Lipase surface diffusion studied by fluorescence recovery after photobleaching

We have analyzed surface diffusion properties of a variant of Thermomyces lanuginosa lipase (TLL) on hydrophilic silica and silica methylated with dichlorodimethylsilane (DDS) or octadecyltrichlorosilane (OTS). For this study a novel method for analysis of diffusion on solid surfaces was developed. The method is based on fluorescence recovery after photobleaching using confocal microscopy. When a rectangular area of the sample was photobleached, fluorescence recovery could be analyzed as one-dimensional diffusion, resulting in simplified mathematical expressions for fitting the data. The method was initially tested by measuring bovine serum albumin diffusion on glass, which led to a diffusion coefficient in good correspondence to earlier reports. For the analysis of TLL diffusion, ellipsometry data of TLL adsorption were used to calibrate fluorescence intensity to surface density of lipase, enabling measurements of the diffusion coefficient at different surface densities. The average diffusion coefficient was calculated in two time intervals after adsorption. Mobile fraction and diffusion coefficient were lowest on the OTS surface, when extrapolated to infinite surface dilution. Moreover, the diffusion rate decreased with time on the hydrophobic surfaces. Our observations can be explained by the surface dependence on the distribution of orientations and conformations of adsorbed TLL, where the transition from the closed to the catalytically active open and more hydrophobic structure is important.     

Light scattering and fluorescence photobleaching recovery study of poly(amidoamine) cascade polymers in aqueous solution

Diffusion coefficients of polyamidoamine cascade polymers (PAMAMs) were measured in aqueous solutions by dynamic light scattering and, after labeling with fluorescein isothiocyanate, by fluorescence photobleaching recovery. The dynamic light scattering results depended weakly on pH at a high salt concentration, but varied strongly with the concentration of added salt in the low-salt limit. The fluorescence photobleaching recovery values were almost independent of salt concentration. The difference between the two techniques is that thermodynamic nonideality strongly affects light scattering at the concentrations that are experimentally accessible. The hydrodynamic sizes from fluorescence photobleaching recovery were somewhat smaller than those from dynamic light scattering in the high-salt limit, despite attachment of the dye. Nevertheless, fluorescently tagged PAMAMs should make suitable markers and diffusion probes. © 1996 John Wiley & Sons, Inc.     

Room-temperature imprinting poly(acrylic acid)/poly(allylamine hydrochloride) multilayer films by using polymer molds

Layer-by-layer assembled polyelectrolyte multilayer films of poly(acrylic acid) (PAA)/poly(allylamine hydrochloride) (PAH) have been successfully patterned by room-temperature imprinting using a Norland Optical Adhesives (NOA 63) polymer mold. The proper amount of water in the PAA/PAH multilayer film can decrease the viscosity of the film and facilitate the imprinting. Many factors, such as imprinting pressure, length of imprinting time, and the structure and size of the patterns in the polymer mold, can produce an influence on the final imprinted pattern structures on multilayer films. A high imprinting pressure of 100 bar and elongated imprinting time of several hours is needed to achieve a patterned PAA/PAH multilayer film with a feature size of several tens of micrometers. With a twice imprinting, grid structures can be successfully produced when a NOA 63 mold having line structures is used. Room-temperature imprinting by using polymer NOA 63 mold provides a facile way to fabricate layered polymeric films with various kinds of pattern structures.     

Salt-induced swelling and electrochemical property change of hyaluronic acid/myoglobin multilayer films

The ionic strength in supporting electrolyte solution had a significant influence on the electrochemical and electrocatalytic behaviors of myoglobin (Mb) in {HA/Mb}n films, which were assembled layer-by-layer on pyrolytic graphite (PG) electrodes with oppositely charged hyaluronic acid (HA) and Mb. The results of cyclic voltammetry (CV), quartz crystal microbalance (QCM), scanning electron microscopy (SEM), rotating disk voltammetry (RDV), and electrochemical impedance spectroscopy (EIS) showed that after incubation with testing solution at high concentration of salt (CKCl), the {HA/Mb}n films swelled and the film permeability was enhanced, suggesting that the external salt ions and accompanied water molecules in the exposure solution are incorporated into the films. Systematic investigation of the type and size effect of counterions in supporting electrolyte solution on the electrochemical responses for the {HA/Mb}n films and the positive shift of the formal potential (E degrees ') with CKCl suggest that it is cationic rather than anionic counterions that control the electrode process of {HA/Mb}n films at PG electrodes with electron hopping mechanism. The salt-induced swelling of {HA/Mb}n films facilitated the transportation of counterions, and then accelerated the electron transfer of Mb in the films with the underlying electrodes, making the film electrodes show better CV responses. The comparative study showed that only Mb layer-by-layer films assembled with "soft" and flexible polyions could demonstrate the salt-induced effect and that the {HA/Mb}n films showed better swelling capability than {PSS/Mb}n films (PSS = poly(styrenesulfonate)) due to the unique character of HA.     

