Las proteínas térmicamente inestables que están presentes en uvas, jugos de uva y vinos podrían llegar a ser insolubles y precipitar causando la formación de turbidez o precipitados indeseables en vinos blancos después del embotellado durante el almacenamiento.La turbidez proteica en vinos blancos es evitada tradicionalmente, mediante la adición de bentonita, aunque esta técnica presenta algunas desventajas tales como efectos negativos sobre las propiedades sensoriales del vino debido principalmente a la remoción de componentes aromáticos o gustativos y por la merma de vino, debido al gran aumento de volumen y poder de sedimentación de la bentonita. Además, el recurso humano, los tiempos de proceso y la descarga de residuos al ambiente sigue siendo una inmensa preocupación, debido a los significativos costos asociados a la salud y seguridad laboral, y así como también las responsabilidades y obligaciones legales de la industria en material de impacto ambiental. Es estimado que los costos del uso de la bentonita en la industria del vino a nivel mundial son del orden de los 300-500 millones de dólares por año. Por lo tanto es necesario el desarrollo de nuevas tecnologías alternativas a la bentonita que sean económicamente viables y que mantengan la calidad del vino, así como también la generación de un menor impacto ambiental. No obstante, nuevas técnicas exitosas a nivel industrial aun no han sido desarrolladas, por que afectan la calidad del vino o porque su aplicación no es viable económicamente bajo normales condiciones de operación de producción de vino. Por lo tanto, es muy atractivo investigar la viabilidad de nuevas prácticas que tengan un menor impacto sobre el ambiente y sean económicamente viables.Por esa razón la principal motivación de nuestra investigación ha sido estudiar la viabilidad industrial de una tecnología alternativa al uso de la bentonita, la cual permita remover proteínas inestables de los vinos blancos usando zirconia como material adsorbente. Además, nosotros nos hemos concentrado en el desarrollo de un proceso continuo que permita conseguir vinos estables proteicamente sin afectar sus propiedades fisicoquímicas y sensoriales, probando diferentes técnicas de regeneración del material adsorbente. Primeramente nosotros hemos estudiado la estructura, morfología y propiedades superficiales de la zirconia, así como también su capacidad de adsorción para remover proteínas inestables de vinos blancos, aplicando tratamientos regenerativos químicos y térmicos. Después hemos comparado las propiedades fisicoquímicas y sensoriales de un vino blanco estabilizado proteicamente mediante zirconia y bentonita.Además, nosotros hemos desarrollado un proceso híbrido integrando un proceso de adsorción en columna y un proceso de microfiltración tangencial de vino, para conocer los efectos de este nuevo proceso sobre el ensuciamiento de la membrana y la estabilidad proteica del vino.Por otro lado, nosotros también hemos aplicado un nuevo proceso de estabilización proteica de vino base para cava, comparando los resultados con el método tradicional usando bentonita como agente estabilizante y observando los efectos de ambos tratamientos sobre la calidad de la espuma y las fracciones proteicas del vino. Finalmente, hemos aplicado el nuevo proceso de estabilización proteica a escala industrial, empacando zirconia sobre una columna fija y realizando el proceso mediante sistema continuo y discontinuo.Los resultados demuestran que la zirconia puede ser regenerada química o térmicamente, que sus propiedades físicas, morfológicas y químicas no son alteradas y que incluso su capacidad de adsorción proteica podría ser aumentada probablemente producto de la adsorción de algunos componentes o centros activos derivados de las proteínas del vino. A través del proceso híbrido ha sido posible conseguir vinos estables proteicamente y aumentar la densidad de flujo del permeado durante la microfiltración del vino. De hecho, hemos observado que la reducción de proteínas mediante la adsorción en columna usando zirconia también ocurre durante la microfiltración tangencial, por lo tanto ambos procesos pueden actuar conjuntamente en la reducción y estabilización proteica.Por otro lado, comparando las propiedades fisicoquímicas y sensoriales de vinos blancos estabilizados proteicamente mediante zirconia y bentonita, podemos afirmar que los mejores resultados son conseguidos usando zirconia como agente estabilizante.La estabilización proteica de vinos blancos en continuo también puede ser útil para estabilizar vinos base para cava, ya que comparando la calidad de la espuma de