Dissertations / Theses on the topic 'Sol-immobilization'

Much attention has been paid to immobilization of enzymes to improve enzyme stability and permit its reuse. Glucose oxidase entrapment in different kinds of silica sol-gel matrices was investigated. The enzyme showed stable activity for 11 uses in the sol-gel with tetramethyl orthosilicate (TMOS) as precursor, and at least 7 uses in the sol-gel induced electrochemically with tetraethyl orthosilicate (TEOS) as precursor. The sol-gel made with TEOS as precursor and HCl as catalyst showed stability in enzyme activity for 11 uses but the activity decreases on the same sol-gel when modified with silica nanoparticles. Factors such as optimal incubation time of glucose solution, the reproducibility between different sol-gels, and the storage time were investigated. Good linearity and analytical results on real samples were obtained. The detection method was based on a colorimetric method for determining the concentration of hydrogen peroxide produced from the oxidation reaction of glucose.

Thesis (Ph. D.)--University of Nebraska--Lincoln, 2004.
PDF text: [2] leaves abstract, vii, 156 leaves dissertation : ill. (some col.). Site viewed on Jan. 25, 2005. Includes bibliographical references.

Im Bereich der Umwelttechnik bieten sich vielversprechende Einsatzmöglichkeiten für Sol-Gel-immobilisierte Mikroorganismen sowohl für die Entfernung als auch für die Detektion von Schadstoffen an. Für einen effizienten Einsatz sind zum einen eine hohe Langzeitaktivität und -vitalität der eingebetteten Zellen als auch eine gute Lagerbeständigkeit wichtig. Neben einer hohen Makroporosität, die sowohl für einen guten Stoffaustausch mit der Umgebung sorgt sowie den Zellen Raum für Zellteilung bietet, ist vor allem auch die Aufrechterhaltung der Feuchtigkeit in der Immobilisierungsmatrix während der Immobilisierung, der Lagerung und des Einsatzes wichtig. Besonders vorteilhaft hat sich hier eine Immobilisierung in dünnen SiO2-Schichten auf Blähtonbruchstücken erwiesen, da dieses Trägermaterial neben einer hohen Makroporosität auch ein hohes Wasserspeichervermögen besitzt. Immobilisiert in dünnen SiO2-Schichten auf Blähton konnten diverse Schadstoff-abbauende Mikroorganismen mehrere Monate auch außerhalb eines flüssigen Mediums unter feuchten Bedingungen gelagert werden, ohne dass ihre Abbauleistung erheblich sank. Ein weiteres Verfahren um Immobilisierungsmaterialien mit überdurchschnittlich hoher Makroporosität bei gleichzeitig hoher Stabilität zu erzeugen, stellt das Freeze-Gelation Verfahren dar. Über einen Gefriertrocknungsschritt können hier zudem die zu immobilisierenden Zellen in eine trocken lagerfähige Form überführt werden, wodurch Handhabung sowie Lagerung und Transport vereinfacht werden. Außerdem kann durch Wahl der Einfrierbedingungen und Zugabe von Füllstoffen zu dem SiO2-Sol entscheidend die Porenstruktur der Immobilisierungsmatrix beeinflusst und den Anforderungen entsprechend eingestellt werden. Allerdings zeigte sich, dass diese Methode nicht für alle Mikroorganismen gleichermaßen geeignet ist. Trotz diverser kryoprotektiver Maßnahmen konnte keine ausreichende Überlebensrate für sensible Stämme wie Aquincola tertiaricarbonis L108 erzielt werden. Neben der Erhöhung der Makroporosität der SiO2-Matrix wurde versucht das Sol-Gel-Verfahren mit einem flexiblen organischen Polymer (Na-Alginat) zu kombinieren, um eine Immobilisierungsmatrix mit einem weichen Kern zu erzeugen, der Zellteilung zulässt. Als zusätzlicher Vorteil des entwickelten Alginat/SiO2-Hybridmaterials erwies sich, dass dieses auch auf einfache Weise mittels Drucktechniken in definierten Spots abgelegt werden kann und so die Zellen in Arrays abgelegt werden können. Das Potential dieser Methodik für die Entwicklung von Ganzzellsensoren wurde am Beispiel der Grünalge Chlorella vulgaris als Modell-Sensorzelle demonstriert und für die Detektion von Atrazin als Modellsubstanz eingesetzt.

O presente projeto teve como objetivo selecionar componentes orgânicos para obtenção de compósitos híbridos pela técnica sol-gel utilizando como precursor tetraetilortossilicato (TEOS) visando avaliar o efeito do agente orgânico na evolução do sol para gel e nas propriedades finais dos suportes para imobilizar a lipase microbiana de Burkholderia cepacia. Foram testados três compostos orgânicos ?-ciclodextrina (?CD), carboximetilcelulose (CMC) e hidroxietilcelulose (HEC) e os resultados obtidos foram comparados com o comportamento já estabelecido para o polivinilálcool (PVA), tanto em termos morfológicos como atividade catalítica em meio aquoso (hidrólise de triacilgliceróis) e meio orgânico (síntese de éster de cadeia curta e longa). A motivação do estudo está diretamente relacionada com a elevada afinidade do suporte híbrido SiO2-PVA pelo glicerol, sendo uma das limitações do uso deste suporte para imobilizar lipases com finalidade de aplicação em reações de transesterificação conduzidas em fluxo contínuo. As características morfológicas e estruturais dos suportes resultantes foram determinadas empregando técnicas convencionais, indicando similaridade das propriedades estruturais, fornecendo suportes com áreas superficiais entre 361,6 a 529,7 m2 g-1 e morfologia amorfa. A diferença marcante entre os suportes foi verificada quanto a capacidade de adsorção do subproduto da reação de transesterificação, sendo verificado que a substituição do polivinilálcool por ?-ciclodextrina reduziu em até 50% a habilidade do suporte hibrido em absorver glicerol. Os componentes derivados de celulose apresentaram resultados similares aos obtidos com o polivinilálcool. Em termos de afinidade pelo glicerol os resultados obtidos permitiram a classificação dos suportes híbridos na seguinte ordem crescente: SiO2-CMC > SiO2-PVA > SiO2-HEC > SiO2- ?CD. Quanto a afinidade dos suportes para imobilização da lipase de B. cepacia, os valores de atividade hidrolítica dos derivados imobilizados, nas condições adotadas, revelaram que todos os suportes tiveram o mesmo tipo de interação com a enzima resultando em valores de recuperação de atividade na faixa de 56 - 63%, valores próximos aos normalmente obtidos pela matriz de SiO2-PVA (68%). Os parâmetros cinéticos (Km e Vmax) determinados indicaram que a lipase imobilizada nos diferentes tipos de suporte, apresentou afinidade semelhante pelo substrato azeite de oliva. Os valores determinados de Km e Vmax da lipase de B. cepacia na forma livre (Km = 410 mM e Vmax =12391 Ug-1) mostraram que independente do suporte, a imobilização reduziu aproximadamente 60% da afinidade da enzima pelo substrato. Em meio orgânico, tanto as reações de esterificação como de transesterificação mediadas pelos derivados imobilizados apresentaram desempenho similar com atividade de esterificação da ordem de 118 ?M.g-1min-1 e rendimentos de formação de ésteres etílicos da ordem de 98,30%. De uma forma geral, os resultados obtidos foram promissores e permitiram selecionar o composto orgânico ?CD para substituir com vantagens o polivinilálcool no preparo de matrizes hibridas para imobilização da enzima lipase. O menor poder hidrofílico desta matriz em relação às outras matrizes testadas pode ser creditado a estrutura cíclica da ?CD que reduziu a capacidade do suporte SiO2-?CD em adsorver glicerol, principal subproduto da reação de transesterificação. Testes em processos conduzidos em fluxo contínuo são sugeridos para comprovação da eficiência dessa matriz.
The objective of this work was to select organic compounds for preparing hybrid composites by the sol-gel technique using tetraethylorthosilicate (TEOS) as precursor aiming at evaluating the effect of the organic agent in the transformation of the sol into gel and in the final properties of the matrices for immobilizing lipase from Burkholderia cepacia. Three organic components ?-cyclodextrin (?CD), carboxymethylcellulose (CMC) and hydroxyethylcellulose (HEC) were tested. The organic component polyvinyl alcohol (PVA) was used as control taking into consideration its morphological properties and the catalytic activity of the immobilized derivative in aqueous medium (triacylglycerols hydrolysis) and organic medium (synthesis of short and long chain esters). The motivation of this work was directly related with the high affinity of the matrix polysiloxane-polyvinyl alcohol (SiO2- PVA) by the glycerol, limiting the use of the immobilized derivative in the transesterification reactions running on a continuous flow. The morphological and structure properties of the resulting matrices was determined using conventional techniques, showing similar structure properties, with surface area values between 361.6 and 529.7 m2g-1 and amorphous morphology. The main difference among the matrices were observed regarding their ability to adsorb glycerol, the main byproduct of the transesterification reaction. It was verify that the replacement of the polyvinyl alcohol by ?-cyclodextrin reduced in about 50% the hybrid composite capacity to adsorb glycerol. On the other hand, the cellulose components showed similar results as the polyvinyl alcohol. Considering as an evaluated parameter the glycerol affinity, the achieved results allowed classifying the hybrid composites as follows: SiO2- CMC > SiO2-PVA > SiO2-HEC > SiO2-?CD. The performance of the hybrid composites for immobilizing lipase from B. cepacia, through the hydrolytic activity reveal that all the matrices show the same interaction with the lipase, resulting in activity recovered values between 56-63%, which is close to the value attained by SiO2-PVA matrix (68%). The kinetic parameters (Km and Vmax) values determined for free lipase (Km = 410 and Vmax = 12391 Ug-1), showed that independely of the immobilization support the affinity of the enzyme for the substrate was reduced by 60%. In organic medium, all the immobilized derivatives showed similar behavior on both esterification and transesterification reactions attaining esterification activity of 118 ?M.g-1min-1 and ethyl ester yields of 98.30%. Overall, the results were promising and allowed selecting the organic component ?CD to replace, with advantages, the use of PVA for preparing hybrid composites. The lower hydrophilic capacity of this matrix compared with the others matrices can be explained by the cyclic structure of the ?CD that reduced the capacity of the SiO2-?CD matrix in the glycerol adsorption, the main byproduct of the transesterification reaction. Assays carried out in packed bed reactor running on a continuous basis are propose to confirm the efficiency of this matrix.