Organization and dynamics of NBD-labeled lipids in membranes analyzed by fluorescence recovery after photobleaching

Lateral diffusion of membrane constituents plays an important role in membrane organization and represents a central theme in current models describing the structure and function of biological membranes. Fluorescence recovery after photobleaching (FRAP) is a widely used approach that provides information regarding dynamic properties and spatial distribution of membrane constituents. On the basis of the unique concentration-dependent fluorescence emission properties of a fluorescently labeled cholesterol analogue modified at the tail region, 25-[N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-methyl]amino]-27-norcholesterol (25-NBD-cholesterol), we have previously shown that it exhibits local organization even at very low concentrations in membranes. In this paper, we address aspects regarding the molecular size and dynamics of such an organized assembly of 25-NBD-cholesterol by monitoring its lateral diffusion characteristics using FRAP. To obtain a comprehensive understanding of the organization and dynamics of 25-NBD-cholesterol in the membrane, we compare its diffusion properties to that of a fluorescent phospholipid analogue 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-(1,3-benzoxadiazol-4-yl)) (NBD-PE). Our results indicate significant differences in the membrane dynamics of these NBD-labeled lipids. Importantly, on the basis of a novel wavelength-selective FRAP approach, our results show that the organization of 25-NBD-cholesterol is heterogeneous, with the presence of fast- and slow-diffusing species which could correspond to predominant populations of monomers and dimers of 25-NBD-cholesterol. The potential application of the wavelength-selective FRAP approach to monitor the organization and dynamics of molecules in membranes therefore represents an exciting possibility.     

Cell adhesion properties of patterned poly(acrylic acid)/poly(allylamine hydrochloride) multilayer films created by room-temperature imprinting technique

Patterned poly(acrylic acid) (PAA)/poly(allylamine hydrochloride) (PAH) multilayer films with line structures of different lateral size and vertical height were fabricated by a room-temperature imprinting technique, and their cell adhesion properties were investigated. The nonimprinted PAA/PAH multilayer films are cytophilic toward NIH/3T3 fibroblasts and HeLa cells whether PAA or PAH is the outer most layer. In contrast, the PAA/PAH multilayer films with a 6.5-microm-line/3.5-microm-space pattern structure are cytophobic toward NIH/3T3 fibroblasts and HeLa cells when the height of the lines is 1.29 microm. By either increasing the lateral size of the patters to 69-microm-line/43-mum-space or decreasing the height of the imprinted lines to approximately 107 nm, imprinted PAA/PAH multilayer films become cytophilic. This kind of transition of cell adhesion behavior derives from the change of the physical pattern size of the PAA/PAH multilayer films and is independent of the chemical composition of the films. The easy patterning of layer-by-layer assembled polymeric multilayer films with the room-temperature imprinting technique provides a facile way to tailor the cellular behavior of the layered polymeric films by simply changing the pattern dimensions.     

Modulation of the functions of osteoblast-like cells on poly(allylamine hydrochloride) and poly(acrylic acid) multilayer films

Deposition of layer-by-layer polyelectrolyte multilayer (PEM) films has been a widely applied surface modification technique to improve the biocompatibility of biomaterials. The objective of this study was to investigate the impact of the deposition of poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA) multilayer films on adhesion, growth and differentiation of osteoblasts-like MG63 cells. PAH and PAA were deposited sequentially onto tissue culture polystyrene at either pH 2.0 or pH 6.5 with 4-21 layers. While the MG63 cells attached poorly on the PAH/PAA multilayer films deposited at pH 2.0, while the cells adhered to the PEM films deposited at pH 6.5, depending on layer numbers. Cell adhesion, proliferation and osteogenic activities (alkaline phosphatase activity, expression of osteogenic marker genes and mineralization) were highest on the 4-layer PAH/PAA film and decreased with increasing layer numbers. On the other hand, the behavior of MG63 cells did not show any difference on the adjacent even and odd layers, except PEM4 and PEM5, i.e. the surface charges of the PAH/PAA multilayer films with over ten layers seem indifferent to osteoblastic functions. The results in this study suggested that the mechanical properties of PEM films may play a critical role in modulating the behavior of osteoblasts, providing guidance for application of PEM films to osteopaedic implants.     

