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.     

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.     

Fabrication of Langmuir-Blodgett film of a fullerene derivative with a cyclic peptide as an anchor

A novel cyclic octapeptide carrying a fullerene unit and poly(ethylene glycol) at the side chain (cyclo8-C 60 + PEG) was synthesized, and its monolayer formation at the air/water interface and on a substrate was studied. Surface pressure-area per molecule isotherms indicated that cyclo8-C 60 + PEG formed a stable monolayer at the air/water interface. The cyclo8-C 60 + PEG monolayers prepared from various spreading volumes (i.e., from various initial areas per molecule) overlapped nicely on a single curve, suggesting that the molecules were uniformly dispersed on the surface without aggregation of the fullerene units. The uniform dispersibility is due to the scaffold effect of the cyclic peptide unit to keep the fullerene units away from each other. The formed monolayer could be quantitatively transferred onto a solid substrate. UV-vis absorption spectroscopy of the Langmuir-Blodgett (LB) monolayer showed that the electronic structure of the fullerene unit was not affected by the formation of the monolayer. Cyclic voltammetry of the LB monolayer in an aqueous solution containing redox species indicated that the LB monolayer was densely packed. Furthermore, reversible redox peaks attributed to the one-electron reduction of the fullerene unit were observed, showing that the redox property of the fullerene unit was also retained in the monolayer. It is thus concluded that the cyclic peptide is a good candidate as a scaffold for stable monolayer formation at the air/water interface and for intact immobilization of the fullerene moiety onto a substrate.     

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.     

Fabrication and structural characterization of the Langmuir–Blodgett films from a new chitosan derivative containing cinnamate chromophores

A new amphiphilic chitosan derivative, octanoylchitosan cinnamate (OCC) was synthesized through regioselective modifications of chitosan. A solution of OCC was spread to water to form a stable monolayer at the air/water interface. The surface pressure (π)–area (A) isotherm indicated that the polymer had a limiting area of about 100 Å² per repeat unit. YZ-type multilayers were deposited onto hydrophobic substrates through Langmuir–Blodgett (LB) technique. The structural features of the LB films were investigated by UV absorption, circular dichroism (CD) and linear dichroism (LD) spectroscopy. The results showed that the intrinsic chirality originating from the helical order of the OCC backbones was maintained in the LB films. Besides, the polymer backbones were uni-axially oriented in the LB film. The ordered structures of OCC assembled in a dilute solution and in a cast film were also investigated and the results were compared with that of the LB film.     

Patterning of photosensitive polyimide LB film and its application in the fabrication of biomolecular microphotodiode array

Ultra thin film of photosensitive polyimide having benzene and sulfonyloxyimide moieties in the main chain was prepared using a Langmuir-Blodgett (LB) technique, and then micro array pattern of the polyimide LB film on a gold substrate was obtained by deep UV lithographic technique. In order to array cytochrome c molecules along the micro-patterned gold substrate, the well-characterized monolayer of cytochrome c was immobilized with a mixed monolayer of 11-mercaptoundecanoic acid (11-MUDA) and decanethiol. The redox activity and electron transfer between cytochrome c molecular center and gold electrode interface for the self-assembled cytochrome c monolayer were investigated by cyclic voltammetry measurement. Biomolecular photodiode consisting of cytochrome c and green fluorescent protein (GFP) onto the patterned gold substrate was fabricated by self-assembly process. The integration and morphology of cytochrome c and GFP were studied from the measurements of atomic force microscopy (AFM) and fluorescence emission. Especially, current-voltage characteristics of the protein multilayers were investigated by scanning tunneling microscopy (STM) and its application in biomolecular photodiode was also examined.     

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.     