Immobilization of fluorescent chemosensors and chromogenic reagents on solid supports for developing optical sensors result in improved analytical performance characteristics such as continuous read-out, increased sensitivity, lower reagent consumption and possibility of using the sensor in solvents where the free molecule displays low solubility. The aim of this study is to immobilize dansylaminophenyl boronic acid (DAPB acid) and diphenylcarbazide (DPC) into various solid supports for the determination of fructose and hexavalent chromium, respectively. DAPB acid reacts with diol containing molecules to produce electron transfer resulting fluorescence quenching. Whereas DPC reacts specifically with chromate to produce a magenta colored complex having absorption maximum at 540 nm. Utilizing sol-gel technology, inorganic polymer matrices which enabled to observe fluorescence and absorbance signal in VIS region has been constructed. Also methylmethacrylate (MMA) and methacrylic acid (MAA), which are known to give transparent organic co-polymers, are chosen as monomers in the synthesis of organic copolymer. Hydrogels such as polyvinyl alcohol and Ca-alginate gel have been utilized for their good optical characteristics in the working range. Several considerations in the construction of host matrix were taken into account, such as the porosity of the polymers, functionalization of the matrix and use of additives for increasing the affinity of the medium toward the dopant molecule and swelling properties of organic polymers. The performances of the immobilizations were evaluated in terms of the transmittance and leaching properties of the host matrix, optical properties of dopant and optical response characteristic of the dopant for the analyte. The sensor applications of the immobilized probe molecule DPC were investigated. Studies regarding the enhancement of the performance of the flow injection analysis method for fructose determination, previously carried out in our laboratory, based on the fluorescence quenching of DAPB acid probe in solution were stated.

The aim of this study was to immobilize the redox enzyme laccase over TiO2-SiO2-Au thin film coated ITO glass substrates in order to prepare electrochemically active surfaces for biosensor applications. Colloidal TiO2-SiO2-Au solution was synthesized by sol-gel route and thin film was deposited onto the substrates by dipcoating method. The cysteamine was utilized as a linker for immobilization of enzyme covalently through gold active sites. Preliminary studies were conducted by using invertase as model enzyme and Pyrex glasses as substrates. The effect of immobilization parameters such as immobilization temperature, concentration of enzyme deposition solution, immobilization time for laccase were examined. Leakage studies were conducted and storage stability of immobilized laccase was determined. Highest laccase activity was achieved when immobilization was performed with 50 µ
g/ml solution at 4°
C for 2 hours. Laccase activity decreased after 4 hours of impregnation in enzyme solution. Laccase leakage was observed in the first usage of substrates and 55% activity decrease was determined in the subsequent use which might be attributed to the presence of uncovalently adsorbed enzyme on the fresh samples. In air and in buffer storage stabilities were also tested. It was found that the activity of samples almost vanished after 6 days regardless of storage conditions. Both enzymes had more activity on ITO substrate.

The aim of this study was to investigate the viability of patterning by immobilization, photocatalytic removal, and re-immobilization steps of the enzyme on photocatalytically active thin films for biosensor fabrication purposes. For this aim, TiO2-SiO2-Au sol-gel colloids were synthesized and deposited on glass substrates as thin films by dip coating. Cysteamine linker was assembled on gold nanoparticles to functionalize thin films with amine groups for immobilization of model enzyme invertase. Effect of immobilization temperature, enzyme concentration of the immobilization solution and immobilization period on invertase immobilization were investigated. The immobilized invertase activity was found independent from the immobilization temperature in the range tested (4oC-room temperature). The optimum enzyme concentration and period for immobilization was determined as 10µ
g/ml and 12 hours respectively. The resulting invertase immobilized thin films showed high storage stability retaining more that 50% of their initial activity after 9 weeks of storage. Photocatalytic enzyme removal and re-immobilization studies were carried out by irradiating the invertase immobilized thin films with blacklight. Upon 30 minutes of irradiation, immobilized invertase was completely and irreversibly inactivated. Initial immobilized invertase activity (before the irradiation) was attained when invertase was re-immobilized on thin films that were irradiated for 5 hours. Thus it was inferred that with sufficient exposure, enzymes can be completely removed from the surfaces which makes the re-immobilization possible. The possibility of enzyme removal with photocatalytic activity and re-immobilization can pave the way to new patterning techniques to produce multi-enzyme electrode arrays.

Bien que les matériaux poreux soient nombreux dans la nature, la synthèse en laboratoirede matériaux présentant une porosité multi-échelle ou hiérarchisée est toujours délicate. Enutilisant la matière molle (émulsions concentrées, auto-assemblages, mésophases lyotropes, etc)et le procédé sol-gel, il est possible d’obtenir une grande variété de matériaux monolithiques, àporosité hiérarchisée, composés d’un squelette silicique. La porosité de ces matériaux peut êtreoptimisée en jouant avec la nature de l’émulsion, le tensioactif utilisé, ou avec l’ajout d’agentd’extérieur comme le sel. En combinant ces méthodes, des matériaux possédant une mésoporositéhexagonale ont été obtenus. Grâce à leur surface riche en silanols, ces matériaux poreux ont étéfonctionnalisés par greffage post-synthèse de molécules organiques. Dès lors, l’immobilisationd’entités biologiques comme les enzymes au sein de la structure poreuse a permis d’utiliser cesmatériaux pour des réactions d’hydrolyse, de synthèse ou de décoloration en milieu aqueux dansune approche de « chimie verte ». Enfin, des micro-organismes ont été piégés dans ces matériauxporeux qui ont été recouverts d’une coque en silice. Les micro-organismes peuvent s’y développersans restriction et leur croissance est très différente de celle observée dans les cultures classiques.La coque en silice, formée en surface, est donc imperméable au passage des bactéries (taillemicrométrique) mais perméable à la diffusion des substrats et des réactifs. Cette diffusion a étémise à profit pour réaliser des réactions enzymatiques en cascade. Ces matériaux se positionnentcomme des biocatalyseurs très prometteurs pour de nombreuses applications
Although porous materials are numerous in nature, the laboratory synthesis of materials withmulti-scale or hierarchical porosity is always difficult. By using soft matter (concentrated emulsions,self-assemblies, lyotropic mesophases, etc.) and the sol-gel process, it is possible to obtaina wide variety of monolithic materials with hierarchical porosity composed of a silicic skeleton.The porosity of these materials can be optimized by playing with the nature of the emulsion,the surfactant used, or with the addition of external agents such as salt. By combining these methods,materials with hexagonal mesoporosity have been obtained. Thanks to their silanol-richsurface, these porous materials have been functionalized by post-synthesis grafting of organicmolecules. Therefore, the immobilization of biological entities such as enzymes within the porousstructure has made it possible to use these materials for hydrolysis, synthesis or discolorationreactions in aqueous media in a "green chemistry" approach. Finally, microorganisms were trappedin these porous materials which were covered with a silica shell. Microorganisms can growthere without restriction and their growth is very different from that observed in conventionalcultures. The silica shell formed on the surface is therefore impermeable to the passage of bacteria(micrometric size) but permeable to diffusion of substrates and reagents. This diffusion wasused to carry out cascade enzymatic reactions. These materials are positioned as very promisingbiocatalysts for many applications

La preparazione di nanoparticelle (NPs) metalliche risulta complessa a causa di diversi parametri operativi in grado di modificare le proprietà del prodotto finale, per questo la ricerca di nuove strategie sintetiche atte a controllare morfologia e geometria di questi sistemi costituisce uno dei temi di maggior interesse scientifico per la chimica della catalisi. La tecnica di sol immobilization permette di ottenere catalizzatori nanostrutturati composti da nanoparticelle stabilizzate da polimeri e depositate su un materiale che funga da supporto. La possibilità di preparare polimeri con proprietà specifiche, ha aperto l’opportunità di sfruttare questi sistemi per modellare le caratteristiche delle nanoparticelle e la loro attività catalitica. In particolare, in quest’attività di ricerca è stato studiato, attraverso una reazione modello, in che modo il peso molecolare ed il grado di idrolisi degli stabilizzanti polimerici a base di poli-vinilalcol, possono influenzare la morfologia e l’attività delle nanoparticelle di oro.

