Fluorescence spectroscopic studies to characterize the internal environment of tetraethyl-orthosilicate derived sol-gel bulk and thin films with aging

Characterization of the internal environment of a sol-gel matrix is an important area of investigation in optical biosensors. In the present study, different sol-gel compositions were prepared by varying the water (H2O) to tetraethyl-orthosilicate (TEOS) ratio (R) from 1 to 16 and the changes in the internal environment of the sol-gel both in bulk and thin films as a function of aging (storage) were investigated using fluorescence spectroscopy. We focussed on the fluorescence characteristics , viz. emission and excited state lifetime of Hoechst 33258 (H258), a bisbenzimidazole derivative, which was used as fluorescence probe entrapped in the TEOS derived sol-gel bulk and thin films. These sols were prepared at a low pH (approximately 2.0) and the thin films were coated by dip coating technique at withdrawal speeds of 1 cm/min and 0.1cm/min. Usually, uniform thin films were obtained at a high speed (1 cm/min) and partially cracked film at a low speed (0.1 cm/min) as observed by fluorescence microscope. These observations did not change during aging. On the contrary, three months long observations on steady-state fluorescence emission measurements on H258 depicted a blue shift from 535 nm to 508 nm at R = 1 in the sol-gel bulk, whereas at higher ratios this was not prominent. At all ratios, dual emission bands were observed in thin films. This may be due to faster sol-gel to xerogel transition during aging depending on the ratio (R). Analysis of the excited state decay profiles of H258 revealed a double exponential fitting having a short (tau1) and a long (tau2) component in both fresh and during aging, in the sol-gel bulk and thin films, indicating heterogeneity in the internal environment. The value of tau1 increased from 0.4 ns to 1.2 ns whereas tau2 attained a value from 3.0 ns to 3.6 ns at R = 1 upon aging in the sol-gel bulk. The corresponding values of tau1 and tau2 in thin films were 0.3 ns and 3.5 ns, respectively. The values of these decay components in thin films did not alter much due to storage, but their relative contributions showed more systematic changes in the thin films. The observed changes could be correlated to rigidification in the bulk depending on the ratio (R). This process was very slow at R > or = 4. The heterogeneity in the internal environment of bulk and thin films upon aging appeared to be different as revealed from analysis of excited-state lifetime. Thus, the bisbenzimidazole derivative H258 appears to be very useful probe for characterizing the internal environment of both the sol-gel bulk and thin films.     

Durable cofactor immobilization in sol-gel bio-composite thin films for reagentless biosensors and bioreactors using dehydrogenases

A new strategy directed to the durable immobilization of NAD(+)/NADH cofactors has been tested, along with a suitable redox mediator (ferrocene), in biocompatible sol-gel matrices encapsulating a bi-enzymatic system (a dehydrogenase and a diaphorase, this latter being useful to the safe regeneration of the cofactor), which were deposited as thin films onto glassy carbon electrode surfaces. It involves the chemical attachment of NAD(+) to the silica matrix using glycidoxypropylsilane in the course of the sol-gel process (in smooth chemical conditions). This approach based on chemical bonding of the cofactor (which was checked by infrared spectroscopy) led to good performances in terms of long-term stability of the electrochemical response. The possibility to integrate all components (proteins, cofactor, mediator) in the sol-gel layer in an active and durable form gave rise to reagentless devices with extended operational stability (i.e. high amperometric response maintained for more than 12h of continuous use under constant potential, whereas the signal completely vanished within the first few minutes of working with non-covalently bonded NAD(+)). To confirm the wide applicability of the proposed approach, the same strategy has been applied to the elaboration of biosensors for D-sorbitol, D-glucose and L-lactate with using D-sorbitol dehydrogenase, D-glucose dehydrogenase and L-lactate dehydrogenase respectively. The analytical characteristics of the glucose sensors are given and compared to previous approaches described in the literature for the elaboration of reagentless biosensors.     

Effect of ethanol on tropomyosin in solution and in reconstituted thin filaments

The effects of ethanol at concentrations below 10% on the conformation of tropomyosin, its end-to-end polymerization, its binding to F-actin, and its effects on actomyosin ATPase activity were studied. Ethanol stabilized the tropomyosin conformation by shifting the helix thermal unfolding profile to higher temperatures, and increased the end-to-end polymerization of tropomyosin. Ethanol-induced changes in the excimer fluorescence of pyrene-tropomyosin indicated that its conformation was stabilized by ethanol both free and bound to F-actin. Effects of tropomyosin and tropomyosin-troponin on actomyosin ATPase activity were measured under conditions for which tropomyosin binding to F-actin increases the activity. Under conditions for which the binding of tropomyosin to F-actin is optimum, in the presence of tropomyosin, the actomyosin ATPase activity decreased as the ethanol concentration increased, further indicating that ethanol induces a structural change in the tropomyosin-F-actin complex. Under conditions for which the binding of tropomyosin to F-actin is weak (low salt or high temperature), addition of ethanol increased the ATPase activity due to increased binding of tropomyosin to F-actin. Thus, ethanol appears to modify actomyosin ATPase activity by increasing the binding of tropomyosin to F-actin and affecting the structure of tropomyosin in the tropomyosin-F-actin filament.     