Self-assembly effect during the adsorption of polynucleotides on stearic acid langmuir-blodgett monolayer

Interaction of polyadenylic acid, poly(A), with stearic acid Langmuir-Blodgett (LB) monolayer was studied in different electrolyte surroundings. For this purpose LB films of stearic acid, transferred on the mica substrate from poly(A) containing subphase, were analyzed with atomic force microscopy (AFM). The density of polynucleotides surface coverage is ruled by the monovalent electrolyte concentration in the subphase that is in good agreement with previous results. Divalent cations in the subphase are needed to stabilize poly(A) molecules on the surface through formation of "salt bridges". At the very low divalent electrolyte concentration polynucleotides adsorb on the LB film to domains in which the effect of self-assembly is observed. Increase of divalent electrolyte concentration leads to the loss of this orientation effect. The explanation of this effect is proposed.     

Second‐harmonic generation from Langmuir–Blodgett film of cyclic peptide carrying two disperse red 1 on fused quartz surface

Cyclic octapeptide carrying one or two nonlinear optical chromophores, disperse red 1 (DR‐1), was synthesized and immobilized on a substrate to attain an active surface for second‐harmonic generation (SHG). Each cyclic octapeptide was transferred on a fused quartz substrate by the Langmuir–Blodgett (LB) method to afford a uniform monolayer. Infrared reflection–absorption spectroscopy of the LB monolayer revealed that the cyclic skeleton lay roughly flat on the surface. The SHG intensity from the monolayer of the cyclic peptide with two DR‐1 units was stronger than that from that with one DR‐1 unit. The difference is discussed in terms of molecular orientation and surface density of the active chromophores. The cyclic peptide is shown here to be an effective scaffold to modify a substrate surface with functional groups of a monolayer with taking stability of the monolayer and orientation of the functional group into consideration. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

The effect of surface chemistry and structure of titanium nitride (TiN) films on primary hippocampal cells

Thin films of TiN were investigated as a candidate microelectrode material for multi-electrode arrays, which are used for recording from electrically active cells in culture. TiN films were deposited onto glass substrates by DC pulsed reactive magnetron sputtering. The structure, phase composition and surface chemistry were studied using X-ray diffraction (XRD), Atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). The biocompatibility of the TiN films was examined morphologically by monitoring neuronal network formation and comparing this to a control substrate. Results indicate that neuronal cell adhesion and growth is influenced by the surface chemistry and associated crystal orientation of the TiN thin films.     

Enzymatic degradation of monolayer for poly(lactide) revealed by real-time atomic force microscopy: effects of stereochemical structure, molecular weight, and molecular branches on hydrolysis rates

The influences of the stereochemical structure, the molecular weight, and the number of molecular branches for poly(lactide) (PLA) on enzymatic hydrolysis rates of PLA monolayers were studied by atomic force microscopy (AFM) and the Langmuir-Blodgett (LB) technique. Monolayers of six kinds of PLA with different molecular weights, stereochemical structure, and numbers of molecular branches were prepared by LB techniques and then characterized by AFM in air. The PLA molecules covered homogeneously with a silicon substrate and did not form lamellar crystals in the monolayer. We determined the initial hydrolysis rate of PLA monolayers in presence of proteinase K by volumetric analysis from the continuous AFM height images. The presence of D-lactyl unit reduced the hydrolysis rate of the monolayer. The hydrolysis rate for the linear PLLA samples increased with a decrease in the molecular weight. In contrast, the rates of erosion for branched PLLA monolayers were independent of the molecular weight of samples. The erosion rate of branched PLLA monolayers was found to be dependent on the average molecular weight of PLLA segment in branched molecules, not on the overall molecular weight of samples. From these results, furthermore, the hydrolysis mode of PLAs by proteinase K is discussed.     