Abstract In this Ph.D. project, the effective parameters in the sol-immobilization method which can affect catalytic reactivity including: capping agent, solvent of synthesis, and support were studied. The synthesized catalysts were employed in the liquid phase hydrogenation reactions of two biomass derived molecules namely furfural and vanillin. In this regard, the role of protective agents, which are used to stabilize colloidal nanoparticles (NPs) in solutions, for Pd NPs supported on carbon support in the liquid phase hydrogenation of furfural was explored. The use of Pd as a hydrogenation catalyst is well documented, as hydrogenation reactions can only be performed by metals that can easily chemisorb hydrogen. Pd is one of such metals capable of dissociate hydrogen even at room temperature. The capping agent adsorbed on the surface of the NPs can alter their activity and selectivity, by modifying the particle size, size distribution, morphology, and stability against leaching and agglomeration. Besides, the effect of the amount of protective agent and the synthesis solvents have been investigated, allowing better insight into the metal-support interaction in Pd/TiO2 catalysts for the hydrogenation of furfural. Then, the effect of using different carbonaceous supports with various chemical-physical properties on the activity and selectivity towards the hydrodeoxygenation of vanillin as a lignin model compound was explored. To better understand the role of the capping agents in controlling the activity and the selectivity of the furfural hydrogenation, a series of carbon-supported Pd nanoparticles were prepared through controlled sol-immobilization method using different capping agents, including polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), and poly(diallyldimethylammonium) chloride (PDDA) containing oxygen and/or nitrogen groups. The catalysts were characterized by different techniques. UV-Vis and Fourier-transform infrared (FTIR) spectroscopy were used to evaluate the interaction between capping agents and Pd precursor, while transmission electron microscopy (TEM) was performed to study the final morphology of the catalyst. Finally, X-ray photoelectron spectroscopy (XPS) was employed to investigate the surface chemistry of the carbon support, the chemical state, and the exposure of supported palladium species. The UV-Vis spectra of the system composed of Pd precursor plus capping agent demonstrated the interaction of the metal with PVA and PVP. In the case of PDDA, changes to the precursor salt complex were noticed through shifts in [PdCl4]2- absorbance bands. The disappearance of the peaks associated with Pd2+ and the observed scattering indicated a complete reduction to Pd0 and the formation of metal nanoparticles. The interaction of the capping agents with the Pd precursor was studied by FTIR spectroscopy. For PVA, the peaks corresponding to the stretching vibrations of C–O–C linkage were observed, which suggested the presence of cross-linked PVA molecules, and the slight modification observed for the peaks in the spectrum of PVA + Na2PdCl4 suggested a weak interaction between the metal precursor and the –OH groups present in PVA. In the case of PVP, a decrease in the intensity of the peak after the addition of Pd was observed which confirmed that both O and N groups are present in PVP interaction with the metal precursor. For PDDA, after the addition of Pd, the intensity of the peaks decreased, which indicated that the activity of the vibrational modes is modified in the mixture, probably due to PDDA–Pd interaction. The morphological characteristics of the synthesized catalysts were evaluated by HRTEM. We noticed that the capping agent has a major effect on the size and distribution of Pd NPs when using activated carbon as the support. All catalysts had an average particle size of 3–4 nm, with the presence of isolated larger particles in the case of PdPDDA/C. The XPS survey data revealed that only Pd, C, N, and O species were present on the surface of the catalysts. Depending on the capping agent used, substantial differences were observed in the relative amount of Pd on the surface: PdPVA/C (1.30 %) > PdPDDA/C (0.76 %) > PdPVP/C (0.50 %). The data also showed a different oxygen content in the samples. PdPVA/C displayed the highest relative amount of O (14.7 % compared to PdPDDA/C (9.70 %) and PdPVP/C (9.10 %)), respectively. The highest oxygen content on the surface of PdPVA/C catalyst is probably due to the presence of –OH groups in PVA. PdPVP/C exhibited the highest N content (2.95 %), higher than PdPDDA/C (1.86 %) due to the presence of a pyrrole-type N species in PVP and dimethyl-ammonium groups in PDDA, while in the PdPVA/C N was not detected on the surface, as expected. The performance of the prepared catalysts was examined for the liquid-phase hydrogenation of furfural (furfural 0.3 M; furfural/metal ratio 500 mol/mol, 5 bar H2, temperature range of 25–75 °C) with 2-propanol as solvent. The catalytic results revealed that the activity of catalysts is correlated to the relative amount of Pd at the surface: PdPVA/C (1.3%) > PdPDDA/C (0.76%) > PdPVP/C (0.50%). At 75 °C the catalysts exhibit similar reactivity. We ascribed this effect to the partial removal of a capping agent during the reaction, thereby exposing a higher number of active sites to the reactant. In the next step, since the best results were obtained using PVA, this latter was chosen as the protective agent, focusing on the effects that its amount and the change of the solvent of synthesis to methanol-water mixtures might have on the preparation of Pd/TiO2 catalysts. This new approach indicates the necessity of using the capping agent, and results also show that the acidification step, which lowers the isoelectric point (IEP) to afford anchoring of the NPs to the surface of the support, can be eliminated while still maintaining the same degree of Pd immobilization (≥ 96%) and particle size control (< 2 nm). These samples were evaluated for furfural hydrogenation; there was an improvement in selectivity towards furfuryl alcohol and tetrahydrofurfuryl alcohol, whereas ether by-products were suppressed. Supported Pd NPs were prepared in accordance with the standard sol-immobilization method, in which the temperature of the chemical reduction was maintained at 1 °C. This temperature was chosen as earlier research in our workgroup reviled the formation of smaller nanoparticles when lower temperatures were used. UV-Vis spectroscopy was performed to characterize the precursor salt and colloidal solutions during NPs preparation. Changes to the precursor salt complex were noticed through shifts in [PdCl4]2- absorbance bands. Catalysts prepared with increasing volumes of MeOH showed shifts in the distinctive ligand to metal charge transfer (LMCT) and d-d transitions. This confirms a change to the Pd metal precursor complex when it is solvated in either the MeOH/H2O mixture or in pure MeOH. One reason for this is the exchange of ligands between the Pd salt complex and the solvent synthesis. The Pd % loading of each catalyst was measured using microwave plasma – atomic emission spectroscopy (MP-AES). All catalysts were characterized using TEM, in order to get information of their average NP sizes and particle size distributions/dispersions. An initial comparison of catalysts, showed that an acidified immobilization step increases the average NP size. The bigger NP size and dispersion was found for the sample prepared by H2O as the solvent, PVA, and acidification step, which it can be ascribed to the interaction of the stabilizer agent (PVA) with acid during the immobilization step. XAFS was performed to investigate both changes to Pd oxidation state (XANES) and the local structural environment with respect to Pd (EXAFS). The results confirmed that by using PVA in the solvent system, the Pd oxidation state contains a greater quantity of Pd2+. Interestingly, by removing the acidification step during the synthesis, an increase in the oxidized Pd state was observed. This was consistent with the TEM data indicating this increase in Pd2+ is related to the observed particle sizes. Pd surfaces are known to form passivating oxide layers when exposed to air with an increase in the temperature needed to form a bulk oxide structure. Therefore, the Pd2+ fraction refers to the quantity of the available Pd surface and hence the particle size. Fitting of the 1st shell Pd K edge EXAFS data was consistent with the trends observed through TEM and XANES characterization. The presence of small Pd NPs is confirmed by the decreased magnitude of Pd-Pd scattering, signified by smaller CNPd-Pd. All catalysts were tested for the hydrogenation of furfural at 50°C. Although the XANES, EXAFS, and TEM data have all confirmed that sample prepared by the pure MeOH contains smaller NPs than the sample prepared by pure H2O, the initial catalytic activity showed that the sample synthesized by H2O has a significant increase in initial TOF h-1. In addition, furfural hydrogenation performed over the bare TiO2 support displayed 98.4 % selectivity to the acetal product (2-(diisopropoxymethyl) furan), at isoconversion, while sample prepared by H2O revealed less selectivity to the acetal (18.8%). To understand these differences in activity/selectivity we performed further HRTEM investigations of the fresh samples. These studies have identified a 'halo' of PVA around NPs produced using MeOH (e.g. PdMeP) and at the interface between NPs and support. We suggest that the poor solubility of PVA in methanol is responsible for this increase in PVA clustering on the catalytic surface/support interface identified with HRTEM. We also propose that the active sites on TiO2 are formed through the spillover of hydrogen onto the support and accounts for the superior yield of acid catalysed products for Pd/TiO2 compared to TiO2 alone. In the case of Pd/TiO2, the catalysts prepared using MeOH result in aggregation of PVA at the Pd-TiO2 interface, which restrict spillover effects and decreases the amount of acid catalysed products. For the catalysts prepared without the addition of PVA, we observed a higher proportion of ethers than for the analogous catalysts prepared with PVA. This further supports our hypothesis that PVA at the metal-support interface limits the spillover of hydrogen. Catalyst recycle tests were carried out over six consecutive hydrogenation cycles. The catalysts prepared without PVA quickly deactivated with almost negligible performance by the sixth consecutive run. The TEM analysis of the spent sample confirmed that when PVA is not present the samples quickly agglomerate and effective surface area of Pd rapidly diminishes. Finally, the effect of support on both activity and selectivity of the catalyst in the hydrogenation of vanillin to vanillyl alcohol and the subsequent hydrodeoxygenation (HDO) to creosol was studied. Four types of carbonaceous materials (three activated carbon: Norit, KB, G60, and a carbon nanofiber: HHT) were used as support for Pd nanoparticles. Catalysts were synthesized with sol immobilisation method using PVA as the capping agent, and tested in the hydrodeoxygenation reaction of vanillin under mild reaction conditions (50 °C, 5 bar H2 and isopropanol as solvent). Catalysts have been extensively characterized by TEM, XPS and BET in order to correlate the surface properties with the catalytic behaviour and selectivity to the target products. In the end, recycle tests were carried out on the most active catalyst to determine the reusability of the material used. BET analysis was conducted on Pd-supported catalysts. Pd/Norit catalyst has the highest surface area (2000 m2/g), whereas Pd/HHT has the lowest surface area (40 m2/g). The average pore radius of all samples was in the range of 2 nm for Pd/KB to 25,4 nm for Pd/HHT. XPS analysis were performed on the synthesized catalysts to determine the oxidation state of the Pd, the graphitization order and the presence and abundance of oxygen functionality. Pd/Norit, Pd/KB and Pd/G60 had a similar amount of C1s species (84.4 %, 87.3 % and 91.1 %, respectively), while Pd/HHT had the highest number of C 1s (98.8 %). Evaluation of O1s species revealed that the Pd/HHT catalyst has the lowest amount of functionalisation (0.9 %), while Pd/Norit the highest one (14.3 %). The results showed that the Pd/HHT catalyst has the highest amount of C=C (82.1 %), therefore the support can be considered highly graphitised. TEM analysis were performed to determine the mean particle size and size distribution. In all samples, the nanoparticles are homogeneously distributed on the support. Pd/Norit and Pd/KB had similar mean Pd particle sizes (2.5 and 2.7 nm respectively). Whereas, Pd/G60 and Pd/HHT displayed higher mean Pd nanoparticle diameters (3.5 and 3.9 nm, respectively). The catalysts were tested in the vanillin HDO reaction. The reaction profile shows the features of a typical consecutive reaction, with vanillyl alcohol as the intermediate product, and creosol as the final product. Interesting, an additional product was detected in the reaction mixture, namely vanillyl isopropyl ether. The ether is produced by reaction of vanillyl alcohol with a molecule of solvent (isopropanol) and it is consumed with time since it is in equilibrium with the alcohol. The results revealed that the rate of conversion of vanillin enhances with increasing degree of graphitisation. These results can be explained by the strong interaction between the graphitic plane of the support and the aromatic ring of the substrate that allows a better interaction with the active metal nanoparticles. At the same time, the activity in the vanillin hydrogenation reaction decreases with an increase in oxygen content at the carbon surface. The support-substrate interaction is responsible for the change in activity; the increase in oxygen functionality actually disrupts the graphical plane structure of the support. The support affected the selectivity at the lower conversion. In fact, at 50 % of conversion, vanillyl alcohol was the main product of Pd/Norit, Pd/HHT and Pd/KB (selectivity in the 73-82 %), while Pd/G60 provided high levels of both vanillyl isopropyl ether and creosol (23 and 21 %, respectively). The formation of ether was associated with the amount of carboxylic support functionality (COOH groups). The recycling tests were carried out on the most active catalyst: Pd/HHT. After five consecutive reactions, the conversion did not significantly decrease, demonstrating the high stability of the catalyst. Comparably, the selectivity of vanillyl alcohol remained almost unchanged.