un vino base para cava tratado con zirconia y bentonita, los resultados demuestran que la calidad de la espuma de aquellos vinos bases es mejor usando zirconia, ya que la adicción de bentonita produce considerables efectos negativos sobe la calidad de la espuma.Finalmente, los resultados obtenidos a escala industrial, muestran que es viable la estabilización proteica de vinos blancos usando zirconia como material adsorbente ya sea mediante un sistema continuo o discontinuo sin afectar la calidad del vino inicial. / Heat-unstable soluble proteins in grapes, grape juices and wines may become insoluble and precipitate causing the formation of undesirable hazes or deposits in white wines after bottling and during storage. Proteins are commonly prevented from forming hazes with bentonite, though this technique does have drawbacks: for example, the sensory properties of the wine are affected adversely because flavour compounds are removed and wine volume is lost as lees because of the swell and settling of the bentonite. In addition, the handling and disposal of spent bentonite continues to be a concern, because it involves high labour input and the associated costs, occupational health and safety issues, and the wine industry's environmental responsibilities and legislative requirements. It is estimated that the cost of bentonite fining to the wine industry worldwide is in the order of US$300-500 m per annum. Alternative fining technologies to bentonite, which are economically viable and maintain wine quality, are currently being sought. However, no successful techniques have been developed to date: all the attempts so far have either affected the quality of the wine or not been economically viable under standard winemaking conditions. Therefore, research on the implementation of new practices that have a less negative impact on the environment and are economically viable is particularly challenging.For this reason the aim of this thesis was to study the industrial viability of an alternative technology to bentonite fining which enables unstable proteins to be removed from white wines using zirconia as the adsorbent material. We also attempted to develop a continuous process, which stabilizes wine protein without having any negative effects on the physicochemical and sensory properties of the wine. Likewise we tried to make the process have a lower environmental impact by testing various regenerative treatments of the adsorbent material.First we studied the structure, morphology and surface properties of the zirconia and its capacity to remove unstable proteins from white wine versus thermal and chemical regeneration treatments. Subsequently we compared the physicochemical and sensory properties of a white wine fined by zirconia and bentonite. To further our understanding of the effect on membrane fouling and wine protein stability, we developed a hybrid process consisting of in-column adsorption with crossflow microfiltration. We also applied this new method to stabilize the base sparkling wine and compared the results with the conventional method of using bentonite as the fining agent to see the effects of the treatments on the foam quality and protein fractions. Finally, we applied the new method on an industrial scale by packing zirconia into a fixed bed column and by using the batch and continuous systems.The results show that the zirconia can be regenerated by thermal and chemical treatments, and that its physical, morphological and chemical properties are not altered. In fact its protein adsorption capacity can increase probably because some compounds or active centres derived from wine proteins are absorbed.The hybrid process was used to increase the permeate flux during crossflow microfiltration and stabilize wine proteins. We observed that proteins were reduced when the zirconia column adsorption was used during the crossflow microfiltration. Therefore both processes may act together.By comparing the physicochemical and sensory analyses of white wine proteins stabilized by zirconia and bentonite, we found that results were best when zirconia was used.The continuous protein stabilization of white wines by zirconia may also be useful for stabilizing proteins in base sparkling wines. Treating base sparkling wines with zirconia definitely gives better foam quality than with bentonite. Finally the results obtained in our industrial scale experiment showed that white wine continuous protein stabilization with zirconia as the adsorbent material is not only viable in both the continuous and batch systems, it also leaves the quality of the wine unchanged.