Characterization of silk fibroin/hyaluronic acid polyelectrolyte complex (PEC) films

This study aimed the characterization of the films casted from the aqueous mixtures of the pH induced complexes between silk fibroin (SF) and hyaluronic acid (HA). The insoluble and transparent films were subjected to scanning electron microscopy (SEM) analyses to show the morphological changes. Thermal analysis of complex films was determined by a differential scanning calorimeter (DSC). The changes in the crystalline state were monitored by X-ray diffractometer (XRD) and Fourier transform infrared spectroscopy (FTIR). It was shown that the complexation between HA and SF was dominantly induced by pH. It was shown that the complex films comprised mixtures of crystalline and non-crystalline regions.     

Enzyme immobilization on poly(ethylene-co-acrylic acid) films studied by quartz crystal microbalance with dissipation monitoring

In this study, we use the quartz crystal microbalance with dissipation monitoring (QCM-D) to study the immobilization of the enzyme horseradish peroxidase (HRP) on poly(ethylene-co-acrylic acid) (PEAA) films. The surface polarity of spin-coated PEAA films was varied by heat treatments in air or in a 30% NaOH aqueous solution leading to COOH-depleted or COOH-enriched surfaces, respectively. Two reaction schemes, direct adsorption and amine coupling, were employed for HRP immobilization on the two surfaces. The shifts in frequency and dissipation, Deltaf and DeltaD, measured by QCM-D and the ratio DeltaD/Deltaf were used to evaluate the binding amount and the conformation of the adsorbed enzyme. It is found that HRP immobilized via covalent linkages forms rigid and little dissipative films. In contrast, directly adsorbed HRP films exhibit a highly dissipative structure. HRP-catalyzed oxidation of the 4-chloro-1-naphthol in the presence of H(2)O(2) was used to characterize the catalytic activity of the HRP films. The results show that the enzymatic activity of the covalently immobilized HRP tends to be higher.     

XPS and wettability characterization of modified poly(lactic acid) and poly(lactic/glycolic acid) films

Poly(lactic acid) (PLA) and poly(lactic/glycolic acid) copolymers (PLGA) are biodegradable drug carriers of great importance, although successful pharmaceutical application requires adjustment of the surface properties of the polymeric drug delivery system to be compatible with the biological environment. For that reason, reduction of the original hydrophobicity of the PLA or PLGA surfaces was performed by applying a hydrophilic polymer poly(ethylene oxide) (PEO) with the aim to improve biocompatibility of the original polymer. PEO-containing surfaces were prepared by incorporation of block copolymeric surfactants, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (Pluronic), into the hydrophobic surface. Films of polymer blends from PLA or PLGA (with lactic/glycolic acid ratios of 75/25 and 50/50) and from Pluronics (PE6800, PE6400, and PE6100) were obtained by the solvent casting method, applying the Pluronics at different concentrations between 1 and 9.1% w/w. Wettability was measured to monitor the change in surface hydrophobicity, while X-ray photoelectron spectroscopy (XPS) was applied to determine the composition and chemical structure of the polymer surface and its change with surface modification. Substantial reduction of surface hydrophobicity was achieved on both the PLA homopolymer and the PLGA copolymers by applying the Pluronics at various concentrations. In accordance with the wettability changes the accumulation of Pluronics in the surface layer was greatly affected by the initial hydrophobicity of the polymer, namely, by the lactide content of the copolymer. The extent of surface modification was also found to be dependent on the type of blended Pluronics. Surface activity of the modifying Pluronic component was interpreted by using the solubility parameters.     