L’uso di molecole stabilizzanti (polimeri, surfattanti o leganti organici) nel corso della sintesi di nanoparticelle in sospensione è fondamentale per permettere il controllo della dimensione della fase attiva e per evitare l’aggregazione dei colloidi nella fase di sintesi e deposizione del sol metallico sul supporto. Nonostante questo, molto spesso, l’effetto dello stabilizzante non è solo quello di modificare le proprietà morfologiche (ad esempio la dimensione) delle nanoparticelle supportata ma anche di cambiare l’interazione della fase attiva con i reagenti dal punto di vista elettronico e diffusionale. La messa a punto di metodologie di sintesi controllate ed efficaci è molto importante. Le tecniche di sintesi utilizzate per la preparazione di catalizzatori a base di metalli nanostrutturati sono innumerevoli, ma una metodologia particolarmente interessante, che garantisce piccole dimensioni delle nanoparticelle ed un’elevata distribuzione del metallo sul supporto, è la tecnica della sol-immobilization. In questo lavoro di tesi è stato studiato come il tipo e la quantità di stabilizzante influisce sulla dimensione della nanoparticella e sull’attività catalitica del catalizzatore, usando come reazione modello l’ossidazione selettiva dell’5-idrossimetilfurfurale (HMF) ad acido 2,5 furandicarbossilico (FDCA).

Nella sintesi di sospensioni colloidali d’oro è fondamentale l’impiego di stabilizzanti organici per prevenire la crescita, l’aggregazione e la coalescenza delle nanoparticelle. Quando queste sospensioni sono utilizzate per la preparazione di catalizzatori, la presenza dell’agente stabilizzante sulla superficie delle nanoparticelle può influenzare molto l’attività catalitica modificando l’interazione dei reagenti con la fase attiva. In alcuni casi l’accessibilità delle molecole alla superficie delle nanoparticelle può essere limitata, ma un’adeguata scelta dello stabilizzante può anche portare ad un miglioramento delle prestazioni catalitiche realizzando catalizzatori con selettività migliori grazie all’effetto elettronico e sterico dello stabilizzante. In questo lavoro è stato studiato l’effetto di alcuni stabilizzanti polimerici, commerciali e di sintesi, sull’attività delle nanoparticelle di Au. In particolare, alcuni catalizzatori a base di oro supportato su carbone attivo, preparati tramite sol-immobilization, sono stati testati nella reazione di ossidazione selettiva del 5-idrossimetilfurfurale ad acido 2,5-furandicarbossilico. I polimeri commerciali impiegati sono stati il polivinilpirrolidone (PVP), il polietilenglicole (PEG) e il polivinilalcol (PVA). Variando alcune caratteristiche durante la sintesi dei catalizzatori, come il rapporto polimero:Au e il peso molecolare del polimero, è stato possibile studiare come lo stabilizzante influenzi la dimensione e le prestazioni catalitiche delle nanoparticelle d’oro. I polimeri di sintesi testati sono stati la polivinilammina (PVAm) e il polivinilalcol fosforilato (PPVA). Questi due particolari polimeri hanno permesso di studiare l’effetto legato all’elemento stabilizzante dell’Au, poiché hanno una struttura simile a quella del PVA, ma legati alla catena polimerica presentano atomi di azoto e gruppi fosfato che si legano all’oro.

Ce travail contribue à l'acquisition des connaissances sur les mécanismes et les facteurs contrôlant le transfert des éléments traces dans le système sol-nappes d'eaux souterraines. Le nombre de plus en plus important de sols contaminés par les éléments traces potentiellement toxiques suite aux activités industries a reçu beaucoup d'attention au cours des deux dernières décennies. Les polluants accumulés dans ces sols peuvent alors être transférés dans différents compartiments de l'environnement en fonction de leur origine, leur spéciation et les conditions physico-chimiques et biologiques du milieu. Ainsi, une des problématiques est la pollution des nappes d'eau souterraine par ces éléments traces. Les flux massiques d'éléments traces dans les sols vers les nappes d'eau souterraines sont un paramètre d'entrée important pour prévoir le devenir des pollutions des aquifères et donc à évaluer le potentiel de la contamination des ressources en eau potable. L’objectif de cette thèse a été de proposer une méthode de mesure des flux en laboratoire qui permette de simuler au mieux les conditions naturelles des transferts des éléments traces vers les nappes. Pour cela, nous avons mis au point une colonne de laboratoire non-saturée. Cette colonne a été testée dans différentes conditions de lixiviation, comparée aux méthodes de lixiviations normées et testée en condition d’immobilisation des éléments traces lors de l’apport d’amendements. Dans un premier temps, la colonne de laboratoire permet de diminuer l’erreur sur l’estimation des flux. Elle permet des mettre en évidence des phénomènes de sorption, désorption et complexation des éléments traces sur les substrats, contrôlant ainsi les transferts verticaux.Dans un deuxième temps nous avons évaluer l’effet d’amendements minéraux sur la mobilité des éléments traces sur deux sols contaminés par des extractions chimiques. Nous avons ensuite étudié la lixiviation de ces éléments suite à l’apport d’amendements: de l’hydroxyapatite et de la Grenaille d’acier dans ces deux sols en utilisant les colonnes de sol développés précédemment. L'étude de la localisation des éléments traces sur les minéraux nouvellement formés suite à l'apport de ces amendements minéraux et leur interaction avec les constituants minéraux d'origine des sols (microscopie couplé à des spectromètres de fluorescence X) nous a permis de comprendre et de déterminer les réactions mis en jeu au cours de la lixiviation de ces éléments. Ainsi, l’apport d’hydroxyapatite (HA) et de Grenaille d’acier (GA) ont permis de diminuer significativement les concentrations en Cd, Zn dans les lixiviats. En revanche, l’apport de HA et GA aux sols augmente significativement la libération de As (dans le cas de HA) et Pb suite a l’apport de GA et HA. Les phases minérales porteuses de ces éléments traces, ont pu être caractériser et ainsi les mécanismes responsables de l’immobilisation ou du relargage ont pu être identifiés
This work contributes to the knowledge of the mechanisms and factors controlling the transfer of trace elements, particularly in the soil- groundwater pathway. Extensive soil contamination with potentially toxic traces elements from various industries has in many industrialized countries received significant attention over the last two decades. Mass fluxes of trace elements in soils to groundwater are important input parameter for predicting the fate of pollution of aquifers and thus to assess the potential for contamination of drinking water resources. The objective of this study was to propose a method for measuring the fluxes in laboratory to simulate the natural conditions. For this, we have developed an unsaturated soil column. This column was then tested in various leaching conditions (compared to standardized leaching methods and tested under conditions of immobilization of trace elements). At first, the laboratory column reduces the error in the estimation of flux. And allows to highlight sorption, desorption and complexation of trace elements on the substrates. In a second step we evaluate the effect of mineral amendments on the mobility of trace elements in two contaminated soils (extraction), the study their location on the newly formed minerals (microscopy coupled with X-ray fluorescence spectrometers) and finally the leaching of these. The addition of hydroxyapatite (HA) and Steel Shot (GA) have significantly reduced the concentrations of Cd, Zn and As (in the case of the contribution of GA). In contrast, the addition of HA and GA in soils significantly increases the release of As (in the case of HA) and Pb following the addition of GA and HA). Mineral phases carrying these trace elements have been well characterized and the mechanisms responsible for the retention or release have been identified

