RESUMEN
The film formation of novel ω-aminoalkylcellulose carbamates on gold surface was studied by means of quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM). The influence of the pH value of the buffer solution, the concentration, the degree of polymerization, and the structure (spacer length) of the polymers on the coating was investigated. The layer formation was explained based on the pKa value and the degree of substitution of the ω-aminoalkylcellulose carbamates determined by potentiometric titration. This work provides novel supporting materials that might be applied in field of immobilization of biomolecules.
Asunto(s)
Carbamatos/química , Celulosa/química , Adsorción , Oro/química , Concentración de Iones de Hidrógeno , Microscopía de Fuerza Atómica , Tecnicas de Microbalanza del Cristal de Cuarzo , Relación Estructura-Actividad , Propiedades de SuperficieRESUMEN
The use of cationic biopolymer surfaces for high protein binding affinity matrices is described. As model proteins, fluorescently labeled bovine serum albumins (FITC-BSA, TRITC-BSA) have been employed. The amount of proteins on such cationically rendered surfaces was quantified by QCM-D. In addition, flexible, transparent, patterned COP slides have been prepared and loaded with proteins ranging from 15 pM to 15 µM TRITC-BSA.
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Biopolímeros/química , Celulosa/química , Quitosano/química , Fluoresceína-5-Isotiocianato/análogos & derivados , Albúmina Sérica Bovina/química , Adsorción , Fluoresceína-5-Isotiocianato/química , Rodaminas/químicaRESUMEN
A cutinase from Thermomyces cellullosylitica (Thc_Cut1), hydrolyzing the synthetic polymer polyethylene terephthalate (PET), was fused with two different binding modules to improve sorption and thereby hydrolysis. The binding modules were from cellobiohydrolase I from Hypocrea jecorina (CBM) and from a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PBM). Although both binding modules have a hydrophobic nature, it was possible to express the proteins in E. coli . Both fusion enzymes and the native one had comparable kcat values in the range of 311 to 342 s(-1) on pNP-butyrate, while the catalytic efficiencies kcat/Km decreased from 0.41 s(-1)/ µM (native enzyme) to 0.21 and 0.33 s(-1)/µM for Thc_Cut1+PBM and Thc_Cut1+CBM, respectively. The fusion enzymes were active both on the insoluble PET model substrate bis(benzoyloxyethyl) terephthalate (3PET) and on PET although the hydrolysis pattern was differed when compared to Thc_Cut1. Enhanced adsorption of the fusion enzymes was visible by chemiluminescence after incubation with a 6xHisTag specific horseradish peroxidase (HRP) labeled probe. Increased adsorption to PET by the fusion enzymes was confirmed with Quarz Crystal Microbalance (QCM-D) analysis and indeed resulted in enhanced hydrolysis activity (3.8× for Thc_Cut1+CBM) on PET, as quantified, based on released mono/oligomers.
Asunto(s)
Actinomycetales/enzimología , Hidrolasas de Éster Carboxílico/metabolismo , Tereftalatos Polietilenos/química , Proteínas Recombinantes de Fusión/metabolismo , Adsorción , Sitios de Unión , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/genética , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Hidrólisis , Modelos Moleculares , Tereftalatos Polietilenos/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Partially and fully regenerated cellulose model films from trimethylsilyl cellulose (TMSC) were prepared by a time dependent regeneration approach. These thin films were characterized with contact angle measurements and attenuated total reflectance infrared spectroscopy (ATR-IR). In order to get further insights into the completeness of the regeneration we studied the interaction of cellulase enzymes from Trichoderma viride with the cellulose films using a quartz crystal microbalance with dissipation (QCM-D). To support the results from the QCM-D experiments capillary zone electrophoresis (CZE) and atomic force microscopy (AFM) were applied. The changes in mass and energy dissipation due to the interaction of the enzymes with the substrates were correlated with the surface wettability and elemental composition of the regenerated films. The highest interaction activity between the films and the enzyme, as well as the highest cellulose degradation, was observed on fully regenerated cellulose films, but some degradation also occurred on pure TMSC films. The enzymatic degradation rate correlated well with the rate of regeneration. It was demonstrated that CZE can be used to support QCM-D data via the detection of enzyme hydrolysis products in the eluates of the QCM-D cells. Glucose release peaked at the same time as the maximum mass loss was detected via QCM-D. It was shown that a combination of QCM-D and CZE together with enzymatic digestion is a reliable method to determine the conversion rate of TMSC to cellulose. In addition QCM-D and AFM revealed that cellulase is irreversibly bound to hydrophobic TMSC surfaces, while pure cellulose is digested almost completely in the course of hydrolysis.
