Using a chitosan nanolayer as an efficient pH buffer to protect pH-sensitive supramolecular assemblies.

It is attractive to control the properties of macro objects and films by employing simple nanolayer composites, as in the case of nanoarchitectured Layer-by-Layer (LbL) coating. In this paper, we use chitosan as a surface-based pH buffer to protect adsorbed supramolecular fibres from pH-mediated disassembly. Protons are generated on a titania surface under illumination at 405 nm leading to an appreciable pH change on the surface. We find that supramolecular polymers that are highly sensitive to pH change will not disassemble after irradiation if a nanolayer of chitosan is present. We propose that chitosan can be used as an efficient pH-responsive protective layer for pH sensitive soft materials.

Structural characterization of a spin-assisted colloid-polyelectrolyte assembly: stratified multilayer thin films.

The assembly of polyelectrolytes and gold nanoparticles yields stratified multilayers with very low roughness and high structural perfection. The films are prepared by spin-assisted layer-by-layer self-assembly (LbL) and are characterized by X-ray reflectivity (XRR), UV-vis spectroscopy, atomic force microscopy (AFM), and transmission electron microscopy (TEM). Typical structures have four repeat units, each of which consists of eight double layers (DL) of poly(sodium 4-styrenesulfonate)/poly(allylamine hydrochloride), one monolayer of gold nanoparticles (10 nm diameter), and another layer of poly(allylamine hydrochloride). XRR scans show small-angle Bragg peaks up to seventh order, evidencing the highly stratified structure. Pronounced Kiessig fringes indicate a low global roughness, which is confirmed by local AFM measurements. TEM images corroborate the layered structure in the growth direction and nicely show the distinct separation of the individual particle layers. An AFM study reveals the lateral gold particle distribution within one individual particle layer. Interestingly, the spin-assisted deposition of polyelectrolytes reduces the roughness induced by the particle layers, leading to self-healing of roughness defects and a rather perfect stratification.

Surface-supported multilayers decorated with bio-active material aimed at light-triggered drug delivery.

In this work, we report on the functionalization of layer-by-layer films with gold nanoparticles, microcapsules, and DNA molecules by spontaneous incorporation into the film. Exponentially growing films from biopolymers, namely, hyaluronic acid (HA) and poly-L-lysine (PLL), and linearly growing films from the synthetic polymers, namely, poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH), were examined for the embedding. The studied (PLL/HA)(24)/PLL and (PAH/PSS)(24)/PAH films are later named HA/PLL and PSS/PAH films, respectively. The HA/PLL film has been found to be more efficient for both particle and DNA embedding than PSS/PAH because of spontaneous PLL transport from the interior of the whole HA/PLL film to the surface in order to make additional contact with embedded particles or DNA. DNA and nanoparticles can be immobilized in HA/PLL films, reaching loading capacities of 1.5 and 100 microg/cm(2), respectively. The capacities of PSS/PAH films are 5 and 12 times lower than that for films made from biopolymers. Polyelectrolyte microcapsules adsorb irreversibly on the HA/PLL film surface as single particles whereas very poor interaction was observed for PSS/PAH. This intrinsic property of the HA/PLL film is due to the high mobility of PLL within the film whereas the structure of the PSS/PAH film is "frozen in". Gold nanoparticles and DNA form micrometer-sized aggregates or patches on the HA/PLL film surface. The diffusion of nanoparticles and DNA into the HA/PLL film is restricted at room temperature, but DNA diffusion is triggered by heating to 70 degrees C, leading to homogeneous filling of the film with DNA. The film has not only a high loading capacity but also can be activated by "biofriendly" near-infrared (IR) laser light, thanks to the gold nanoparticle aggregates on the film surface. Composite HA/PLL films with embedded gold nanoparticles and DNA can be activated by light, resulting in DNA release. We assume that the mechanism of the release is dependent on the disturbance in bonding between "doping" PLL and DNA, which is induced by local thermal decomposition of the HA/PLL network in the film when the film is exposed to IR light. Remote IR-light activation of dextran-filled microcapsules modified by gold nanoparticles and integrated into the HA/PLL film is also demonstrated, revealing an alternative release pathway using immobilized light-sensitive carriers (microcapsules).