Metal nanoparticle catalysts have in the last decades been extensively researched for their enhanced performance compared to their bulk counterpart. Properties of nanoparticles can be controlled by modifying their size and shape as well as adding a support and stabilizing agent. In this study, preformed colloidal gold nanoparticles supported on activated carbon were tested on the reduction of 4-nitrophenol by NaBH4, a model reaction for evaluating catalytic activity of metal nanoparticles and one with high significance in the remediation of industrial wastewaters. Methods of wastewater remediation are reviewed, with case studies from literature on two major reactions, ozonation and reduction, displaying the synergistic effects observed with bimetallic and trimetallic catalysts, as well as the effects of differences in metal and support. Several methods of preparation of nanoparticles are discussed, in particular, the sol immobilization technique, which was used to prepare the supported nanoparticles in this study. Different characterization techniques used in this study to evaluate the materials and spectroscopic techniques to analyze catalytic activities of the catalyst are reviewed: ultraviolet-visible (UV-Vis) spectroscopy, dynamic light scattering (DLS) analysis, X-ray diffraction (XRD) analysis and transmission electron microscopy (TEM) imaging. Optimization of catalytic parameters was carried out through modifications in the reaction setup. The effects of the molar ratio of reactants, stirring, type and amount of stabilizing agent are explored. Another important factor of an effective catalyst is its reusability and long-term stability, which was examined with suggestions for further studies. Lastly, a biochar support was newly tested for its potential as a replacement for activated carbon.

Les opérations d'excavation produisent plusieurs tonnes de sols généralement contaminés par la présence de polluants. Les sols excavés sont considérés comme des déchets et sont soit envoyés en décharge, soit destinés à être réutilisés en fonction du niveau de pollution. Dans tous les cas, les sols doivent être correctement traités afin de : (i) diminuer le relargage de polluants dans l'environnement, et (ii) minimiser les problèmes entrainés dans les projets de génie civil liés aux réactions entre les phases cimentaires et les polluants. Dans cette thèse, nous nous sommes concentrés sur les sulfates et le molybdène (Mo). Concernant les sulfates, nous avons considéré deux problématiques principales : (i) l'attaque sulfatique externe des structures en béton qui sont en contact direct avec des sols sulfatés (ex : barrages, fondations), et (ii) le relargage de sulfates en solution, en plus du gonflement et de la perte de résistance mécanique dans des sols sulfatés destinés à la valorisation (ex : réutilisation dans la construction de routes). Dans le cas du Mo, il peut se retrouver en solution, entraînant alors des risques importants pour l'environnement. Par conséquent, dans cette thèse, nous avons étudié la réaction du béton au contact des sulfates et la stabilisation des sulfates dans les sols en utilisant des liants alternatifs afin de réduire leur pollution et envisager leur réutilisation. De plus, nous nous sommes intéressés à l'interaction du Mo avec des liants alternatifs et leur capacité à stabiliser le Mo. Dans un premier temps, nous avons étudié la capacité de sept bétons différents à résister à l'attaque sulfatique externe dans des conditions expérimentales similaires. Le ciment Portland ordinaire a présenté des expansions élevées (>0,1%) en raison de la formation d'ettringite en excès provoquée par la réaction entre les aluminates et les sulfates. Pour le ciment Portland sans C_3A, des expansions plus faibles ont été mesurées, mais l'apparition de fissures à plus long terme a été attribuée à la formation de gypse. Par ailleurs, les liants alternatifs ont présenté de faibles expansions, de l'ordre de 0,01 à 0,03%, expliquées par l'absence de C_3A et de portlandite, en plus de la formation d'ettringite lors de l'hydratation (cas des liants ettringitiques) et de l'absence de calcium (cas du géopolymère à base de métakaolin). Dans un deuxième temps, nous avons comparé la capacité de quatre liants à stabiliser les sulfates dans des sols sulfatés. Les liants ayant une teneur élevée en C_3A ont entrainé des expansions élevées (>5%) à cause de la formation d'ettringite en excès. Ces liants ont également rélargué des métaux lourds en solution du fait de leur teneur élevée en clinker. En revanche, les liants contenant du laitier ont conduit à de faibles expansions (<2%), la rétention des sulfates a été d'environ 89%, et avec un relargage limité de métaux lourds.[...]
Excavation operations produce several tons of soil generally contaminated by the presence of pollutants. Excavated soil is considered as waste and it can be either sent to landfill or destined for reuse depending on the level of pollution. In any case, soil should be properly treated in order to: (i) decrease the release of pollutants into the environment, and (ii) minimize the problems involved in civil engineering applications due to the reactions between cementitious phases and pollutants. In the context of this thesis, we focused on sulfates and molybdenum (Mo). Concerning sulfates, we considered two main issues: (i) external sulfate attack of concrete structures, which are in direct contact with sulfate-rich soil (e.g. dams, foundations), and (ii) the release of sulfates into solution in addition to the swelling and mechanical strength loss in sulfate-rich soil intended for valorization (e.g. reuse in road construction). In the case of Mo, its release into solution is also a serious concern as it can lead to significant risks for the environment. Therefore, in this thesis, we investigated the reaction of concrete in contact with sulfates, and the stabilization of sulfates by using alternative binders for pollution reduction and for reuse of soil. Additionally, we studied the interaction of Mo with alternative binders and their capacity to stabilize Mo. First, we studied the capacity of seven different concretes to resist external sulfate attack under similar experimental conditions. It was found that ordinary Portland cement had high expansions (>0.1%) due to the formation of ettringite in excess caused by the reaction between aluminates and sulfates. Portland cement without C_3A presented lower expansions but gypsum was found to be responsible of cracking at later ages. Meanwhile, alternative binders had low expansions in the range of 0.01-0.03% explained by the absence of C3A and portlandite, in addition to the formation of ettringite during hydration (case of ettringite binders) and the absence of calcium (case of the geopolymer-based metakaolin). Second, we compared the capacity of four different binders to stabilize sulfates in a sulfate-spiked soil. Binders having high C_3A content led to high volume expansions (>5%) caused by the formation of ettringite in excess. These binders also released heavy metals into solution due to their high clinker content. In contrast, binders containing ground granulated blast furnace slag (GGBS) led to low expansions (<2%), sulfate retention was about 89% and lower heavy metals contents were detected in solution. Sulfate solubility was controlled by ettringite, which did not lead to expansion probably due to the low kinetics of precipitation in addition to the absence of portlandite, which is often related to expansive ettringite.[...]

Silica gels containing incorporated heteropolyacids (HPAs) were synthesized in acidic media by co-condensation of tetraethoxysilane (TEOS) with phosphotungstic or phosphomolybdic acids using sol-gel technique. Effect of the synthesis conditions on their structure and morphology was studied. Yields of modified materials were some lower as compared to non-modified silica gels. All materials were mesoporous but contained micropores in their structures. Presence of bands of Keggin’s structures in FT-IR spectra along with absence of XRD patterns of crystalline HPAs confirmed their fine incorporation into silica network. Particle sizes of modified materials were 800-1100 nm excepting for W-containing sample obtained with trimethylstearylammonium chloride. This unusual effect was attributed to stabilization of primary silica nanoparticles by interaction between surfactant and HPA. High ratio HPA/TEOS resulted in partial loss of porosity. Obtained results might be used for optimization of synthesis of effective catalysts and adsorbents containing HPAs in mesoporous structure.