In this work, a gradient of interpenetrating polymer networks, consisting of anionic and cationic polymers, has been investigated with respect to protein resistant properties and swelling characteristics at different pH and ionic strength conditions. The swelling and protein adsorption have been studied using \emph{in situ} spectroscopic ellipsometry(SE) and imaging surface plasmon resonance(iSPR) respectively. It has been shown that, by altering the buffer pH, the region of lowest protein adsorption on the surface could be moved laterally. The swelling has similarly been shown to respond to both changes in pH and ionic strength. Additionally, the arise of surface charge and the polymer swelling in solution, both a consequence of the ionisation of fixed charges on the polymer, have been indicated to occur at different buffer pH. The studied polymer systems show promising properties for future applications in, for example, the biosensor area, where the surface chemistry can be tailor-made to work optimally in a given environment.

Thrombus formation and blood coagulation is a major problem associated with blood contacting products such as catheters, vascular grafts and artificial hearts. An intense research is being conducted towards the synthesis of new hemocompatible materials and mdifications of surfaces with biological molecules. In this study, polyurethane (PU) films were synthesized in medical purity from diisocyanate and polyol without using any other ingredients and the chemical, thermal and mechanical properties were characterized by solid state NMR, FTIR, GPC, mechanical tests, DMA and TGA. The surfaces of PU films were modified by covalent immobilization of different molecular weight heparins / low molecular weight heparin (LMWH) and unfractionated heparin (UFH) and these surfaces were examined by ESCA, ATR-FTIR, AFM and contact angle goniometer. Cell adhesion studies were conducted with whole human blood and examined by SEM. The effects of different types of heparins on blood protein adsorption and on platelet adhesion were analyzed by electrophresis and SEM, respectively. The surfaces of the UFH immobilized polyurethane films (PU-UFH) resulted in lesser red blood cell adhesion in comparison to LMWH immobilized polyurethane film surfaces (PU-LMWH). When the PU films were treated with blood plasma, the surfaces modified with two different heparin types showed a clearly different protein adsorption behavior especially in the early stage of blood plasma interaction. PU-LMWH samples showed about three times less protein adsorption compared to PU-UFH samples. The morphologies of platelets adhered on material surfaces demonstrated differences / such as PU-UFH had clusters with some pseudopodia extensions, while PU-LMWH had round shaped platelets with little clustering. PU surfaces modified by immobilization of LMWH and UFH, demonstrated promising results for the improvement of non-thrombogenic devices and surfaces.

For the development of smart surfaces high attention is focused on stimuli-responsive polymers. Since type and rate of response to environmental stimuli can be regulated by chain length, composition, architecture and topology, polymer films offer a variety of opportunities to develop such stimuli-responsive surfaces. Here polymer brush surfaces designed for a controlled adsorption of proteins and a switchable activity of immobilized enzymes are presented. The work is focused on temperature as well as pH-sensitive binary brushes, consisting of poly(N-isopropylacrylamide) (PNIPAAm) and poly(acrylic acid) (PAA), and their swelling behavior as well as their protein adsorption affinity is compared to the corresponding homopolymer brushes. All polymer brushes are covalently grafted by ester bonds to an anchoring layer of poly(glycidyl methacrylate), that itself is grafted via ether bonds to a silicon surface. Methodical investigations of layer thickness and refractive index of the brushes in the swollen state and after protein adsorption are carried out with in-situ spectroscopic ellipsometry, varying the brush composition and the solution parameters pH, salt concentration and temperature. The ellipsometric findings are correlated to results of contact angle, atomic force microscopy and zeta-potential measurements as well as colorimetric assays of enzyme activities at the brush surface. Furthermore the swelling of PNIPAAm brushes and protein adsorption at PAA Guiselin brushes are investigated in more detail with attenuated total reflexion Fourier-transform infrared spectroscopy and quartz crystal microbalance with dissipation, respectively.