Effect of layer-by-layer electrostatic assemblies on the surface potential and current voltage characteristic of metal-insulator-semiconductor structures.

In the present Article, the Kelvin probe method for surface potential measurement is introduced to study polyelectrolyte multilayer coatings deposited on silicon plates. Metal-insulator-semiconductor (MIS) structures with polyelectrolyte layers as insulator were fabricated. The polyelectrolyte layer deposition on the surface of silicon plates led to a change of the current-voltage characteristics connected with resistance changes of the MIS structures. Poly(ethylenimine) (PEI) monolayer formation resulted in resistance decrease, and the following increase of the polyelectrolyte layer number led to MIS structure resistance increase. The results are interpreted as an interplay between accumulation of majority carriers (electrons) near the semiconductor surface and resistance increase due to insulating polyelectrolyte adsorption, and both effects can be discriminated by varying the polyelectrolyte layer thickness.

Competitive adsorption from mixed hen egg-white lysozyme/surfactant solutions at the air-water interface studied by tensiometry, ellipsometry, and surface dilational rheology.

The competitive adsorption at the air-water interface from mixed adsorption layers of hen egg-white lysozyme with a non-ionic surfactant (C10DMPO) was studied and compared to the mixture with an ionic surfactant (SDS) using bubble and drop shape analysis tensiometry, ellipsometry, and surface dilational rheology. The set of equilibrium and kinetic data of the mixed solutions is described by a thermodynamic model developed recently. The theoretical description of the mixed system is based on the model parameters for the individual components.

Experimental evidence of the electrostatic contribution to the bending rigidity of charged membranes.

We address the issue of the origin of the bending rigidity of a charged membrane formed from amphiphilic molecules. Electrostatic effects are investigated by direct measurement of the force necessary to deform a catanionic membrane as function of the ionic strength of the medium by means of an atomic force microscope (AFM). Using continuum mechanical modeling of membrane deformation, we derive the bending rigidity of the catanionic membranes and monitor for the first time its decrease in response to increasing salt concentration.

Interactions of cytochromes b5 and c with phospholipid monolayers.

Monolayers of charged and neutral phospholipids at the air/water interface containing the cytochromes b5 and c are studied by film balance techniques and by fluorescence microscopy. A new technique is introduced to obtain a defined and homogeneous protein distribution within the membrane. It is shown that both proteins preferentially partition into the fluid membrane phases coexisting with solid lipid domains, thus allowing formation of periodic protein distributions. Protein reconstitution in protein/lipid ratios up to 1:50 does not change the pressure, pi c, corresponding to the main lipid transition but changes the slope in the pressure/area isotherms. It also affects the pressure-induced lipid crystallization, in that the monolayer can be viewed as segregated into a protein-free and a protein-enriched phase. Whereas penetration of cytochrome c into the monolayer is highly dependent on lipid head group charge, this does not hold for cytochrome b. In both cases, monolayer penetration is monotonously reduced with increasing surface pressure, pointing to the dependence of hydrophobic protein-lipid interactions on hydrocarbon chain density.

Monitoring the location profile of fluorophores in phosphatidylcholine bilayers by the use or paramagnetic quenching.