Light olefins/paraffins sorption in poly(lactic acid) films

The sorption behavior of small molecules like ethane and ethylene in poly (lactic acid) (PLA) was studied in the temperature interval from 283 to 313 K using a Quartz Crystal Microbalance (QCM). The effect of the polymer structure on the solubility selectivity of PLA films with respect to these two gases was studied using polymer with two different L:D ratios (98:2 and 80:20). Furthermore, the polymer films were submitted to different thermal treatments to address the influence of crystallinity and morphology of the noncrystalline fraction on the sorption behavior. The sorption results obtained indicate that ethylene solubility coefficient in annealed PLA 98:2 is about 26% higher than that of ethane and 41% higher in PLA 98:2 melted. The dual‐mode sorption model describes well the sorption isotherms behavior, which is concave concerning the pressure axis. The fully amorphous PLA presents the better selectivity for the studied gases, since the crystallinity seems to produce a negative effect on the selectivity. © 2008 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 46: 1312–1319, 2008     

Polarized fluorescence spectra of poly(ethylene terephthalate) films

The polarized excitation and fluorescence spectra of lowcrystalline, isotropic poly(ethylene terephthalate) (PET) film samples were measured in the glassy state at room temperature. Whereas the emission anisotropy r₀ of the excitation spectrum, recorded at the fluorescence maximum, changes sharply from 0.35 to 0 with decreasing wavelength in the region around 317 nm, the polarization of the fluorescence spectrum of PET is independent of wavelength. The fluorescence polarization of PET remains constant, if the temperature is increased up to 22 °C above T_g until the light scattering due to the crystallization causes complete depolarization. The photophysical behaviour supports the existence of a dilute solution of groundstate - stable sandwich dimers in the non-crystalline regions of PET.     

Photochromic inorganic-organic multilayer films based on polyoxometalates and poly(ethylenimine)

Novel photochromic inorganic-organic multilayers composed of polyoxometalates and poly(ethylenimine) have been prepared by the layer-by-layer (LbL) self-assembly method. The growth process, composition, surface topography, and photochromic properties of the multilayer films were investigated by UV-visible and Fourier transform infrared spectroscopy, atomic force microscopy, electrospin resonance (ESR), and X-ray photoelectron spectroscopy (XPS). Irradiated with ultraviolet light, the transparent films changed from colorless to blue. Moreover, the blue films showed good reversibility of photochromism and could recover the colorless state gradually in air, where oxygen plays an important role in the bleaching process. On account of the ESR and XPS results, parts of W6+ in multilayers were reduced to W5+, which exhibited a characteristic blue; a possible photochromic mechanism can be speculated. This work provides basic guideline for the assembly of multilayers with photochromic properties.     

Fabrication and characterization of multilayer films from amphiphilic poly(p-phenylene)s

Over the past two decades, considerable efforts have been devoted to the development of conjugated polymeric materials for electronic applications due to the tunability of their properties through variation of their chemical structure. The LB technique is one of the most effective and precise methods for controlling the organization and thereby the properties of polymer films at the nanoscale for device fabrication. A detailed study was performed on the Langmuir-Schaefer (LS) monolayer and Langmuir-Blodgett-Kuhn (LBK) multilayer formation of newly designed conjugated poly(p-phenylene)s (C(n)PPPOH), incorporated with alkoxy groups with different chain lengths (C(6)H(13)O-, C(12)H(25)O-, and C(18)H(37)O-) and hydroxyl groups on the polymer backbone. The monolayer formed at the air-water interface was characterized using surface pressure-area isotherms, including hysteresis measurements. The films were then transferred to different hydrophilic solid substrates and analyzed using surface plasmon resonance spectroscopy, UV-vis spectroscopy, fluorescence spectroscopy, and AFM measurements. The results showed that the polymer with a short alkoxy chain (C(6)PPPOH) forms uniform monolayers at the air-water interface and can be transferred as multilayer films compared to C(12)PPPOH and C(18)PPPOH. The observed film thicknesses measured by SPR and AFM studies were similar to the theoretical value obtained in the case of C(6)PPPOH, whereas this was not the case with the other two polymers. The present study shows that the polymer C(n)PPPOH with short alkoxy chain can be transferred onto different solid substrates for device fabrication with molecular level control.     

Photocurrent generation in multilayer self-assembly films fabricated from water-soluble poly(phenylene vinylene)

A novel, water-soluble, cationic PPV derivative poly[(2,5-bis(3-bromotrimethylammoniopropoxy)-phenylene-1,4-divinylene)-alt-1,4-(2,5-bis(2-(2-hydroxyethoxy)ethoxy))phenylene vinylene] (BH-PPV) has been synthesized by a Heck coupling reaction. Multilayered assemblies of the BH-PPV and the sodium salt of hexa(sulfobutyl)fullerenes (C(60)-HS) were fabricated successfully by an alternate deposition technique. The multilayer structures were studied by UV/Vis spectroscopy, small angle X-ray diffraction, and atomic force microscopy. The photoinduced charge transfer property of the self-assembled multilayer film was also measured by a three-electrode cell technique. A steady and rapid cathodic 5.5 microA cm(-2) photocurrent response was measured as the irradiation of the multilayer film was switched on and off. Importantly, the response of on/off cycling is prompt and reproducible. A possible mechanism for the electron-transfer process is proposed.     

