New catalytic properties of monoamine oxidase immobilized in Langmuir-blodgett films with amphiphilic polyelectrolytes

Langmuir-Blodgett (LB) films of monoamine oxidase (MAO) have been formed on the surface f a polypropylene membrane using amphiphilic polyelectrolytes. The enzyme activity of such protein-polyelectrolyte films was measured by a Clark electrodes. It was shown that in LB films thus formed the use of amphiphilc polyelectrolytes, MAO activity was higher than in polyelectrolyte-free LB films. Immobilization of MAO with branched polyethylenimine modified on 12% by laurylchain led to pronounced changes in its catalytic properties. The dependence of the enzyme's kinetic parameters on amphiphilic polyelectrolyte structures was discussed. (c) 1994 John Wiley & Sons, Inc.     

Redox-active cellulose Langmuir-Blodgett films containing beta-carotene as a molecular wire

Redox-active Langmuir-Blodgett (LB) films containing dihydrophytyl ferrocenoate (DFc) and beta-carotene (betaC) were fabricated by use of 6-O-dihydrophytylcellulose (DHPC) as a matrix. A mixture of DFc-DHPC formed a stable monolayer. Atomic force microscopy images revealed that the DFc molecules were dispersed uniformly throughout the surface in the ratio DFc:DHPC = 2:8 at 30 mN m-1. The DFc-DHPC monolayer was transferred successfully onto a substrate, yielding Y-type LB films. Cyclic voltammograms for the DFc-DHPC LB films on an indium tin oxide (ITO) electrode exhibited a well-defined surface wave. The voltammograms of the DFc-DHPC LB films exhibited 60-40% redox-active ferrocene moieties, whereas those of the DFc-DHPC-betaC LB films exhibited 90-70%. X-ray diffraction patterns indicated that the distance between layers was independent of betaC molecules incorporated into the LB films. Consequently, these results suggested that betaC can function as a molecular wire.     

Langmuir-Blodgett monolayer films of bacterial photosynthetic membranes and isolated reaction centers: preparation, spectrophotometric and electrochemical characterization.

The Langmuir-Blodgett (LB) film technique has been successfully applied to the construction of stable and photo-active films of chromatophore membranes and isolated reaction centers from two species of photosynthetic bacteria, Rhodobacter sphaeroides and Rhodopseudomonas viridis. LB films of these preparations were characterized at the air/water interface through compression isotherms and film stabilities. Films deposited on glass slides were analyzed by spectrophotometric and redox potentiometric techniques. The results obtained indicate that the in vivo properties of the photosynthetic apparatus in the deposited films are essentially unchanged. Furthermore, the pigments and redox cofactors in the films are highly oriented and offer a unique opportunity for structural and functional studies of the kind described in the accompanying paper (Biochim. Biophys. Acta 1057 (1991) 258-272).     

Enzymatic activities and infrared studies of glutamate dehydrogenase immobilized on Langmuir-Blodgett films

Summary A technique is described for the immobilization of active glutamate dehydrogenase (GDH) on behenic acid Langmuir-Blodgett (LB) films. The optimization of the immobilization conditions shows that the activities of GDH bound on hydrophobic and hydrophilic LB films were similar and decreased dramatically when the immobilized enzyme was dried. The GDH binding was followed by Fourier transform infrared (FTIR) spectroscopy. Modifications of GDH conformation and LB film structure were observed during the enzyme binding. After GDH activity test, a partial dissociation of behenic acid occurred and the -sheet band of the enzyme increased by comparison with the -helix band.Abbreviations LB Langmuir-Blodgett - FTIR spectroscopy Fourier transform infrared spectroscopy - GDH glutamate dehydrogenase - TEA triethylamine     

Langmuir-Blodgett films of a novel cellulose derivative with dihydrophytyl group: the ability to anchor beta-carotene molecules