Superacidic mesoporous materials containing covalently embedded PTA were synthesized by sol-gel method. Tetraethyl orthosilicate (TEOS) and phosphotungstic acid (PTA) were used as precursors in the synthesis, ionic and nonionic surfactants were used as pore-forming agents, the reaction proceeded in acidic media. TEM images revealed mesoporous structure with embedded PTA clusters. FT-IR spectra of obtained materials contained characteristic bands of PTA at 957 cm-1. Synthesized catalysts had high BET surface area and high concentration of acidic sites. Alkylation of 1,3,5-trimethylbenzene by 1-decene demonstrated high catalytic activity. The catalyst obtained with Pluronic P123 as a template was the most effective and resulted in highest conversion of 1-decene into alkylated products. Covalent embedding of PTA clusters in addition to thermal and chemical stability of synthesized catalysts enabled their recyclability. Catalysts remained active during subsequent cycles of alkylation.

Quantum dots are semiconductive particles whose properties are highly influenced by the presence of at least one electron. Cadmium selenide quantum dots were synthesized via colloidal synthesis. Contrary to previous preparations, more focus was placed on the temperature rather than the duration of time at which they form. A series of colored solutions were obtained because the excited quantum dots of various sizes emitted specific wavelengths of light. The emission spectra showed that the temperature-dependent quantum dots were successfully synthesized. The quantum dots were also immobilized on various surfaces, and the luminescence properties were examined. The quantum dots that were immobilized in sol-gels through chemiluminescence (CL) analyses were found to be stable and were able to maintain their luminescence properties with extensive uses and long-term storage. Linear calibration curves were obtained for concentrations of hydrogen peroxide from 1.75 x 10-4 M to 1.75 x 10-2 M in TCPO-CL.

Lo scopo di questo lavoro è stato quello di analizzare e di ottimizzare la degradazione fotocatalitica del blu di metilene, partendo innanzitutto da un’accurata ricerca bibliografica mirante all’individuazione di possibili migliorie da apportare ad alcuni dei fattori partecipanti al processo. Le modifiche apportate a tali fattori sono state, inoltre, testate sperimentalmente sia dal punto di vista radiometrico che delle performance chimiche di degradazione del blu di metilene, allo scopo di ottenere un confronto e una possibile correlazione tra i due diversi aspetti. I film di biossido di titanio (TiO2), usati come fotocatalizzatori, una volta applicati a fibre ottiche utilizzate come mezzi, sono state attivate ​​con irradiazione UV-LED. Diverse sono le soluzioni di rivestimento utilizzate e sottoposte a confronto: tra queste sono state considerate soluzioni a dispersione e sol-gel, tutte applicate alle fibre ricorrendo alla tecnica di rivestimento a immersione. Gli aspetti che sono stati messi in luce dalle sperimentazioni riguardano la resistenza meccanica del rivestimento e la dipendenza dalla lunghezza dello stesso, le modifiche applicate al reattore e alle fibre ottiche, i rivestimenti multipli e la dipendenza dalla potenza energetica fornita. Per ciascuno di questi sono stati conseguiti risultati interessanti, anche se la resa quantica ottenuta è risultata essere modesta e senz’altro migliorabile ulteriormente. Il confronto diretto tra il rivestimento a immersione tramite una dispersione rispetto al metodo sol-gel ha rivelato una stabilità meccanica e un’efficienza di resa quantica del secondo molto più elevata. Come metodo di rivestimento, il sol-gel si è dimostrato vantaggioso rispetto al semplice rivestimento a dispersione: la durata dello strato sol è significativamente maggiore, il che ne rende preferibile l'uso nel trattamento delle acque reflue.

碩士
長庚大學
化工與材料工程研究所
98
Lipase from Candida Rugosa was immobilized by entrapment in sol-gel matrix with superparamagnetic nanoparticles to catalyze the transesterification in hexane. To get the optimum value of specific activity, the experiment design was utilized. This assay includes two part experiment design, which one is to forecast the optimum value of specific activity of different immobilized conditions, another is that of different reaction condition. The parameter which is taken into consideration of immobilized condition includes the molar ratio of water and silane, amount of PTMS, addition of Enzyme, amount of additive-PVA and hydrolyzed time of silane. The result showed a quite apparent influence of amount of PTMS and hydrolyzed time of silane to specific activity when proceed enzyme immobilization. The parameter which thinked to be the factor of impacting specific activity contains the amount of additional water , concentration of Geraniol and reaction temperature. The result showed a apparent raise of specific activity after the enzyme was entrapped in sol-gel matrices. It is 8 times of immobilized enzyme to free enzyme. Moreover, the immobilized enzyme exhibits great reusability and thermal stability. To sum up, the enzyme after immobilized by sol-gel matrices represents a more stable conformation and activity then free enzyme.

Although enzymes show great potential for use in industrial applications, their implementation from a practical perspective is still somewhat limited by various shortcomings in the area of enzyme immobilization. The use of silica sol-gels for protein entrapment has been studied extensively over the past 15 years or so. However, our understanding of the interactions between the immobilization matrix and the entrapped biomolecules is still relatively poor. Non-invasive in situ spectroscopic characterization is a promising approach to gain a better understanding of the fundamentals governing sol-gel immobilization. This thesis describes the application of Fourier transform infrared (FTIR) microscopy, two dimensional (2D) FTIR and fluorescence spectroscopy to characterize the immobilization matrix, entrapped model proteins and their interactions. Hydroperoxide lyase (HPL [E.C. 4.1.2.]) was chosen as a potential model protein for sol-gel entrapment. HPL activity was evaluated by use of factorial experimental design investigating the effects of KCl and Triton X-100 on HPL activity with 13-hydroperoxy-octadecadienoic acid (LA-OOH) and the novel water soluble 13-hydroperoxy-octadienoyl sulfate (LS-OOH) as substrates. The highest HPL activity was achieved under aqueous conditions with high salt and low surfactant concentrations and LA-OOH as the substrate. A significant interaction between salt and surfactant as well as salt and substrate was identified and a hypothesis to explain the basis of the interaction phenomena is presented. To analyze sol-gels with spectroscopic techniques, a sample format amenable to these techniques was needed. Therefore, a spin-coating technique for the preparation of aluminum or glass supported sol-gel thin films containing immobilized protein and a varying degree of the organically modified precursor propyltrimethoxysilane (PTMS) was developed. This approach produced samples that were suitable for chemical mapping using FTIR microscopy or fluorescence spectroscopic investigations. A data analysis method was developed to extract information on chemical speciation and distribution from FTIR data matrices obtained through FTIR microscopy. Results indicate that sol-gel thin films are not homogeneous on the microscopic level. Instead, they are heterogeneous with a clustering in the distribution of the model proteins studied (lysozyme [E.C. 3.2.1.17], lipase [E.C. 3.1.1.3] and bovine serum albumin (BSA)) at the scale investigated. The appearance of these clusters was found to depend on the type of protein entrapped, as well in some cases on the composition of the sol-gel. Moreover, the PTMS distribution was positively correlated with the protein distribution in the case of lipase and negatively correlated in the case of lysozyme and BSA. Additionally, sol-gels with a higher PTMS content appeared to conserve protein structure in areas where lipase clustered. Lysozyme and BSA, on the other hand, seemed to retain their structures in high concentration clusters better at lower PTMS content. A hypothesis taking into account the surface hydrophobicity of the proteins and the sol-gel composition as the basis for these phenomena is proposed. Fluorescence spectroscopy revealed that the PTMS content of the sol-gels had a direct effect on the physical properties of the immobilized proteins as evidenced by a blue shift of the intrinsic tryptophan (TRP) fluorescence. Temperature-dependent fluorescence spectroscopy revealed that the amount of TRP quenching was inversely proportional to the PTMS content of the sol-gel, suggesting that there were varying amounts of water available for quenching for the different immobilized enzyme systems. Analysis of the sol-gels by 2D FTIR spectroscopy with a focus on the amide A region using Gaussian peak deconvolution revealed two different species of water for the 50 % PTMS thin film sol-gels with BSA that could be described as fully and not fully H-bonded. It was also found that these species of water showed different removal profiles during thermal treatment. 2D FTIR of the amide I region followed by absorbance difference spectrum evaluation revealed that the temperature stability of the three model proteins was also sol-gel composition dependent. A hypothesis that the surface characteristics of the proteins determine the nature of the composition dependence is presented.

碩士
長庚大學
化工與材料工程學系
99
In this study，magnetic nanoparticles were used as carriers for Immobilization of thrombolytic drug to expand a magnetic targeted drug delivery system. Magnetic nanoparticles prepared by sol-gel method were characterized by TEM, FTIR, SQUID, light-scattering, TGA and XRD,.Next,the silicon oxide nanoparticles functionalized with 3-aminopropyltriethoxysilane (APTES) which provides –NH2 fuctional group for conjugating with thrombolytic agents after activation with glutaraldehyde.Optimum systhesis conditions have been identified. Recombinent tissue plasminogen activator (rt-PA) were immobilized to the best magnetic silicon oxide nanoparticles. The optimum drug loading is reached when 0.5 mg rt-PA is conjugated with 5 mg magnetic silicon oxide nanoparticles where 91% drug is attached to the carrier.Immobilized drug could retain it’s activity to 86.5% compared with free drug.

Thesis (Ph. D.)--University of Georgia, 2004.
Directed by William S. Kisaalita. Includes articles submitted to Applied microbiology and biotechnology, Biosensors & bioelectronics, and Journal of envrronmental science and health. Includes bibliographical references.