A range of mesoporous materials based on SBA-15, KIT-6 and FDU-12 have been prepared using neutral block copolymers Pluronic P123 and F127 and characterised using methods including electron microscopy and nitrogen adsorption. Typically the materials have a hexagonal (p6mm) or cubic (Fm3m and Ia-3d) symmetry and pore geometry and are rendered porous by either calcination or solvent extraction. Organic functional groups were incorporated into the silica walls of the materials by co-condensation in the form of propyl thiols and additives in the form of alkanes were added to control pore size and geometry. The effects of temperature, additives, organic functionalisation, synthesis time and sol-gel composition were investigated and the resulting materials were tested as supports for protein adsorption, enzyme immobilisation, and drug delivery. Two FDU-12 materials of differing entrance and cavity sizes were used to adsorb a range of proteins with molecular weight 17 to 160 kDa to determine if there was a size exclusion effect. It was seen that the larger pore material was able to adsorb proteins of a larger size (molecular weight 105 kDa) and an exclusion effect was observed when the dimension of the proteins became too great (larger than 130 kDa). There was no clear trend for the smaller pore material where each protein was adsorbed to some extent by the material but apart from the smallest protein, myoglobin, mainly on the surface and not within the pores. The adsorption of the lipase B from Candida Antartica, CALB, was studied on a range of mesoporous supports with their templates removed by either calcination or extraction. The effect of pore size and functionalisation was investigated in terms of maximum loading and rate of loading. By functionalising the KIT-6 material the maximum loading of CALB was reduced from 45.5 to 32 mg/g whereas functionalising the FDU-12 material increased the maximum from 33 to 42.5 mg/g. The activity of the immobilised CALB was measured by enantioselective transesterification of (R)-1-phenylethanol in methyltetrabutyl ether (MTBE). The effect of loading, surface functionalisation and reusability in organic media were investigated. Functionalisation with propyl thiol was seen to increase the rate of conversion after 30 minutes for both KIT-6 and FDU-12 materials. Selected FDU-12 and KIT-6 materials with window sizes from 6 to 12 nm and with and without functionalisation were used to carry out a drug release study using Bovine serum albumin (BSA). BSA was loaded onto the material and the uptake quantified using nitrogen adsorption, elemental analysis, and thermogravimetric analysis. The release of BSA into simulated body fluid at 37 ºC was measured using HPLC. Functionalisation was seen to have little effect. The type of cubic morphology controlled the rate at which the BSA was released. The KIT-6 3D channel material exhibited a burst release initially followed by a steady release of BSA whereas the mesocage FDU-12 material had a slower and more linear release profile, closer to that desired.

No description available.

Mesoporous silica materials are versatile platforms for biological catalysis, isolation of small molecules for detection and separation applications. The design of mesoporous silica supports for tailored protein and biomolecule interactions has been limited by the techniques to demonstrate biomolecule location and functionality as a function of pore size. This work examines the interaction of proteins and lipid bilayers with engineered porous silica surfaces using spherical silica particles with tunable pore diameters (3 – 12 nm) in the range relevant to biomolecule uptake in the pores, and large particle sizes (5 - 15 µm) amenable to microscopy imaging The differentiation of protein location between the external surface and within the pore, important to applications requiring protein protection or catalytic activity in pores, is demonstrated. A protease / fluorescent protein system is used to investigate protein location and protection as a function of pore size, indicating a narrow pore size range capable of protein protection, slightly larger than the protein of interest and approaching the protease dimensions. Selective functionalization, in this case exterior-only surface functionalization of mesoporous particles with amines, is extended to larger pore silica materials. A reaction time dependent functionalization approach is demonstrated as the first visually confirmed, selective amine functionalization method in protein accessible supports. Mesoporous silica nanoparticles are effective supports for lipid bilayer membranes and membrane associated proteins for separations and therapeutic delivery, although the role of support porosity on membrane fluidity is unknown. Transport properties of bilayers in lipid filled nanoparticles as a function of pore diameter and location in the particle are measured for the first time. Bilayer diffusivity increases with increasing pore size and is independent of bilayer location within the core, mid or cap of the particle, suggesting uniform long range bilayer mobility in lipid filled pores. Application of lipid bilayers on mesoporous silica was examined for membrane associated proteins A unique method to adhere functional proteins in lipid bilayers on mesoporous silica particles is established using vesicles derived from cell plasma membranes and their associated proteins. This method of membrane protein investigation retains proteins within native lipid membranes, stabilizing proteins for investigation on supports.