Spin probes differing in the position of their paramagnetic centre are used to quench the fluorescence of pyrene derivatives and chlorophylls incorporated into dimyristoyl phosphatidylcholine membranes. Pyrene butyric acid and pyrene decanoic acid with known orientation relative to the membrane surface are investigated. The quenching efficiency of fatty acid spin probes is dependent on the position of the nitroxide radical group in the fatty acid chain. Using this short range interaction we developed a spectroscopic method to chlorophyll-containing vesicles, we were able to characterize the orientation of the porphyrin ring within the membrane. Moreover, the chlorophyll fluorescence is also quenched by a water-soluble spin label. Therefore the porphyrin ring appears to be orientated in the polar head group region of the lipid layer, but not to be protruding out into the water phase. This conclusion is confirmed by the use of pyrene derivatives. Fluorescence quenching by a water-soluble spin label within the lipid matrix is observed even in the rigid state of the membrane. Fluorescence lifetime measurements suggest the existence of two different quenching mechanisms: (1) a static quenching occurring below the lipid phase transition temperature, and (2) an additional dynamic quenching taking place in the fluid state of the lipid bilayer.

Structure and temperature behavior of metallo-supramolecular assemblies.

A detailed structural analysis of a Langmuir-Blodgett (LB) multilayer composed of a polyelectrolyte-amphiphile complex (PAC) is presented. The PAC is self-assembled from metal ions, ditopic bis-terpyridines, and amphiphiles. The vertical structure of the LB multilayer is investigated by X-ray reflectometry. The multilayer has a periodicity of 57 A, which corresponds to an architecture of flat lying metallo-supramolecular coordination polyelectrolyte (MEPE) rods and upright-standing amphiphiles (dihexadecyl phosphate, DHP). In-plane diffraction reveals hexagonal packing of the DHP molecules. Using extended X-ray absorption fine structure (EXAFS) experiments, we prove that the central metal ion is coordinated to the terpyridine moieties in a pseudo-octahedral coordination environment. The Fe-N bond distances are 1.82 and 2.0 A, respectively. Temperature resolved measurements indicate a reversible phase transition in a temperature range up to 55 degrees C. EXAFS measurements indicate a lengthening of the average Fe-N bond distance from 1.91 to 1.95 A. The widening of the coordination cage upon heating is expected to lower the ligand field stabilization, thus giving rise to spin transitions in these composite materials.

Surface viscoelastic properties of floating polyelectrolyte multilayers films: a capillary wave study.

A capillary wave technique was used to study the viscoelastic properties of floating polyelectrolyte multilayers of (PSS/PAH)(n) at the air-water interface. Oppositely charged polyelectrolyte layers were adsorbed onto two different Langmuir monolayers, either the lipid dimethyldioctadecylammonium bromide (DODAB) or the block copolymer poly(styrene-b-sodium acrylate) (PS-b-PAA). The results allow to propose a schematic representation of the multilayers in three zones: Zone I as a precursor, representing the adhesion between the Langmuir monolayer and the bulk polyelectrolyte multilayer. Zone II forms a bulk or core zone of the multilayer. Zone III as an outer zone in direct contact with the aqueous phase. The results show an increase of the elasticity after the formation of four polyelectrolyte layers accompanied by an apparent negative viscosity. This behaviour was interpreted as a translation of elasticity dominance from zone I to zone II. The Young modulus of seven layers was in the same order of magnitude as observed for planar polyelectrolyte multilayer films.

Development of equilibrium domain shapes in phospholipid monolayers.

Fluorescence micrographs of monolayers of L-alpha-dipalmitoylphosphatidylcholine (DPPC) at the air water interface are analyzed. Ordered phase domains in coexistence with the fluid lipid phase change their shapes with time and with lateral pressure. With domains fixed under an electrode and well separated from their neighbours a peculiar instability of the boundary lines is observed. It is ascribed to the onset of an electrostatically induced shape transition. Elaborating the boundary regions where domain fusion and fission, respectively, are observed leads to a model on in-plane dipole orientation.

Miscibility of DPPC and DPPA in monolayers at the air/water interface.