A novel cellulose derivative, 6-O-dihydrophytylcellulose (DHPC), was first synthesized via a ring-opening polymerization and allowed to self-assemble onto an air-water interface. Langmuir-Blodgett (LB) films were characterized with atomic force microscope (AFM), UV-vis spectroscopy, and Fourier transform infrared spectroscopy. The surface pressure-area (pi-A) isotherms for DHPC and beta-carotene (betaC) mixture indicated strong interaction between these compounds to pack well. Thus, DHPC has the ability to anchor betaC in the monolayer. It was proved that a betaC-DHPC monolayer was transferred successfully onto a substrate, yielding Y-type LB films by UV spectroscopic analysis. The transmission and reflection-absorption IR spectra (RAS) indicated that the dihydrophytyl chains had almost trans-zigzag conformation and were oriented nearly perpendicular to the substrate. AFM section analysis revealed the thickness per layer to be 2.32 nm. Consequently, DHPC was found to be an appropriate matrix to fabricate the mixed LB films containing betaC.     

Immobilization of urease on poly(N-vinyl carbazole)/stearic acid Langmuir-Blodgett films for application to urea biosensor

Urease was immobilized in mixed monolayers of poly(N-vinyl carbazole) (PNVK) and stearic acid (SA) formed at an air-water interface. The monolayers were transferred onto indium-tin-oxide (ITO) coated glass plates using Langmuir-Blodgett (LB) film deposition technique. Urease immobilized on PNVK/SA LB films, characterized using FTIR and UV-visible spectroscopy, was found to exhibit increased stability over a wide pH (6.5-8.5) and temperature (25-50 degrees C) range. Potentiometric measurements on these urease electrodes were carried out using an ammonium ion analyzer. Two values for K(m)(app) were obtained at lower and higher concentrations of substrate urea.     

Langmuir and Langmuir-Blodgett films of organophosphorus acid anhydrolase

In this paper, we describe the preparation and characterization of Langmuir and Langmuir-Blodgett (LB) monolayers of the enzyme organophosphorus acid anhydrolase (OPAA). Langmuir films of OPAA were characterized on different subphases, such as phosphate, ammonium carbonate, and bis-tris-propane buffers. Monolayers at the air-water interface were characterized by measuring the surface pressure and surface potential-area isotherms. In situ UV-vis absorption spectra were also recorded from the Langmuir monolayers. The enzyme activity at the air-water interface was tested by the addition of diisopropylfluorophosphate (DFP) to the subphase. LB films of OPAA were transferred to mica substrates to be studied by atomic force microscopy. Finally, a one-layer LB film of OPAA labeled with a fluorescent probe, fluorescein isothiocyanate (FITC), was deposited onto a quartz slide to be tested as sensor for DFP. The clear, pronounced response and the stability of the LB film as a DFP sensor show the potential of this system as a biosensor.     

Do properties of bovine serum albumin at fluid/electrolyte interface follow the Hofmeister series?--An analysis using Langmuir and Langmuir-Blodgett films

Folding and solubility of proteins are dependent on their state of hydration. How does a protein-bovine serum albumin (BSA) behave in the presence of Hofmeister electrolytes, especially at interfaces? Langmuir films of bovine serum albumin (BSA) in the presence of different Hofmeister electrolytes at air/solution interface and as Langmuir-Blodgett films (LB films) at solid/solution interface have been studied using the surface pressure-molecular area (pi-A) isotherms and surface energy parameters. Changes in secondary structure have been analyzed using circular dichroism (CD) and fluorescence spectroscopy. Hydrodynamically coupled water fraction of BSA in different environments has been estimated using quartz crystal microbalance (QCM) and related to the secondary structural changes. Molecular modeling of BSA in different environments showed that the protein has a compact structure at the interface compared to vacuum. The contact areas estimated using molecular modeling agreed with the experimental results. The results show that the properties of BSA at the interface follow the Hofmeister series with NaF leading to maximum compaction in the protein. Further, in addition to ion specific solvation and different ion size, water structure alteration and the bound water fractions contribute importantly to the Hofmeister effect.     