碩士
長庚大學
化工與材料工程研究所
92
Due to its excellent piezoelectric properties, lead zirconium titanates (PZT) are the most promising and the most practical piezoelectric film materials for sensing applications. In this thesis, we grew PZT films on the Si wafers and also investigated the performances of the surface acoustic wave devices made by PZT films deposited on the Si(100) wafers. To employ the SAW devices in bio-sensor applications, we utilized the immobilization of self-assembled monolayers (SAM) within the delay line area of PZT SAW devices. Some modification processes were also used to make the biosensors more sensitive and reliable. According to the experimental results, the weight of molecules immobilized at each of modification steps could be found. They are 601,30,25,109 ng for the SAM modification ,active modification, POD modification and SAv modification steps respectively. In addition, we could also recognize the weights of Biotin-BSA molecules and anti-BSA molecules immobilized after SAv modification process. Their weights are 20ng and 18ng respectively.

Im Bereich der Umwelttechnik bieten sich vielversprechende Einsatzmöglichkeiten für Sol-Gel-immobilisierte Mikroorganismen sowohl für die Entfernung als auch für die Detektion von Schadstoffen an. Für einen effizienten Einsatz sind zum einen eine hohe Langzeitaktivität und -vitalität der eingebetteten Zellen als auch eine gute Lagerbeständigkeit wichtig. Neben einer hohen Makroporosität, die sowohl für einen guten Stoffaustausch mit der Umgebung sorgt sowie den Zellen Raum für Zellteilung bietet, ist vor allem auch die Aufrechterhaltung der Feuchtigkeit in der Immobilisierungsmatrix während der Immobilisierung, der Lagerung und des Einsatzes wichtig. Besonders vorteilhaft hat sich hier eine Immobilisierung in dünnen SiO2-Schichten auf Blähtonbruchstücken erwiesen, da dieses Trägermaterial neben einer hohen Makroporosität auch ein hohes Wasserspeichervermögen besitzt. Immobilisiert in dünnen SiO2-Schichten auf Blähton konnten diverse Schadstoff-abbauende Mikroorganismen mehrere Monate auch außerhalb eines flüssigen Mediums unter feuchten Bedingungen gelagert werden, ohne dass ihre Abbauleistung erheblich sank. Ein weiteres Verfahren um Immobilisierungsmaterialien mit überdurchschnittlich hoher Makroporosität bei gleichzeitig hoher Stabilität zu erzeugen, stellt das Freeze-Gelation Verfahren dar. Über einen Gefriertrocknungsschritt können hier zudem die zu immobilisierenden Zellen in eine trocken lagerfähige Form überführt werden, wodurch Handhabung sowie Lagerung und Transport vereinfacht werden. Außerdem kann durch Wahl der Einfrierbedingungen und Zugabe von Füllstoffen zu dem SiO2-Sol entscheidend die Porenstruktur der Immobilisierungsmatrix beeinflusst und den Anforderungen entsprechend eingestellt werden. Allerdings zeigte sich, dass diese Methode nicht für alle Mikroorganismen gleichermaßen geeignet ist. Trotz diverser kryoprotektiver Maßnahmen konnte keine ausreichende Überlebensrate für sensible Stämme wie Aquincola tertiaricarbonis L108 erzielt werden. Neben der Erhöhung der Makroporosität der SiO2-Matrix wurde versucht das Sol-Gel-Verfahren mit einem flexiblen organischen Polymer (Na-Alginat) zu kombinieren, um eine Immobilisierungsmatrix mit einem weichen Kern zu erzeugen, der Zellteilung zulässt. Als zusätzlicher Vorteil des entwickelten Alginat/SiO2-Hybridmaterials erwies sich, dass dieses auch auf einfache Weise mittels Drucktechniken in definierten Spots abgelegt werden kann und so die Zellen in Arrays abgelegt werden können. Das Potential dieser Methodik für die Entwicklung von Ganzzellsensoren wurde am Beispiel der Grünalge Chlorella vulgaris als Modell-Sensorzelle demonstriert und für die Detektion von Atrazin als Modellsubstanz eingesetzt.:Inhalt Abstract 1 Danksagung/Vorwort 2 Inhalt 4 Abkürzungsverzeichnis 6 Abbildungsverzeichnis 8 Tabellenverzeichnis 11 1 Einleitung und Motivation 12 1.1 Zielstellung der Arbeit 14 2 Grundlagen 16 2.1 Sol-Gel-Verfahren 16 2.1.1 Geschichte des Sol-Gel-Verfahrens 16 2.1.2 Chemische Grundlagen des Sol-Gel-Verfahrens 18 2.1.3 Prekursoren (Vorläufermaterialien) 19 2.1.3.1 Alkoxysilane (Siliciumalkoxide) 19 2.1.3.2 Alkalisilikate 23 2.1.4 Modifizierungsmöglichkeiten der SiO2-Matrix 24 2.1.4.1 Chemische Modifizierung 24 2.1.4.2 Physikalische Modifizierung 25 2.1.4.3 Organisch-anorganische Hybridmaterialien 26 2.1.4.4 Alginat/SiO2-Hybridmaterialien 26 2.2 Sol-Gel-Immobilisierung von Mikroorganismen 28 2.2.1 Sol-Gel-Beschichtung von Trägermaterialien 30 2.2.2 Sol-Gel-Immobilisierung in (Hydro-)Gelkörpern 33 2.2.2.1 Sonderform: Freeze-Gelation-Formkörper 35 2.2.3 Sol-Gel-Immobilisierung mittels Drucktechniken 37 3 Material und Methoden 38 3.1 Mikroorganismen 38 3.2 Freeze-Gelation-Verfahren 39 3.2.1 Zellimmobilisierung 39 3.2.2 Kryoprotektektive Maßnahmen 40 3.2.3 Charakterisierung der Freeze-Gelation-Formkörper 40 3.2.4 Aktivitätsuntersuchung – Schadstoffabbau 41 3.3 Beschichtung von Trägermaterialien 42 3.3.1 Synthese des SiO2-Sols 42 3.3.2 Vorbehandlung der Trägermaterialien 42 3.3.3 Zellimmobilisierung 42 3.3.4 Charakterisierung der Trägermaterialien 43 3.3.5 Aktivitätsuntersuchung – Schadstoffabbau 44 3.4 Alginat/SiO2-Hybridgele 44 3.4.1 Synthese amino-funktionalisierter SiO2-Sole 44 3.4.1.1 Charakterisierung der amino-funktionalisierten SiO2-Sole 45 3.4.2 Vernetzung von Na-Alginat mit amino-funktionalisiertem SiO2-So (Erzeugung von Alginat/SiO2-Hydrogelen) 45 3.4.2.1 Vorbehandlung der Trägermaterialien 45 3.4.2.2 Charakterisierung der Alginat/SiO2-Hydrogele 46 3.4.3 Zellimmobilisierung 47 3.4.3.1 Charakterisierung der immobilisierten Zellen 48 3.4.4 Aktivitätsuntersuchung – Schadstoffdetektion 48 3.4.4.1 PAM-Fluorometer: Atrazin 48 4 Ergebnisse und Diskussion 50 4.1 Lösungsstrategien 50 4.2 Freeze-Gelation-Formkörper 54 4.3 Sol-Gel-Beschichtung von Trägermaterialien 65 4.4 Alginat/SiO2-Hybridmaterialien 74 4.5 Zusammenfassung der Ergebnisse 93 5 Schlussfolgerung und Ausblick 99 6 Literaturverzeichnis 102 7 Verzeichnis eigener Publikationen 109 ANHANG 111