Surface immobilization of probe molecules in surface based assays is a key area of research in the continued development of immunoassay microarrays. Interest continues to grow in microarray based immunoassays given their potential as a high throughput technique for immunodiagnostics. Therefore, it is important to thoroughly study and understand the implications of interfacial chemistry and immobilization conditions on the performance of the assay. This thesis presents a body of work that examines the impact of probe density, interfacial chemistry, and enhancement factors for arrays read with surface plasmon resonance (SPR) imaging. An array of structurally similar Salmonella disaccharides was immobilized at varying densities and the interface formed was thoroughly investigated to determine the properties of the interface. The arrays were then used with SPR imaging to evaluate the binding of an antibody specific for one disaccharide of the three stereoisomers on the array. A dilute disaccharide surface was found to provide optimal antibody binding. Higher densities result in steric hindrance of antibody binding by not allowing the disaccharide to insert into the antibody binding pocket. The role of interfacial chemistry in antibody attachment was studied to determine optimum conditions. The study examined physical adsorption, covalent attachment, and affinity capture. It was found that covalent attachment provided the most stable attachment and resulted in the lowest levels of antigen detection. Both the physical adsorption and affinity capture provided larger antigen binding capacity and therefore more sensitive antigen detection. The covalent attachment was chosen to evaluate an enhanced assay with the incorporation of gold nanoparticles. These particles provided detection limits that were an order of magnitude improved over those excluding the nanoparticles. A novel surface chemistry for antibody immobilization in SPR imaging studies was evaluated. This involved the electrochemical driven formation of mono- to multilayers of diazonium benzoic acid films. The studies showed the ability to control the thickness of the films formed and also the ability of the antibody chips to capture antigen from solution.

Esta tese apresenta o estudo do efeito do solvente acetato de etila e acetona no comportamento em solução dos polímeros acetato butirato celulose (CAB) e acetato butirato carboximetil celulose (CMCAB) e nas características dos seus filmes finos obtidos pela técnica de revestimento rotacional ou por adsorção. As medidas de viscosidade e espalhamento de raio-X a baixo ângulo (SAXS) mostraram que o acetato de etila é um melhor solvente para CAB e CMCAB do que a acetona. A caracterização dos filmes foi feita através de medidas de elipsometria, microscopia de força atômica (AFM), espectrocospia vibracional por geração de soma de freqüências (SFG) e medidas de ângulo de contato. Os filmes de CMCAB obtidos por revestimento rotacional são mais espessos quando preparado em acetona do que em acetato de etila. Imagens de AFM mostraram que os filmes de CMCAB oriundos de soluções em acetato de etila são mais homogêneos e lisos do que aqueles preparados a partir de acetona. Medidas de SFG comprovaram a forte afinidade da acetona com SiO2/Si, mostrando que esse solvente cria uma nova camada para os filmes de CAB e CMCAB. Os valores de energia superficial calculados para CAB e CMCAB foram semelhantes ~ (49,0 ± 0,5) mJ/m², sendo a contribuição da componente dispersiva maior que a da componente polar. A adsorção das proteínas lisozima, albumina do soro bovino (BSA), concanavalina A e jacalina foram mais pronunciadas sobre os filmes de CMCAB do que sobre CAB. Indicando que a presença do grupo carboximetil (CM) contribui significativamente no processo de adsorção das biomoléculas. O efeito da rugosidade dos filmes de CAB e CMCAB sobre o processo de adsorção das proteínas foi estudado. No caso do CMCAB, a adsorção das proteínas foi mais pronunciada