Microdomains in mixed monolayers of oleanolic and stearic acids: thermodynamic study and BAM observation at the air-water interface and AFM and FTIR analysis of LB monolayers

Monolayers of oleanolic acid (OLA) mixed with stearic acid (SA) were studied at the air-water interface. The surface pressure-area (pi-A) isotherms, measured over the whole composition range, and BAM observations were used to investigate the phase behaviour and self-organization of these components in a two-dimensional structure. Pure OLA forms a very compressible monolayer, and BAM observation revealed the coexistence of large and irregular solid domains of different thickness dispersed in a gas matrix, compatible with the two most probable orientations of the OLA molecule at the interface. Mixtures of OLA/SA form condensed monolayers from low surface pressures and the thermodynamic analysis indicates that OLA molecules, in the presence of the long-chain SA, orient with the major axis almost perpendicular to the interface. Langmuir-Blodgett (LB) monolayers of pure SA and mixtures were further characterized by atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR). AFM images of LB mixed monolayers evidenced microphase separation, not observable by BAM. The SA rich domains are 4-6A thicker than those rich in OLA. The FTIR spectra of mixed LB films on CaF2 substrates showed that OLA does not perturb the all-trans conformation of the SA long alkyl chains, up to a mole fraction of 0.4. The carbonyl-stretching band of OLA suggests that the carboxylic groups of neighbour OLA molecules are involved in hydrogen bonds, forming dimers, as in pure solid phase OLA. These interactions seem to prevail over the OLA-water hydrogen bonds.     

Preparation and characterization of monolayer and multilayer Langmuir-Blodgett films of a series of 6-O-alkylcelluloses

Monolayer and multilayer Langmuir-Blodgett (LB) films of 6-O-alkylcelluloses with various chain lengths were prepared and studied. The surface pressure (pi)-area (A) isotherms of 6-O-alkylcelluloses exhibited characteristic behaviors depending on the length of the alkyl chain and temperature. 6-O-Stearylcellulose on the subphase formed a homogeneous monolayer at 10 mN m(-1). By transfer ratio, FT-IR, and contact angle measurements, it was proved that the monolayer of 6-O-stearylcellulose on the water surface was transferred successfully onto a substrate by a vertical dipping method to form a Z-type LB film. The transmission and reflection absorption IR spectrum indicated that the hydrocarbon chains had all-trans rotamers and were oriented nearly perpendicular to the surface in the film. AFM section analysis revealed that the thickness per layer was calculated to be 2.35 nm. These results suggested that the hydrocarbon chains were inclined at an angle of about 25.3 degrees to have high packing density in the alkyl region.     

UV-induced reaction kinetics of dilinoleoylphosphatidylethanolamine monolayers

The UV-induced reactivity of dilinoleoylphosphatidylethanolamine (DLiPE) Langmuir and Langmuir-Blodgett films has been studied by in situ measurements of the changes in the mean molecular area, UV-vis and Fourier transform infrared spectroscopy, and atomic force microscopy (AFM). Optimum orientation and packing density of the DLiPE molecules in the monolayer were achieved by adding uranyl acetate to the subphase. A first-order reaction kinetic model was successfully fitted to the experimental reaction kinetics data obtained at a surface pressure of 30 mN/m. Topographical studies of LB films by AFM were performed on bilayer structures as a function of subphase composition and UV irradiation time. The orientational effect of the uranyl ions on the monolayer molecules was observed as an enhanced homogeneity of the freshly prepared monomeric LB films. However, the long-term stability of these films proved to be bad; clear reorganization and loss of a true monolayer structure were evidenced by the AFM images. This instability was inhibited for the UV-irradiated films, indicating that the UV irradiation gave rise to a cross-linked structure.     

Langmuir-Blodgett monolayer films of the Rhodopseudomonas viridis reaction center: determination of the order of the hemes in the cytochrome c subunit.

The Langmuir-Blodgett (LB) film technique has been applied to produce oriented and photo-active films of isolated reaction center cytochrome c complexes (RC-cyt c) and chromatophore membranes from the photosynthetic bacterium Rhodopseudomonas viridis. Linear dichroism (LD) and redox potentiometry have been used to identify the four cytochrome c hemes of the RC-cyt c complex. Resolved angular orientations of the four hemes in LB films of both isolated RC-cyt-c complexes and of chromatophore membranes permit the reduction of the 24 possible arrangements to two. Additional structural and functional information from other sources allows us to propose a model which best accounts for all the available experimental data.     

Complex formation between chlorophyll a and cytochrome c: surface properties at the air-water interface. Absorbance, fluorescence and fluorescence-lifetime in Langmuir-Blodgett films.