博士
國立中山大學
化學系研究所
92
Based on the linear relationship between concentration of H2O2 and the decrease of electrochemiluminescence (ECL) intensity in a Ru(bpy)32+/TPA system, procedures for the indirect determination of glucose with a flow injection analysis were developed. By passing solutions of glucose through a FIA system containing a glucose oxidase (GOx) immobilized sol-gel column and an ECL system of Ru(bpy)32+ and TPA, glucose can be determined optimally with a detection limit of 1.0 μM in a linear dynamic range of 1.0 – 200.0 μM. A repetitive injection of glucose (100 μM) and human serum solutions gave satisfactory reproducibility with relative standard deviations of 1.3 (N=31) and 3.9 % (N=42) respectively. Interference due to the presence of ascorbic acid, uric acid or other reducible agents in solution can be corrected by passing sample solutions through another sol-gel column that contained no GOx. From the agreement between the contents of glucose in human serum and soft drink analyzed by the developed method and those obtained by the spectroscopy method based glucose assay kit and satisfactory recovery of glucose from interferent containing solutions, the feasibility of the developed method for real sample analysis was confirmed. One of the major purposes of this study was to develop new immobilization approaches and flow cell designs for the fabrication of regenerable ECL-based sensors with improved sensitivity, convenience and long-term stability. Silica particulates were used as immobilization support in ECL sensors for TPA and NAD(P)H and in biosensors for glucose and glucose-6-phosphate（G6P）. The first ECL flow cell was fabricated from a glass tube, and a platinum wire was used as working electrode held at +1.3 V. The volume of the flow cell was about 50 μL. An Ag/AgCl electrode and a piece of Pt wire were used as the reference and counter electrode respectively and placed downstream of the working electrode. Ru(bpy)32+ immobilized silica particulates with 1/3 silica sol content showed the best performance for TPA determination, and the sensitivity of TPA determination was dependent upon the amount of Ru(bpy)32+ immobilized in silica particulates. The lowest level of analyte detected for TPA was 0.02μM, and linear range was from 0.02μM to 5μM. Up to a certain concentration level, it was found that Ru(bpy)32+ was tightly held in silica particulates and did not leach out into aqueous solutions, even with continuous flow for up to ten hours. Ru(bpy)32+ immobilized silica particulates were characterized of well activity and high stability; that stored at 0℃ exhibited its original activity for up to one year. The second ECL flow cell was fabricated from a piece of epoxy block supported Pt electrode (1 × 2 cm) as counter electrode, a piece glass window and a polyethylene spacer with 78 μL cell volume, two 2.0-cm length of 0.6-mm diameter platinum wires were used as working electrodes held at +1.1 V, and an Ag/AgCl electrode as reference electrode. All three electrodes were incorporated within the main body of the cell. One of the biosensor design packed Ru(bpy)32+ incorporated silica particulates in the ECL flow cell, and a glucose dehydrogenase (GDH) immobilized silica sol-gel column is placed between the sample injection valve and the flow cell. The ECL response to samples containing glucose and cofactor (NADP) results from the Ru(bpy)33+ ECL reaction with NADPH produced by glucose dehydrogenase. This ECL biosensor was shown applicable for both NAD+- and NADP+- dependent enzymes, where NADH detection ranged from 0.50μM – 5.0 mM NADH and NADPH detection ranged from 1.0μM - 3.0 mM NADPH. Glucose can be determined in a linear dynamic range of 5.0 - 500 μM. Another biosensor design immobilized glucose-6-phosphate dehydrogenase（G6PDH）onto the Ru(bpy)32+ -doped silica particulates through silica chemistry and then packed these particulates into the ECL flow cell. By passing samples containing G6P and cofactor (NAD) through the ECL flow cell, G6P can be determined in a linear dynamic range of 10.0 μM-1.0 mM. The regenerable ECL biosensor was characterized of good reproducibility and well stability for flow injection analysis. A repetitive injection of NADH (100 μM) and G6P（500μM）gave satisfactory reproducibility with relative standard deviations of 2.8 %（N=105）and 2.8 % (N=40) respectively.

碩士
國立中正大學
化學研究所
90
Abstract Sol-gel technique has been used for the immobilization of lactate dehydrogenase (LDH) and β-nicotinamide adenine dinucleotide (NAD+). In this study, two approaches have been used to immobilize LDH in the sol-gel matrices. First, the immobilization of LDH is based on the electrostatic interaction between the silica material and LDH via a cationic polymer. Since polyethyleneimide (PEI) is a cationic polymer with positive charges, it can be adsorbed on the negatively charged surface of LDH. Hence, when PEI is added in the silica matrices, Km,s = 0.52 ± 0.04 mM, T.O.N = 6.50 ± 0.01 min-1. In addition, an organofunctional silica alkoxide precursor, TESPGA, was also used to modify the sol-gel matrices. This precursor contains a glucose-like group and is expected to enhance the compatibility of LDH with silica material. As a result, when TESPGA is used as the precursor, Km,s = 0.39 ± 0.001 mM, T.O.N = 21.70 ± 5.82 min-1. In both cases, the apparent affinity increase and the apparent activity decreases as compared to that of the solution form (Km,s =0.767 ± 0.011 mM, T.O.N = 1.81×103 ± 0.27×103 min-1. Second, the immobilization of LDH is based on covalent linking of LDH with APTES. Although immobilization based on electrostatic interaction can enhance the affinity between LDH and lactate, it dose not prevent LDH to be leached from the silica matrices. On the other hand, by immobilization of LDH in the silica matrices with covalent linking, it is able to immobilize LDH in the silica matrices without leaching. The apparent affinity between LDH and lactate, Km,s = 0.27 ± 0.01mM, is similar to that of the first approach. However, the apparent activity of LDH decreases, T.O.N = 7.11 ± 0.13min-1. Even though the apparent activity is not as high as that in solution form, the activity is enough for the development of a continuous sensing method for optical biosensing of lactate.

The objective of this work was to develop a novel enzyme immobilization scheme for supported lipase sol-gels and to evaluate the potential of the immobilized biocatalyst for the production of biodiesel in a packed bed reactor. Two sources of lipase (EC 3.1.1.3 triacylglycerol hydrolase) were used in this study and the transesterification of methanol and triolein to produce glycerol and methyl oleate was used as a model reaction of biodiesel production. A commercially available form of immobilized lipase, Novozym® 435, was used as a basis for comparison to the literature. Upon establishing a lipase sol-gel formulation technique, the experimental methodology for the transesterification reaction using Novozyme® 435 was developed. Subsequently, a series of inert materials were considered based on their suitability as supports for immobilized lipase sol-gels and the synthesis of methyl oleate. The value of a supported lipase sol-gel is to improve the activity and stability of the enzyme and develop an immobilized biocatalyst that is practical for use under packed bed reactor conditions. Of the six support materials considered (6-12 mesh silica gel, Celite® R633, Celite® R632, Celite® R647, anion exchange resin, and Quartzel® felt), the diatomaceous earth supports (Celite® R633, R632 and R647) exhibited high enzymatic activity, were thermally stable, and possessed high sol-gel adhesion. From the three types of diatomaceous earth considered, Celite® R632 supported lipase sol-gels were identified as the most promising supported lipase sol-gels for methyl oleate production via transesterification. Upon further evaluation, the Celite® R632 lipase sol-gels were found to achieve high methyl oleate percent conversions, glycerol-water absorption was only significant at glycerol levels higher than 75%, and the immobilized lipase had high stability upon storage at 4°C for 1.5 years. To determine the effects of methanol and glycerol inhibition as well as temperature on the reaction kinetics, a ping-pong bi-bi kinetic model was developed and validated over a range of methanol concentrations and temperatures. The optimal methanol concentration for the conditions tested was in the range of 1.3 M to 2.0 M, and increased with increasing temperature. The model developed was consistent with the experimental data and confirmed that glycerol inhibition and the presence of products had significant effects on the reaction kinetics. The methyl oleate production capabilities of the Celite® supported lipase sol-gel were investigated using a packed bed reactor and compared with Novozym® 435 under similar operating conditions. A kinetic and mass transfer based model was developed for the reactor system using a novel efficiency correlation to account for the effect of glycerol on the enzymatic activity. Increasing the flow rate (1.4 mL/min to 20 mL/min) increased the reaction rate, presumably due to the reduction of the glycerol inhibition effect on the immobilized biocatalyst. The Celite® supported lipase sol-gel was found to have superior performance over Novozym® 435 both under batch stirred tank reaction conditions and in a packed bed reactor (83% conversion for Celite® sol-gel vs. 59% conversion for Novozym® 435 at 20 mL/min in the packed bed reactor). Based on the results obtained, Celite® supported lipase sol-gels exhibited good performance for the transesterification of triolein with methanol to produce methyl oleate in both batch and packed bed reactors, and warrant further exploration for the enzymatic production of biodiesel.

Charles University, Faculty of Pharmacy in Hradec Králové Department of: Pharmaceutical Technology Consultant: PharmDr. Ondřej Holas, Ph.D. Student: Helena Badalcová Title of Thesis: Immobilization of protein macromolecules onto polymer carriers: An overview Since the 70s, the immobilised enzymes have been getting the attention of not only scientific and laboratory workers, but also industrial companies. Enzymes are unique biocatalysts, which are distinguished by their specificity, environment-friendliness and the ability to react under mild conditions can be easily subject of denaturation or inhibition. With regard to the usually high cost of purchase, the use of these enzymes could often be disadvantageous. Immobilization techniques offer an efficient solution to this problem and greatly simplify the use of enzymes in industry and research. Compared to the free forms, immobilized enzymes show greater activity, stability and allow repeated use as well as easier separation from products. This thesis contains an overview of the basic methods of immobilization - physical absorption and covalent bonds to the carrier, entrapment, encapsulation and carrier- free techniques using cross-linking. Finally, we outline possible biomedical applications as well as the use of immobilised enzymes in biosensors.

Study of biosensors has become an essential part of research in biotechnology. Biosensors as fast, portable, highly sensitive, and low-cost bioanalytical detection devices have been utilized in many fields of human activity. The first part of the presented work focuses on electrochemical biosensors for rapid environmental screening of herbicides as water pollutants. A sol-gel immobilization method for a photosystem II (PSII) complex is studied in order to enhance the sensitivity and the signal strength and stability of a PSII-based biosensor. Computer simulations of a PSII biosensor are employed with the aim to find out how the immobilization membrane properties influence the biosensor parameters. Newly developed immobilization by a thin-layer membrane based on the results of computer simulations and revised measurement protocols are presented. The second part of the work is devoted to synthesis and electrochemical detection of newly developed metal labels for electrochemical immunosensors. The synthesis of dendrimer-encapsulated silver nanoparticles and biorecognition properties of biotin-nanocomposite conjugates are discussed. For detection of synthesized labels, a microfluidic detector was manufactured and tested and different approaches to packing of a microfluidic chip employing polydimethylsiloxane (PDMS) were investigated. Newly designed microstructures for a microfluidic separator of magnetic beads (MBs) were studied by computer simulations. The separator was made and trapping of MBs for the further employment in MBs-based immunoassays are presented