Fabrication and photoelectric response of poly(allylamine hydrochloride)/PM thin films by layer-by-layer deposition technique

Thin films of poly(allylamine hydrochloride) (PAH) and bacteriorhodopsin (bR) embedded in purple membrane (PM) have been prepared by layer-by-layer (LBL) self-assembly technique. The results obtained by UV-Vis spectroscopy and atomic force microscopy (AFM) analysis showed that the biological activity of bR was preserved and PM fragments could be well oriented onto the ITO substrate. A photo-electrochemical cell with the structure of ITO/(PAH/PM)(n)/electrolyte (0.5M KCl)/Pt was fabricated and studied. The photocurrent peaks of (PAH/PM)(6) corresponding to light-on and light-off were about 200 and 100 nA/cm(2), respectively, with the former enhanced 30% higher than that of the reference films made of (PDAC/PM)(6).     

Optical organophosphorus biosensor consisting of acetylcholinesterase/viologen hetero Langmuir–Blodgett film

The fiber-optic biosensor consisting of an acetylcholinesterase (AChE)-immobilized Langmuir–Blodegtt (LB) film was developed to detect organophosphorus compounds in contaminated water. The sensing scheme was based on the decrease of yellow product, o-nitrophenol, from a colorless substrate, o-nitrophenyl acetate, due to the inhibition by organophosphorus compounds on AChE. Absorbance change of the product as the output of enzyme reaction was detected and the light was guided through the optical fibers. The enzyme portion of the sensor system was fabricated by the LB technique for formation of the enzyme film. AChE-immobilized LB film was formed by adsorbing the enzyme molecules onto a viologen monolayer using the electrostatic force. The proposed kinetics for irreversible inhibition of organophosphorus compounds on AChE agreed well with the experimental data. The surface topography of AChE-immobilized LB film was investigated by atomic force microscope (AFM). The immobilized AChE had the maximum activity at pH 7. The proposed biosensor could successfully detect the organophosphorus compounds upto 2 ppm and the response time to steady signal of the sensor was about 10 min.     

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.     

Qualitative and quantitative analysis of the secondary structure of cytochrome C Langmuir-Blodgett films

A qualitative and quantitative analysis of the conformation of Langmuir-Blodgett (LB) dried films of cytochrome C on silicon wafers was performed by Fourier transform ir (FTIR) spectroscopy. A deconvolution procedure was applied to the amide I band analysis, in order to determine the percentage of the different secondary structures. Qualitative analysis was performed by examining difference spectra. Films obtained by spreading protein solutions at pH 7.4 and 1, dried at 25 and 100°C, on silicon wafers were also examined in order to detect spectral components associated with denatured protein domains, and to compare them with cytochrome C LB films. FTIR spectroscopy showed that the following important changes characterise LB film spectra: (a) the α-helix component is higher (its percentage is 57 and 54%) than the one estimated in dried film obtained by spreading the solutions at pH 7.4 on a silicon substrate (43%), (b) there is an increase in the intensity of bands attributed to protonated carboxy group bands, involved and not involved in the formation of hydrogen bonds, and a decrease in those attributed to deprotonated carboxy groups, (c) the intensity of several bands attributed to aromatic amino acids and aliphatic chains increases, and (d) bands due to O(SINGLEBOND)H stretching vibrations of crystallization water are present. These conformational changes could be induced by protein-protein interaction caused by the close packing of molecules that occurs during LB film formation; it cannot be excluded that they may be accompanied by partial changes in the tertiary structure of the protein. A preferential orientation of protein molecules in LB films is also a possibility. © 1997 John Wiley & Sons, Inc. Biopoly 42: 227–237, 1997     

Humicola lanuginosa lipase hydrolysis of mono-oleoyl-rac-glycerol at the lipid-water interface observed by atomic force microscopy

A new type of planar lipid substrate for Humicola lanuginosa lipase (HLL) has been prepared by depositing a monolayer of 1-mono-oleoyl-rac-glycerol (MOG) on top of a monolayer of dipalmitoyl-phosphatidylcholine (DPPC) on mica by the Langmuir-Blodgett (LB) technique. The bilayer was subsequently exposed to HLL in a liquid cell of an atomic force microscope (AFM) allowing the time course of the lipolytic degradation to be observed. By analysing a series of AFM images, we find that enzymes are preferentially activated at the edge of nano-scale defects present in the bilayer prior to enzyme injection, while defect-free areas of the substrate are surprisingly stable towards enzyme degradation. The initial rate of hydrolysis is found to be proportional to the perimeter length, P, of the initial nano-scale defects as well as the bulk enzyme concentration, c(HLL); d(lipid)/dt=k P c(HLL). We estimate the specific rate of MOG hydrolysis by HLL to be 2.5x10(4) MOG molecules/(minute x molecule of HLL).     