The binding of cytochrome c to an insoluble monolayer of chlorophyll a was studied. Surface pressure (II), surface potential (delta V) and [14C]cytochrome c surface-concentration (gamma) isotherms were measured versus molecular area (sigma) in mixed films. Compared to the successive-addition method, this procedure allows the formation of homogeneous mixed films. The cytochrome c is incorporated into a chlorophyll a monolayer, compressed at a surface pressure of 20 mN.m-1. On expansion, the quantity of protein incorporated into the monolayer gradually increases. Subsequent compression-expansion cycles result in similar isotherms, distinct from that measured during the first expansion. All surface properties measured, but more specifically the surface radioactivity of [14C]cytochrome c, indicate the irreversibility of protein incorporation into the chlorophyll a monolayer. In fact, surface properties of the binary film are completely different from the properties of either of the pure components. As a result, calculated values of surface potentials for mixed films using the additivity law deviate from experimentally measured potentials. The absorption and fluorescence spectra of mixed films transferred onto a solid substrate by the Langmuir-Blodgett technique, indicate a dilution effect of chlorophyll a by cytochrome c. However, the dilution effect cannot be detected by the fluorescence lifetimes of pure chlorophyll a and mixed chlorophyll a-cytochrome c films, both shorter than 0.2 ns. This provides support for the existence of an energy-transfer mechanism between chlorophyll a monomer and chlorophyll a aggregates which could serve as an energy trap. The role of the protein could be related to that of the matrix.     

Lactose biosensor based on Langmuir-Blodgett films of poly(3-hexyl thiophene)

An amperometric lactose biosensor was developed by immobilizing lactase (EC 3.2.1.23) and galactose oxidase (GaO) (EC 1.1.3.9) in Langmuir-Blodgett (LB) films of poly(3-hexyl thiophene) (P3HT)/stearic acid (SA) for estimation of lactose in milk and its products to prevent "lactose intolerance". The enzyme immobilized LB film was used as working electrode and platinum as reference electrode. The enzyme electrodes show a linearity 1-6 g/dL of lactose and have a shelf life more than 120 days. The reusability of electrode was found ten times with 3% loss in current response. The enzyme electrode was characterized by Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM) and kinetic parameters such as pH, temperature and stability. The working electrode may be used for the estimation of lactose/galactose in food and biological fluids.     

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 dipalmitoylphosphatidylglycerol (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 CO and NH groups from HRP being oriented perpendicular to the surface, parallel to the DPPG acyl chains, i.e. the α-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.     

Lateral organization of GM1 in phase-separated monolayers visualized by scanning force microscopy

Phase separation of glycolipids in lipid mono- and bilayers is of great interest for the understanding of membrane function. The distribution of the ganglioside GM1 in sphingomyelin (SM)/1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC), SM/1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DOPC) and SM/cholesterol/POPC Langmuir-Blodgett (LB) monolayers transferred at 36 mN/m has been studied by scanning force microscopy. Besides lateral organization of the glycolipid in LB monolayers as deduced from topography, material properties have been investigated by phase imaging, pulsed force mode and force modulation microscopy. It was shown that GM1 preferentially clusters in an ordered lipid matrix, i.e. the SM phase in the case of the SM/POPC and SM/DOPC mixture or in the ordered phase of POPC/SM/cholesterol monolayers. At higher local concentrations, three-dimensional protrusions enriched in GM1 occur, which may represent a precursor for the formation of micelles budding into the aqueous subphase. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00249-002-0232-4.     

Atomic force microscopy and force spectroscopy study of Langmuir-Blodgett films formed by heteroacid phospholipids of biological interest