Effect of calcium concentration on the structure of casein micelles in thin films

The structure of thin casein films prepared with spin-coating is investigated as a function of the calcium concentration. Grazing incidence small-angle x-ray scattering and atomic force microscopy are used to probe the micelle structure. For comparison, the corresponding casein solutions are investigated with dynamic light-scattering experiments. In the thin films with added calcium three types of casein structures, aggregates, micelles, and mini-micelles, are observed in coexistence with atomic force microscopy and grazing incidence small-angle x-ray scattering. With increasing calcium concentration, the size of the aggregates strongly increases, while the size of micelles slightly decreases and the size of the mini-micelles increases. This effect is explained in the framework of the particle-stabilizing properties of the hairy layer of kappa-casein surrounding the casein micelles.     

Effect of pressure on the sol-gel transition of carrageenans

The effects of pressure on the sol-gel transition of κ- and ι-carrageenans were studied in KCl solutions under high pressures up to 3000 kg/cm². The carrageenan gels were destabilized by pressure: the pressure depression of melting temperature, (dT/dP)_m, was ?5.7 × 10^?3 and ?4.0 × 10^?3 K cm²/kg independent of KCl concentration for κ- and ι-carrageenans, respectively. The enthalpy, entropy and volume changes accompanying the gel formation were calculated from the Eldridge-Ferry's plots and the Clausius-Clapeyron equation. The volume change per unit cross-link (two disaccharide residues) was estimated to be (2.5 ～ 4.9) and (1.7 ～ 3.4) ml/mol for κ- and ι-carrageenans, respectively. The compressibility of both carrageenan molecules appeared to be larger by (1.6 ～ 2.6) × 10^?12 (κ-form) and by (0.8 ～ 1.3) × 10^?12cm²/dyn (i-form) in gel state as compared with in sol state These increases in volume and compressibility on gelation were attributed to a reduction of water of hydration from the carrageenan molecules, which is mainly due to a replacement of the polymer-water hydrogen bond by the polymer-polymer hydrogen bond. These results seemed not inconsistent with the idea that a double helix structure of carrageenan gels may persist in solution as well as in the solid state.     

Laboratory and field studies on thin paint films

An account is presented of some aspects and results of a collaborative project currently being undertaken as part of the European Collaborative Action COST 520 programme in Working Group 3. The project involves the response of biocide- and non-biocide-containing thin paint films to microbial colonisation under laboratory and field conditions.From the results of exposure studies at four sites, two in the UK and two in Norway, it was found that some microorganisms were common to both locations.Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI TOF MS) was used in a novel way to establish characteristic mass spectral fingerprints of different fungal genera and different species of the same fungal genus. The results show that databases can be produced which provide convincing evidence for the application of this technique in taxonomy.Scanning electron microscopy was used to visualise Aureobasidium pullulans colonising the paint film. Evidence of hyphal penetration and disruption of the paint binder is suggested.Surface roughness measurement was used to investigate the effect of exposure on the surface topography of the paint. It was found that changes in the surface roughness increased over the duration of the experiment.Fourier transform infrared spectroscopy was used in the reflectance mode to detect chemical changes in the surface of the paint film. It was found that no positional or absorbance changes in the spectrum of the paint film were detected as a result of inoculating the film in a vermiculite bed system. However, some spectra did suggest that surface changes had occurred as a result of a reduction in diffuse scattering from the surface.     

Effect of magnesium ions on the structure of DNA thin films: an infrared spectroscopy study

Utilizing Fourier transform infrared spectroscopy we have investigated the vibrational spectrum of thin dsDNA films in order to track the structural changes upon addition of magnesium ions. In the range of low magnesium concentration ([magnesium]/[phosphate] = [Mg]/[P] < 0.5), both the red shift and the intensity of asymmetric PO₂ stretching band decrease, indicating an increase of magnesium-phosphate binding in the backbone region. Vibration characteristics of the A conformation of the dsDNA vanish, whereas those characterizing the B conformation become fully stabilized. In the crossover range with comparable Mg and intrinsic Na DNA ions ([Mg]/[P] ≈ 1) B conformation remains stable; vibrational spectra show moderate intensity changes and a prominent blue shift, indicating a reinforcement of the bonds and binding in both the phosphate and the base regions. The obtained results reflect the modified screening and local charge neutralization of the dsDNA backbone charge, thus consistently demonstrating that the added Mg ions interact with DNA via long-range electrostatic forces. At high Mg contents ([Mg]/[P] > 10), the vibrational spectra broaden and show a striking intensity rise, while the base stacking remains unaffected. We argue that at these extreme conditions, where a charge compensation by vicinal counterions reaches 92–94%, DNA may undergo a structural transition into a more compact form.     