sobre o filme rugoso do que sobre o filme mais liso. Entretanto, para os filmes de CAB a rugosidade não teve um efeito significativo na adsorção das proteínas / The effect of ethyl acetate and acetone on the solution behavior of cellulose acetate butyrate (CAB) and carboxymehtylcellulose acetate butyrate (CMCAB) and on the characteristics of films obtained either by spin coating or adsorption was investigated. Viscosity and small angle X-ray scattering (SAXS) measurements showed that ethyl acetate is a better solvent than acetone for CAB e CMCAB. Films were characterized by means of ellipsometry, atomic force microscopy (AFM), sum frequency generation (SFG) and contact angle measurements. Spin-coated films of CMCAB from ethyl acetate solutions were thicker than those deposited from acetone solutions. AFM images revealed that CMCAB spin coated films from ethyl acetate solutions were homogeneous and flat. However, films obtained from solutions in acetone were very rough. SFG spectra showed that acetone binds strongly to SiO2/Si wafers, creating a new surface for CAB and CMCAB films. Surface energy values determined for spin-coated CAB and CMCAB were similar ~ (49,0 ± 0,5) mJ/m² with the dispersive component larger than the polar component. The adsorption of lysozyme, bovine serum albumin (BSA), concanavalin A and jacalin was more pronounced onto CMCAB films than that onto CAB films. Indicating that carboxymethyl group favored the adsorption process. The influence of surface roughness of CAB and CMCAB on protein adsorption has been investigated. In the case of CMCAB, protein adsorption was morepronounced onto rough films than that onto flat films. However, the roughness of CAB films exerted no significant influence on proteins adsorption

In the present thesis, we propose a modification of silicone surfaces using the controlled deposition of amphiphilic block copolymers from aqueous colloidal dispersions. The surface modifiers are based on poly(dimethylsiloxane) (PDMS) as the hydrophobic part, in order to allow a good compatibility with PDMS artefacts, and poly(glycerol monomethacrylate) (PGMMA) as the hydrophilic block, since this polymer has demonstrated good biocompatibility and low cell attachment. The hydroxyl groups present on PGMMA offer the possibility of further surface functionalization. We have demonstrated the convenience of preparing well-defined amphiphilic block copolymers of PDMS and PGMMA (which we refer to as Sil-GMMA polymers) via atom transfer radical polymerization using a protection/deprotection route (i.e. the silylation of GMMA alcohols groups). Depending on the ratio between hydrophobic and hydrophilic blocks, Sil-GMMA copolymers can self-assemble into micellar and other colloidal structures. Diffusion ordered nuclear magnetic resonance experiments have shown that those micelles did not interact with albumin, suggesting a “stealth” behaviour. Once a library of Sil-GMMA polymers with various block ratio was prepared, the adsorption of Sil-GMMA colloidal dispersions in water/ethanol on PDMS surfaces by simple physisorption was studied. As expected, high PDMS content favoured Sil-GMMA adsorption on silicone surfaces. The presence of our surface modifiers on silicone surfaces was confirmed by a decrease in water contact angle and spectroscopy techniques. We have shown that the surface coatings were stable upon storage in water. Additionally, fibrinogen adsorption was decreased by Sil-GMMA adsorption while albumin adsorption appeared to increase. The preparation of surfaces repellent to fibrinogen and interacting with a “passivating” protein such as albumin is promising. At the same time, this thesis also reports preliminary investigations on the use of enzymes in order to incorporate new functionality to GMMA containing polymers. Although enzymatic activity was observed when using PGMMA instead of glycerol with two different enzymes (glycerol kinase and glycerol dehydrogenase), PGMMA conversions were always low (< 2%).