Langmuir-Blodgett (LB) films of two heteroacid phospholipids of biological interest 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), as well as a mixed monolayer with chi(POPC)=0.4, were transferred onto mica in order to investigate by a combination of atomic force microscopy (AFM) and force spectroscopy (FS) their height, and particularly, their nanomechanical properties. AFM images of such monolayers extracted at 30 mN m(-1) revealed a smooth and defect-free topography except for the POPE monolayer. Since scratching such soft monolayers in contact mode was proved unsuccessful, their molecular height was measured by means of the width of the jump present in the respective force-extension curves. While for pure POPC a small jump occurs near zero force, for the mixed monolayer with chi(POPC)=0.4 the jump occurs at approximately 800 pN. Widths of approximately 2 nm could be established for POPC and chi(POPC)=0.4, but not for POPE monolayer at this extracting pressure. Such different mechanical stability allowed us to directly measure the threshold area/lipid range value needed to induce mechanical stability to the monolayers. AFM imaging and FS were next applied to get further structural and mechanical insight into the POPE phase transition (LC-LC') occurring at pressures >36.5 mN m(-1). This phase transition was intimately related to a sudden decrease in the area/molecule value, resulting in a jump in the force curve occurring at high force ( approximately 1.72 nN). FS reveals to be the unique experimental technique able to unveil structural and nanomechanical properties for such soft phospholipid monolayers. The biological implications of the nanomechanical properties of the systems under investigation are discussed considering that the annular phospholipids region of some transmembrane proteins is enriched in POPE.     

Fabrication of Large-scale Nanostructure by Langmuir-Blodgett Technique

Monolayer of polymer latex spheres was prepared at the air/water interface and deposited onto glass slides through Langmuir-Blodgett （LB） technique. Large-scale, high quality hexagonally close-packed domains were found in scanning electron microscopic pictures. Details of the monolayer-forming ability were discussed. Suitable surface characteristics of the colloidal particles, especially the hydrophilic and hydrophobic properties, are the keys for the formation of ordered monolayer films. The film can be transferred onto various kinds of substrates, even high curvature surface articles, such as fibers, decorations etc, can also be used as substrates. The advantages of this fabrication method of polymer latex spheres monolayer are fast, flexible, simple and very neat.     

Characterization of Langmuir-Blodgett films of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphat idylcholine by FTIR-ATR

Monomolecular films of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphatidylc holine (PPDPC) were transferred from an air/water interface onto a germanium attenuated total reflection crystal by the Langmuir-Blodgett (LB) technique. The assemblies were thereafter investigated by Fourier transform infrared-attenuated total reflection (FTIR-ATR) spectroscopy. To determine the molecular organization in the deposited layers we monitored the CH2 and C = O stretching and the CH2 bending regions of the infrared spectra of these lipids in detail. Using Fourier self-deconvolution technique, the carbonyl stretching mode was resolved into two models corresponding to the conformational differences in the ester linkages of the phospholipid sn-1 and sn-2 acyl chains. By varying the temperature of the subphase and using different surface pressures, we were able to transfer different conformational states of DPPC onto a germanium ATR crystal. Deposition of DPPC at 40 mN m-1 and at 15 degrees C or at 20 mN m-1 and at 35 degrees C results in LB-assemblies in ordered or disordered states, respectively, as judged by the IR spectra. These structures in LB films correspond to the state of DPPC in liposomes below and above the temperature of the order-disorder phase transition. Irrespective of the surface pressure and subphase temperature used during the deposition, an ordering process was found in DPPC films when the number of the transferred layers was increased from one to five. The pyrene-labelled phosphatidylcholine analogue, PPDPC, behaved differently from DPPC. In the case where one to three layers of PPDPC transferred at 35 mN m-1 and at 20 degrees C only conformational structures resembling those in fully hydrated liposomes above the main transition temperature were observed.     

Phase behaviour of binary mixtures involving tristearin, stearyl stearate and stearic acid: thermodynamic study and BAM observation at the air-water interface and AFM analysis of LB films

The binary mixtures involving tristearin (TS), stearyl stearate (SS) and stearic acid (SA) were studied by surface pressure-area (pi-A) measurements and by Brewster angle microscopy (BAM), at the air-water interface, and the Langmuir-Blodgett (LB) monolayers, transferred onto mica substrates, were analysed by AFM. The thermodynamic analysis indicated miscibility in the whole composition range for the system SA/TS, and partial miscibility for systems SA/SS and TS/SS. This behaviour was further confirmed by BAM observation and AFM analysis of LB films. The AFM imaging of collapsed monolayers revealed domains with a multilayered structure varying with system and composition. The layers thickness determined by cross section analysis are consistent with estimated molecular lengths and conformations proposed for the molecules, assuming nearly perpendicular or tilted orientations of the hydrocarbon chains to the interface.