Asunto(s)
Biocatálisis , Celulosa/química , Compuestos de Trimetilsililo/química , Materiales Biocompatibles/química , Celulasa/química , Celulosa/análogos & derivados , Electroforesis Capilar , Proteínas Fúngicas/química , Glucosa/química , Hidrólisis , Interacciones Hidrofóbicas e Hidrofílicas , Ensayo de Materiales/métodos , Microscopía de Fuerza Atómica , Microscopía de Interferencia , Modelos Biológicos , Tecnicas de Microbalanza del Cristal de Cuarzo/métodos , Factores de Tiempo , Trichoderma/enzimología , HumectabilidadRESUMEN
In presented study a new approach using QCM-D for biocompatibility determination was introduced. The adsorption of fibrinogen on PET and modified PET surfaces was monitored in situ using QCM-D. Protein layer thicknesses were estimated on the basis of a Voight based viscoelastic model. The hydrophilicities and morphologies of the surfaces were investigated using a goniometer and AFM. The results showed that PET surfaces coated with sulphated polysaccharides are more hydrophilic and more fibrinogen-repulsive than non-modified PET surfaces. QCM-D equipped with QTools modelling software is well-applicable to the characterisation of surface properties and can be optimised for biocompatibility determination.
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Fibrinógeno/química , Polietilenglicoles/química , Tecnicas de Microbalanza del Cristal de Cuarzo/métodos , Programas Informáticos , Sustancias Viscoelásticas/análisis , Adsorción , Anticoagulantes/química , Materiales Biocompatibles/química , Humanos , Ensayo de Materiales/métodos , Microscopía de Fuerza Atómica , Modelos Moleculares , Tereftalatos Polietilenos , Polisacáridos/química , Propiedades de Superficie , Sustancias Viscoelásticas/químicaRESUMEN
The preparation of thin films of chitosan-silane hybrid materials by combining sol-gel processing and spin coating is reported. A variety of silanes can be used as starting materials for the preparation of such thin films, namely tetraethoxysilane, tri-tert-butoxysilanol, trimethylethoxysilane, p-trifluoromethyltetra-fluorophenyltriethoxysilane, trivinylmethoxysilane, (methoxymethyl)trimethyl-silane, and hexamethoxydisilane. These silanes are subjected to a sol-gel process before they are added to acidic chitosan solutions. The chitosan:silane ratio is kept constant at 6:1 (w/w) and dilutions with ethanol are prepared and spin coated. Depending on the degree of dilution, film thickness can be controlled in a range between 5 and 70 nm. For the determination of additional surface properties, static water contact angle measurements and atomic force microscopy have been employed.
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Materiales Biocompatibles/síntesis química , Quitosano/química , Nanotecnología/métodos , Silanos/química , Animales , Materiales Biocompatibles/química , Braquiuros/química , Geles/síntesis química , Geles/química , Microscopía de Fuerza Atómica , Nanoestructuras/química , Reproducibilidad de los Resultados , Propiedades de Superficie , Factores de TiempoRESUMEN
The adsorption behavior of cellulose-4-[N-methylammonium]butyrate chloride (CMABC) on two hydrophilic substrates is studied, namely nanometric cellulose model thin films and silicon dioxide substrates. The adsorption is quantified in dependence of electrolyte concentration and pH value using a quartz crystal microbalance with dissipation (QCM-D). In case of CMABC, at high ionic strengths (25-100 mM NaCl) high adsorption is observed at pH 7 (Δf(3): -15 to -17 Hz) while at lower ionic strengths (1-10 mM) less CMABC (Δf(3): -2 to -12 Hz) is deposited on the cellulose surfaces as indicated by the frequency changes using QCM-D. A change in pH value from 7 to 8 reveals an increase in adsorption. Atomic force microscopy shows that the coating of cellulose thin films with CMABC changes the morphology from a fibrillar to a particle like structure on the surface. The surface wettability with water increases with an increasing amount of CMABC on the surface compared to neat cellulose model films. At lower pH values (3 and 5), CMABC does not adsorb onto the cellulose model thin films. XPS is used to validate the results and to determine the nitrogen content of the surfaces. In addition, adsorption of CMABC onto another hydrophilic and negatively charged substrate, silicon dioxide coated quartz crystals, cannot be detected at different pH values and electrolyte concentrations as proven by QCM-D.
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Aminobutiratos/química , Celulosa/análogos & derivados , Celulosa/química , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Dióxido de Silicio/químicaRESUMEN
Small angle X-ray scattering (SAXS) is employed to characterize the inner structure and shape of aqueous nanocrystalline cellulose suspensions using the generalized indirect Fourier transformation (GIFT). The use of the GIFT approach provides a single fitting procedure for the determination of intra- and interparticle interactions due to a simultaneous treatment of the form factor P(q) and the structure factor S(q). Moreover, GIFT allows for the determination of particle charges and polydispersity indices. As test material, aqueous nanocrystalline cellulose suspensions (aNCS) prepared by the H2SO4 route have been investigated and characterized (SAXS, dynamic light scattering, zeta potential).