Effect of pressure‐assisted thermal annealing on the optical properties of ZnO thin films

ZnO thin films were prepared by the polymeric precursor method. The films were deposited on silicon substrates using the spin‐coating technique, and were annealed at 330 °C for 32 h under pressure‐assisted thermal annealing and under ambient pressure. Their structural and optical properties were characterized, and the phases formed were identified by X‐ray diffraction. No secondary phase was detected. The ZnO thin films were also characterized by field‐emission scanning electron microscopy, Fourier transform infrared spectroscopy, photoluminescence and ultraviolet emission intensity measurements. The effect of pressure on these thin films modifies the active defects that cause the recombination of deep level states located inside the band gap that emit yellow–green (575 nm) and orange (645 nm) photoluminescence. Copyright © 2012 John Wiley & Sons, Ltd.     

Nonlinearity in the I-V characteristics in thin lipid films: Effect of AC and DC fields

The current-voltage characteristics of bilayer lipid membranes of oxidized cholesterol separating two bathing solutions have already been extensively studied under a DC electric field. The observed deviation from linearity at high field has been explained by field-induced pore formation, which then act as ion channels in the membrane. Using thin films of oxidized cholesterol and of dipalmitoyl phosphatidylcholine, we have reported for the first time similar deviation from nonlinearity in the DC I-V characteristics when the applied field is above 40 V/cm. Upon application of an AC field, the conductivity increases as square of frequency, while the nonlinear nature of the I-V characteristic curve is still retained at all frequencies up to 5,000 Hz. Our results indicate that besides pore formation, the intrinsic electrical properties of the constituent lipid molecules are also responsible for the observed nonlinearity.     

Biosensor based on enzyme-catalysed degradation of thin polymer films

A biosensor based on the enzyme-catalysed dissolution of biodegradable polymer films has been developed. Three polymer-enzyme systems were investigated for use in the sensor: a poly(ester amide), which is degraded by the proteolytic enzyme alpha-chymotrypsin; a dextran hydrogel, which is degraded by dextranase; and poly(trimethylene) succinate, which is degraded by a lipase. Dissolution of the polymer films was monitored by Surface Plasmon Resonance (SPR). The rate of degradation was directly related to enzyme concentration for each polymer/enzyme couple. The poly(ester amide)/alpha-chymotrypsin couple proved to be the most sensitive over a concentration range from 4 x 10(-11) to 4 x 10(-7) mol l(-1) of enzyme. The rate of degradation was shown to be independent of the thickness of the poly(ester amide) films. The dextran hydrogel/dextranase couple was less sensitive than the poly(ester amide)/alpha-chymotrypsin couple but showed greater degradation rates at low enzyme concentrations. Enzyme concentrations as low as 2 x 10(-11) mol l(-1) were detected in less than 20 min. Potential fields of application of such a sensor system are the detection of enzyme concentrations and the construction of disposable enzyme based immunosensors, which employ the polymer-degrading enzyme as an enzyme label.     

Replication of butterfly wing and natural lotus leaf structures by nanoimprint on silica sol-gel films

An original and low cost method for the fabrication of patterned surfaces bioinspired from butterfly wings and lotus leaves is presented. Silica-based sol-gel films are thermally imprinted from elastomeric molds to produce stable structures with superhydrophobicity values as high as 160 degrees water contact angle. The biomimetic surfaces are demonstrated to be tuned from superhydrophobic to superhydrophilic by annealing between 200 degrees C and 500 degrees C.     

Electron microscopic study of 3H-leucine transport between the internal body environment and the enteral environment

Using electron microscopic autoradiography, a space-time characteristics of 3H-leucine transport, from the enteral to the inner environment, and from blood plasma to the enteral environment, has been given. Some gradient of intracellular leucin accumulation is determined, from the tip cells of a villus to epithelocytes of the basal membrane. Amino acid transport from blood plasma to the lumen of the intestine is accomplished mainly via the cytoplasm and intercellular space of epithelocytes of the villus basement. It is proposed that amino acid transport through membranes of microvilli and through basal-lateral membranes of cells being on various levels along the villus may start at different amino acid concentrations which may be, presumably, to different concentration and different conjugation of enzyme-transport complexes in these membranes.     

The effect of 2,4-dinitrophenol on the properties of thin phospholipid films

Summary The properties of a system consisting of a thin phospholipid film separating two electrolyte solutions containing 1mm 2,4-dinitrophenol have been studied. Both the variation of electrical conductance as a function of pH, keeping the pH the same on both sides of the membrane, and the nonlinear variation of electrical potential difference as a function of pH difference across the membrane have been explained in terms of lipid-soluble complexes of the typeXP ₂ ^– whereX is a cation andP dinitrophenate. The maximum conductance was found to be 1.4×10^–5 mhos cm^–2 at pH 4.2.     

Effect of ethanol on glycylsarcosine absorption

Glycylsarcosine (GlySar) absorption by the rat intestine is not altered by acute ethanol administration (luminal perfusion of a 0.7 M ethanol solution) or by chronic consumption of a 15% ethanol solution in drinking water. Both total absorption, per entire rat small intestine, and specific absorption per mg dry weight of mucosa, were unaffected by ethanol. During the absorption of GlySar, glycine, produced by hydrolysis of the peptide in the cytosol of the intestinal cells, appears in the intestinal lumen. During acute ethanol administration the luminal appearance of glycine is decreased probably due to a reduction in intracellular hydrolysis of the dipeptide.