Bioelectronic device consisting of cytochrome c/poly-L-aspartic acid adsorbed hetero-Langmuir-Blodgett films

A bioelectronic device consisting of protein-adsorbed hetero-Langmuir-Blodgett (LB) films was investigated. Four kinds of functional molecules, cytochrome c, viologen, flavin, and ferrocene, were used as a secondary electron acceptor (A2), a first electron acceptor (A1), a sensitizer (S), and an electron donor (D), respectively. To fabricate the cytochrome c adsorbed hetero-LB film, poly-L-aspartic acid was used as the bridging molecule. The hetero-LB film was fabricated by subsequently depositing ferrocene, flavin, and viologen onto the pretreated ITO glass. Cytochrome c-adsorbed hetero-LB films were prepared by the adsorption of cytochrome c onto the poly-L-aspartic acid treated-LB films by intermolecular electrostatic attraction. Finally, the MIM (metal/insulator/metal) structured molecular device was constructed by depositing aluminum onto the surface of the cytochrome c-adsorbed hetero-LB films. Hetero-LB films were analyzed by Atomic Force Microscopy (AFM), and cytochrome c adsorption onto the films confirmed. The photoswitching function was achieved and the photoinduced unidirectional flow was in accordance with the rectifying characteristics of the molecular device. The direction of energy flow was in accordance with the energy level profile across molecular films. Based on the measurement of the transient photocurrent of the molecular device efficient directional flow of photocurrent through the redox potential difference was observed. The photodiode characteristics of the proposed bio-electronic device were verified and the proposed molecular array mimicking the photosynthetic reaction center could be usefully applied as a model system for the development of the bio-molecular photodiode.     

Novel thymine-functionalized polystyrenes for applications in biotechnology. 2. Adsorption of model proteins

This paper investigates the adsorption of bovine serum albumin (BSA) and bovine hemoglobin (BHb) model proteins onto novel thymine-functionalized polystyrene (PS-VBT) microspheres, in comparison with polystyrene (PS) microspheres. Maximum adsorption was obtained for both proteins near their corresponding isoelectric points (pI at pH = 4.7 for BSA and 7.1 for BHb). FTIR and adsorption isotherm analysis demonstrated that, although both proteins were physisorbed onto PS through nonspecific hydrophobic interactions, adsorption onto the functionalized copolymers occurred by both physisorption and chemisorption via hydrogen bonding. FTIR analysis also indicated conformational changes in the secondary structure of BSA and BHb adsorbed onto PS, whereas little or no conformation change was seen in the case of adsorption onto PS-VBT. Atomic force microscopy (AFM), consistent with the isotherm results, also demonstrated monolayer adsorption for both proteins. AFM images of BSA adsorbed onto copolymers with 20 mol % surface VBT loading showed exclusively end-on orientation. Adsorption onto copolymers with lower functionality showed mixed end-on and side-on orientation modes of BSA, and only the side-on orientation was observed on PS. The AFM results agreed well with theoretically calculated and experimentally obtained adsorption capacities. AFM together with calculated and observed adsorption capacity data for BHb indicated that this protein might be highly compressed on the copolymer surface. Adsorption from a binary mixture of BSA and BHb onto PS-VBT showed good separation at pH=7.0; approximately 90% of the adsorbed protein was BHb. The novel copolymers have potential applications in biotechnology.