RESUMEN
The adsorption of carboxymethyl cellulose (CMC), one of the most important cellulose derivatives, is crucial for many scientific investigations and industrial applications. Especially for surface modifications and functionalization of materials, the polymer is of interest. The adsorption properties of CMC are dependent not only on the solutions state, which can be influenced by the pH, temperature, and electrolyte concentration, but also on the chemical composition of the adsorbents. We therefore performed basic investigation studies on the interaction of CMC with a variety of polymer films. Thin films of cellulose, cellulose acetate, deacetylated cellulose acetate, polyethylene terephthalate, and cyclo olefin polymer were therefore prepared on sensors of a QCM-D (quartz crystal microbalance) and on silicon substrates. The films were characterized with respect to the thickness, wettability, and chemical composition. Subsequently, the interaction and deposition of CMC in a range of pH values without additional electrolyte were measured with the QCM-D method. A comparison of the QCM-D results showed that CMC is favorably deposited on pure cellulose films and deacetylated cellulose acetate at low pH values. Other hydrophilic surfaces such as silicon dioxide or polyvinyl alcohol coated surfaces did not adsorb CMC to a significant extent. Atomic force microcopy confirmed that the morphology of the adsorbed CMC layers differed depending on the substrate. On hydrophobic polymer films, CMC was deposited in the form of larger particles in lower amounts whereas hydrophilic cellulose substrates were to a high extent uniformly covered by adsorbed CMC. The chemical similarity of the CMC backbone seems to favor the irreversible adsorption of CMC when the molecule is almost uncharged at low pH values. A selectivity of the cellulose CMC interaction can therefore be assumed. All CMC treated polymer films exhibited an increased hydrophilicity, which confirmed their modification with the functional molecule.
RESUMEN
A method for the immobilization of functional molecules on cellulose surfaces was developed. The irreversible deposition of the water-soluble polyelectrolyte carboxymethyl cellulose (CMC) on solid cellulose surfaces was used as a basis for this immobilization. CMC was modified using aminofluorescein (AMF) as a model compound for a functional molecule. The carbodiimide mediated coupling efficiency of AMF to CMC was studied in detail, and the functional conjugates were isolated. A quartz crystal microbalance with dissipation was employed to study the immobilization of the functionalized CMC onto cellulose model films in situ. The influence of the carbodiimide concentration, the degree of substitution, and the molecular weight of CMC on the immobilization process was investigated. Atomic force microscopy was used to characterize the changes in the surface morphology of the modified cellulose films. Finally, microspotted arrays of AMF-CMC conjugates were prepared with the knowledge obtained from the basic interaction studies. The successful deposition of AMF-CMC conjugates onto cellulose surfaces was proven by fluorescence microscopy. The conjugation of functional molecules to CMC and the subsequent deposition of these products on cellulose can be seen as a versatile method to immobilize these molecules for applications in the field of microarrays and other sensor surfaces. It offers the possibility to introduce new properties on a variety of cellulosic materials.
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Owing to the complex influences of several experimental conditions on the in vitro alteration of blood, there is still a lack of viable in vitro tests and methods for blood compatibility evaluation of biomaterials. The aim of this research was to study a new approach for the haemocompatibility assessment of differently modified PET surfaces using the quartz crystal microbalance with dissipation unit (QCM-D) technique and measure the mass increase caused by clot formation under physiological conditions. For this purpose some of the most frequently applied in vitro methods for haemocompatibility determination, i.e., clotting time measurement and observation of red blood cells' mobility, were applied and their accuracy and sensitivity compared to the new QCM-D approach. Haemocompatibility was evaluated for non-modified poly(ethylene terephthalate) (PET) surfaces and PET surfaces coated with dextran sulphate and heparin. The basic anti-coagulant properties of heparin and dextran sulphate were analysed by means of their activated partial thromboplastine time (APTT). PET, as well as different polysaccharides coatings were chosen for this study due to their promising biocompatible properties and numerous possibilities for biomedical applications. The results showed that the new QCM-D technique to study clot formation in contact with PET surfaces under physiological environment was the most informative and accurate for in vitro haemocompatibility assessment. Although the results achieved with the other two methods were in good correlation, they did not provide such a high level of sensitivity.
