is deposited on the alkylated surface of the first alkanethiol monolayer through three different methods, such as the Langmuir-Blodgett (LB) technique, painted method and painted-frozen method. The response of K + sensors produced by a painted or painted-frozen lipid monolayer on an alkanethiol alkylated gold electrode is larger than that by the LB method, which is due to the difference in fluidity of the three kinds of bilayers. Selectivity coefficients KK+, Na+, KK+, Li+, KK+, Ca2+ and KK+, Mg2+ are 10(-4), 10(-4), 2 x 10(-5) and 3 x 10(-5) respectively, and there is no obvious difference among different fabricating methods. A linear response toward the potassium ion was found in the range from 10(-1) M to 10(-5) M with the detection limit of 10(-6) M. The sensor has a slope of 60 mV per decade. Meanwhile, the longevity of the sensor was improved obviously for at least two months at about -10 degrees C. The higher stability shows the possibility to fabricate a practical biosensor.

2D NMR and FT-Raman spectroscopic studies on the interaction of lanthanide ions and Ln-DTPA with phospholipid bilayers

The interactions of lanthanide ions and the Ln-DTPA (DTPA = diethylenetriaminepentaacetate) complex with di palmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine (DPPE) bilayers are studied by 2D NOESY and FT-Raman spectroscopy. Proton NMR spectroscopic results show that lanthanide ions combine with phosphate groups in the polar region of the outer layer of DPPC liposomes, leading to the separation in chemical shift of the proton signal of N(CH3)(3) The conformational change of the O-C-C-N+ backbone from the gauche conformer to the trans one is not found; i.e., the orientation of the polar headgroup is still parallel to the surface of the bilayers. The Ln-DTPA complex at low concentration in a pH 7.4 solution localizes far away from bilayers and thereby has little effect on the structure of bilayers. The FT-Raman spectroscopic results indicate that lanthanide ions affect strongly the fluidity of acyl chains of DPPE bilayers while the Ln-DTPA complex affects it slightly.