Monolayers of mixtures of 1,2-dipalmitoylphosphatidylcholine (DPPC) as the substrate and 1,2-dipalmitoylphosphatidic acid (DPPA) as the product of the hydrolysis reaction catalyzed by phospholipase D (PLD) were investigated in the presence of Ca2+. The miscibility behavior and the microstructure of mixed domains have been studied by grazing incidence X-ray diffraction (GIXD), Brewster angle microscopy and film balance measurements. The phase diagram reveals partial miscibility on both sides and a wide miscibility gap, which becomes narrower at high pressure. At low pressure, the segregation of condensed DPPA-rich domains in a fluid-like DPPC matrix was detected already at small DPPA concentrations and their structure was determined. A small amount of DPPC mixed into the segregated DPPA domains induces the transformation from rectangular to an oblique unit cell and increases the tilt angle in the condensed domains. At high pressure, two types of condensed phase domains were found: DPPC-rich and DPPA-rich. A drastic reduction of the tilt angle in the DPPC-rich domains with increasing amount of DPPA was observed. The decrease of the tilt angle must be connected with a change of the head group conformation of DPPC in such mixed domains.

Miscibility of lipoteichoic acid in dipalmitoylphosphatidylcholine studied by monofilm investigations and fluorescence microscopy.

The miscibility of the bacterial amphiphile lipoteichoic acid, a constituent of the cytoplasmic membrane of Gram-positive bacteria, in dipalmitoylphosphatidylcholine has been investigated by classic monofilm measurements and fluorescence microscopy at the air-water interface of monofilms obtained by spreading mixtures of both amphiphiles on a water subphase. The isotherms indicated miscibility of both lipids at concentrations up to 30 mol% lipoteichoic acid, whereas at higher concentrations immiscibility was detected. Increasing the lateral pressure over a certain value, lipoteichoic acid is squeezed out of the monofilm. By fluorescence microscopy the influence of lipoteichoic acid on the domain shape of condensed dipalmitoylphosphatidylcholine phases has been studied. The balance between hydrophobic and hydrophilic forces in the mixtures of both amphiphiles is discussed.

Adsorption behavior of oxyethylated surfactants at the air/water interface.

Literature data for homologous series of nonionic and ionic surfactants, such as C(n)OH, C(n)EO(m), Tritons, C(n)SO4Na, and C(n)EO(m)SO4Na, were analyzed on the basis of the Frumkin and reorientation models. The thermodynamic quantities obtained from the reorientation model agree best with the experimental data. They yield the standard free energy of adsorption which can be split into contributions from the alkyl chain, and the polar head group. An additional contribution to the increment of the polar group deltaG0(P) from m EO groups deltaG0(EO(m)) is extracted which can be accounted for by deltaG0(EO(m)) = -2.35 square root(m) kJ/mol. The standard free energy of adsorption is well understood by the increment method used.

Selective staining by the fluorochrome, 5,5-diphenyl-9-ethyl-DiOC2(3). I. Physicochemical studies of dye-dye and dye-tissue interactions.

The 5,5'-diphenyl-9-ethyl-oxacarbocyanine (5,5'-diphenyl-9-ethyl-DiOC2(3); CD) has properties suitable for histological investigations including the spectral range for absorption and fluorescence emission, the values of the corresponding molar coefficients and fluorescence quantum yield. Furthermore, CD remains relatively unchanged over the entire pH range and interacts with protein beta-sheets. The latter fact is detectable spectroscopically as a bathochromic shift. In water-containing media such as the histological stain and washes, CD exists as a monomer, a dimer and in two aggregated states. These differ in their binding affinity to tissue sections, in their solubility in water, alcohol and water/alcohol mixtures, and in their UV/VIS absorption and fluorescence emission. The ratio of the various CD states and the contrast of selectively stained tissue areas can be controlled via the staining conditions and the sequence of the washes. Furthermore, mounting in a xylene-based medium produces a solvatochromic spectral shift of the CD monomer, which leads to a marked elevation in phase contrast.

Selective staining by the fluorochrome 5,5-diphenyl-9-ethyl-oxacarbocyanine. II. Application to paraffin embedded nervous tissue.