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Ensayo de Materiales/métodos , Tereftalatos Polietilenos/efectos adversos , Tereftalatos Polietilenos/química , Tecnicas de Microbalanza del Cristal de Cuarzo/métodos , Adsorción/efectos de los fármacos , Animales , Coagulación Sanguínea/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Eritrocitos/citología , Eritrocitos/efectos de los fármacos , Fibrinógeno/química , Humanos , Masculino , Tiempo de Tromboplastina Parcial , Polisacáridos/química , Sulfatos/química , Propiedades de Superficie , Adulto JovenRESUMEN
The wettability and surface free energy (SFE) of partly and fully regenerated cellulose model surfaces from spin coated trimethylsilyl cellulose were determined by static contact angle (SCA) measurements. In order to gain detailed insight into the desilylation reaction of the surfaces the results from SCA measurements were compared with data from other surface analytical methods, namely thickness measurements, X-ray photoelectron spectroscopy (XPS) and attenuated total reflectance infrared spectroscopy (ATR-IR). Additionally, the influence of ultra high vacuum treatment (UHV) during XPS measurements on the water wettability and surface morphology of regenerated cellulose thin films was investigated. The wetting of polar and non-polar liquids increased with prolonged regeneration time, which is reflected in the higher SFE values and polarities of the films. After UHV treatment the water SCA of partly regenerated films decreases, whereas fully regenerated cellulose shows a higher water SCA. Therefore it is assumed that volatile desilylation products tend to adsorb on partly regenerated films, which strongly influences their wettability.
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Celulosa/química , Reciclaje , Celulosa/análogos & derivados , Propiedades de Superficie , Compuestos de Trimetilsililo/química , Vacio , HumectabilidadRESUMEN
In the present study, blood-compatible PET surfaces were prepared by coating with anticoagulant cellulose sulfates that were synthesized homogeneously in ionic liquids. The adsorption behavior of polysaccharides on PET films was investigated using QCM-D. It was demonstrated that pre-coating with different amino-group-containing polysaccharides improves the affinity toward cellulose sulfate. Moreover, the effect of different degrees of sulfation on the adsorption process was evaluated. Based on these results, several layer-by-layer coated PET foils were prepared that showed significantly improved blood compatibility compared to the initial untreated material.
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Anticoagulantes/química , Celulosa/análogos & derivados , Materiales Biocompatibles Revestidos/química , Ensayo de Materiales , Polietilenglicoles/química , Adulto , Celulosa/química , Humanos , Masculino , Tereftalatos PolietilenosRESUMEN
The determination of pH in highly alkaline solutions and concrete materials is extremely important for monitoring or predicting the corrosion processes of reinforced concrete structures and to follow the hydration process of Portland cement, fly-ash, micro silica and other materials used in concrete manufacturing. The corrosion of reinforced concrete structures and the hydration of pozzolanic materials are long-term processes, which means, that appropriate durable, and resilient pH electrodes are needed, for direct implantation regarding solid concrete bodies. The purpose of this work was to characterise the potentiometric and surface properties of tungsten electrodes after exposure to extreme alkaline solutions. The tungsten wire surface was activated at 800 °C for 30 min within an oxygen flow. The formation of homogenous and compact multiple layers of WO3 crystals was observed using X-ray diffraction and scanning electron microscopy. X-ray diffraction of those tungsten electrodes exposed to saturated calcium hydroxide solution or the pore-water of cement-based materials during 10 months, indicated partly dissolved WO3. Two new compounds appeared on the electrodes surfaces; pure tungsten and CaWO4. The presence of tungsten was affecting any potentiometric response in acidic pH region (2-5) but in pH 5-12 region the response still remained linear with a slope of 42 ± 2 mV/pH unit. The W/WO3 electrode was suitable for the long-term monitoring of corrosion processes in concrete-based materials according to the pH changes as it has stable and repeatable responses to alkaline solutions (pH > 12). All the tested interferring ions had no significant influence on electrode potential. The W/WO3 electrode is simple, robust, inexpensive, and temperature resistant and can be applied in potentiometric titrations as well as in batch and flow-injection analysis. The prepared electrode is a highly promising pH sensor for the monitoring of pH changes in highly alkaline capillary water of concrete.
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The objective of the presented research was to examine the potential of capillary electrophoresis (CE) for the analysis of structurally different reactive dyestuffs, their activation and posterior hydrolysis, with special focus on optimization of the working conditions. Preliminary, the effect of various additives to the background electrolyte on the resolution improvements versus migration time of Reactive Black 5 as a model dyestuff was investigated. Based on these results, the electropherograms of eight commercially interesting reactive dyestuffs of various chemical structures and their converted forms upon alkaline pH were carried-out. In order to examine the behavior of the reactive dyestuff during the dyeing process, the dye-bath absorbance was monitored throughout the Reactive Black 5 exhaustion, and the conversion of the dye's form was highlighted using the CE technique. The obtained results unequivocally prove that CE could offer a fast and efficient detection method of structurally different reactive dyestuffs, as well as their hydrolysis products in the dye-baths and effluents later on.