Enhanced interlayer interaction in cellulose single nanofibre and poly(l-lactic acid) layered films by plasma-initiated surface grafting of poly(acrylic acid) onto poly(l-lactic acid) films

The surface of a poly(l-lactic acid) (PLLA) film was modified with poly(acrylic acid) (PAA) by plasma-initiated polymerization to increase the interaction between PLLA and cellulose single nanofibres (CSNF). The surface wettability of the PAA grafted PLLA film (PLLA-PAA film) was investigated by contact angle measurements. Modification of the PLLA film with PAA decreased the contact angle from 61° to 50°. The surface morphologies of the PLLA film, PLLA-PAA film and CSNF-coated PLLA-PAA film were studied by atomic force microscopy. The interaction between the CSNF and PLLA layers was strengthened by incorporation of a PAA layer onto the PLLA films and it is higher than 2N as proved by a peeling test. This is probably because the carboxyl groups of PAA form hydrogen bonds with the hydroxyl groups of CSNF.     

Surface roughness in Langmuir-Blodgett multilayer films studied by AFM and X-ray diffraction

Langmuir-Blodgett multilayer films consisting of up to 100 layers of cadmium-substituted arachidic acid on silicon wafers have been studied. The samples were deposited from a water subphase containing Cd²⁺ ions by using an alternate-layer Langmuir-Blodgett (LB) trough under computer control. Long-range orientational order was observed by atomic force microscopy on the surface of films with a small number of layers. X-ray reflectivity curves showed regularly spaced Bragg peaks arising from multilayer structure. The periodicity of the LB film is in agreement with twice the estimated molecular length, which confirms that the multilayer structure has a bilayer (Y) configuration. We demonstrate that the growth process of the LB films must have been stochastic. Our results are in good agreement with the Eden model of growth.     

Layer-by-layer self-assembly and electrocatalytic properties of poly(ethylenimine)-silicotungstate multilayer composite films

Hybrid multilayer films composed of poly(ethylenimine) and the Keggin-type polyoxometalates [ SiW₁₁O₃₉ ]^{8 -} ( SiW₁₁ ) {\left[ {{\hbox{Si}}{{\hbox{W}}_{{11}}}{{\hbox{O}}_{{39}}}} \right]^{{8} - }}\left( {{\hbox{Si}}{{\hbox{W}}_{{11}}}} \right) and [ SiW₁₁Co^II( H₂O )O₃₉ ]^{6 -} ( SiW₁₁Co ) {\left[ {{\hbox{Si}}{{\hbox{W}}_{{11}}}{\hbox{C}}{{\hbox{o}}^{\rm{II}}}\left( {{{\hbox{H}}_2}{\hbox{O}}} \right){{\hbox{O}}_{{39}}}} \right]^{{6} - }}\left( {{\hbox{Si}}{{\hbox{W}}_{{11}}}{\hbox{Co}}} \right) were prepared on glassy carbon electrodes by layer-by-layer self-assembly, and were characterized by cyclic voltammetry and scanning electron microscopy. UV-vis absorption spectroscopy of films deposited on quartz slides was used to monitor film growth, showing that the absorbance values at characteristic wavelengths of the multilayer films increase almost linearly with the number of bilayers. Cyclic voltammetry indicates that the electrochemical properties of the polyoxometalates are maintained in the multilayer films, and that the first tungsten reduction process for immobilized SiW₁₁ and SiW₁₁Co is a surface-confined process. Electron transfer to [ Fe( CN )₆ ]^{3 - /4 -} {\left[ {{\hbox{Fe}}{{\left( {\hbox{CN}} \right)}_6}} \right]^{{3} - /{4} - }} and [ Ru( NH₃ )₆ ]^{3 + /2 +} {\left[ {{\hbox{Ru}}{{\left( {{\hbox{N}}{{\hbox{H}}_3}} \right)}_6}} \right]^{{3} + /{2} + }} as electrochemical probes was also investigated by cyclic voltammetry. The (PEI/SiW₁₁Co)_n multilayer films showed excellent electrocatalytic reduction properties towards nitrite, bromate and iodate.