We present a simple and rapid method to label specific structures of neural tissue in paraffin sections. After incubation of deparaffinized sections in the cyanine dye 5,5'-diphenyl-9-ethyl-oxacarbocyanine (DEOC), three different washing and mounting procedures were performed. Incubation in DEOC followed by washes in ethanol and water and mounting in glycerol-gelatin resulted in selective labeling of myelin in the central and peripheral nervous systems. Weak labeling of myelin and axons and staining of nuclei of neurons was seen after incubation in DEOC followed by washes in ethanol and xylene, and mounting in Eukitt. Nuclear proteins, the endoplasmic reticulum and the Golgi apparatus were stained strongly and exclusively after incubation in DEOC, washes in water, ethanol and xylene, and mounting in Eukitt. Thus the absorption pattern of DEOC is changed significantly by solvents applied after the fluorochrome. In any case, fluorescence did not fade even after repeated and intense fluorescence microscopy over a period of 6 months.

Evaluation of the role of polyelectrolyte deposition conditions in growth factor release.

Polyelectrolyte multilayer coatings were prepared from solutions of poly(methacrylic acid) and poly-l-histidine. Bone morphogenetic protein 2 (BMP-2) was adsorbed onto the surface of anodized titanium and polyelectrolyte multilayer coatings were built up on top of the BMP-2. The effect of deposition conditions on coating properties and preosteoblast response was measured by comparing coatings prepared under natural conditions to those prepared from solutions at pH = 6.0 and solutions containing 0.1 M NaCl. High levels of BMP-2 release were achieved, with coatings prepared from pH = 6.0 solutions releasing 86 ng cm-2 and coatings prepared from solutions containing 0.1 M NaCl releasing 114 ng cm-2 over 25 days. Enhanced preosteoblast differentiation was observed on coatings prepared from modified solutions; however, this increased differentiation was apparent for BMP-2-eluting and control coatings. Additionally, a positive relationship between surface roughness and differentiation was observed, which may account for increased differentiation for systems that do not release BMP-2.

Coupling of pyrroloquinoline quinone dependent glucose dehydrogenase to (cytochrome c/DNA)-multilayer systems on electrodes.

The redox protein cytochrome c (cyt c) assembles into electro-active multilayers on gold electrodes by the help of deoxyribonucleic acid (DNA) as a negatively-charged building block. The feasibility of this electro-active system as a novel interface for the immobilization of enzymes on electrodes is investigated in this study. Therefore the known reaction of cyt c and PQQ-GDH is confined to the immobilized state of both molecules. We find that electron-transfer from the substrate via PQQ-GDH and cyt c molecules, towards the electrode occurs; thus the system can be considered as an artificial signal chain. First, a monolayer of cyt c is prepared on a thiol-modified gold electrode and investigated with PQQ-GDH in solution. Cyclic voltammetric measurements prove that a small catalytic current occurs in the presence of the substrate. Next, both proteins are immobilized. We use the layer-by-layer deposition technique to assemble cyt c with DNA in multiple layers and a terminal layer of PQQ-GDH: (cyt c/DNA)(n)/PQQ-GDH. It is found that a catalytic current flows when glucose is present, proving that this system relies on inter-protein electron-transfer. The current intensity can be increased from 0.1nA, at the monolayer system, up to 3.7nA, at the (cyt c/DNA)(4)/PQQ-GDH electrode. This bi-protein multilayer system can follow different glucose concentrations in a linear dynamic range between 25nM and 0.5µM at its pH optimum, i.e. pH 6. Therefore this system is of limited importance for sensing but it represents a new biomimetic signal chain by arranging proteins in multiple layers on electrodes, making direct electron exchange feasible.

Patchiness of embedded particles and film stiffness control through concentration of gold nanoparticles.

Patchy particles are fabricated using a method of embedding-into and extracting-from thick, biocompatible, gel-like HA/PLL films. Control over the patchiness is achieved by adjusting the stiffness of films, which affects embedding and masking of particles. The stiffness is adjusted by the concentration of gold nanoparticles adsorbed onto the surface of the films.