Rozprawy doktorskie na temat „Selective enrichment”

Thesis (Ph. D.)--University of Maryland, College Park, 2008.
Thesis research directed by: Dept. of Chemistry and Biochemistry. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.

The BCNU + O6benzylguanine (O6BG) driven selective enrichment strategy was first established for enhanced transplantation of hematopoietic stem cells. This study describes a novel application of this BCNU + O6BG driven selective enrichment strategy in skeletal muscle stem cell transplantation. Furthermore, this study addresses the three main limitations observed in previously reported skeletal muscle stem cell transplantation strategies. Limitation of ineffective donor cells which lack the ability for successful engraftment was overcome by using a heterogeneous population of donor cells which are present during a normal skeletal muscle regeneration response. The limitation of donor cell death upon transplantation as a result of competition from the endogenous stem cells of the host muscles was overcome by elimination of host muscle stem cells with BCNU + O6BG treatment. Efficiency of elimination of host muscle stem cells was further demonstrated by the complete inhibition of a regeneration response up to 3 months in injured, BCNU + O6BG treated muscles. The limitation of localised engraftment as a result of intramuscular injection of donor cells was also addressed. The transplanted donor cells demonstrated the ability to migrate via systemic circulation. This characteristic of the donor cells would allow the transplantation of cells via intraarterial or intravenous delivery which would overcome the limitation of localised engraftment. Finally, application of the BCNU + O6BG driven selective enrichment strategy in skeletal muscle stem cell transplantation demonstrated enhanced engraftment. This is the first reported attempt of enhanced stem cell transplantation in a solid tissue achieved upon application of the BCNU + O6BG driven selective enrichment strategy. This study provides the basis for application of the BCNU + O6BG driven selective enrichment strategy in other tissues where stem cell transplantation is considered.

Doctor of Philosophy (PhD)
The BCNU + O6benzylguanine (O6BG) driven selective enrichment strategy was first established for enhanced transplantation of hematopoietic stem cells. This study describes a novel application of this BCNU + O6BG driven selective enrichment strategy in skeletal muscle stem cell transplantation. Furthermore, this study addresses the three main limitations observed in previously reported skeletal muscle stem cell transplantation strategies. Limitation of ineffective donor cells which lack the ability for successful engraftment was overcome by using a heterogeneous population of donor cells which are present during a normal skeletal muscle regeneration response. The limitation of donor cell death upon transplantation as a result of competition from the endogenous stem cells of the host muscles was overcome by elimination of host muscle stem cells with BCNU + O6BG treatment. Efficiency of elimination of host muscle stem cells was further demonstrated by the complete inhibition of a regeneration response up to 3 months in injured, BCNU + O6BG treated muscles. The limitation of localised engraftment as a result of intramuscular injection of donor cells was also addressed. The transplanted donor cells demonstrated the ability to migrate via systemic circulation. This characteristic of the donor cells would allow the transplantation of cells via intraarterial or intravenous delivery which would overcome the limitation of localised engraftment. Finally, application of the BCNU + O6BG driven selective enrichment strategy in skeletal muscle stem cell transplantation demonstrated enhanced engraftment. This is the first reported attempt of enhanced stem cell transplantation in a solid tissue achieved upon application of the BCNU + O6BG driven selective enrichment strategy. This study provides the basis for application of the BCNU + O6BG driven selective enrichment strategy in other tissues where stem cell transplantation is considered.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2020
Cataloged from PDF of thesis.
Includes bibliographical references (pages 182-200).
Rapid and reliable detection of ultralow-abundance nucleic acids and proteins in complex biological media may greatly advance clinical diagnostics and biotechnology development. Because of the slow mass transport and weak binding kinetics at ultralow concentration of target biomolecules, enrichment of target biomolecules plays an essential role in the detection of ultralow-abundance biomolecules. Currently, nucleic acid tests rely on enzymatic processes for target amplification (e.g. polymerase chain reaction), which have many inherent issues restricting their implementation in diagnostics. On the other hand, there exist no protein amplification techniques, greatly limiting the development of protein-based diagnosis.
By learning from the desired and undesired features of existing techniques, we designed the blueprint of the next-generation biomolecule enrichment technique, which should ideally be universally applicable to all kinds of biomolecules and be capable of specifically enriching only the target biomolecules among the background biomolecules by billion-fold rapidly. Electrokinetic concentration is a promising candidate for the next-generation biomolecule enrichment technique, because of its simple architecture and ease of operation, high concentration speed, universal applicability, and the rich physics of the system that may enable the development of new functionalities. We defined a technical roadmap of engineering the primitive electrokinetic concentration technique toward the next-generation biomolecule enrichment technique. We start by deciphering the mechanism of electrokinetic concentration (Chapter 2), which is instrumental in the rational design and innovation of the system.
We next developed specific enrichment of target biomolecules in the electrokinetic concentrator based on electrophoretic mobility-based separation and mobility engineering of affinity binders (Chapter 3). We went on to realize the billion-fold enrichment capability of electrokinetic concentrator by massive parallelization and hierarchical cascading of unit electrokinetic concentrators (Chapter 4). After that, we demonstrated the engineered electrokinetic concentrator as an integrated, self-contained platform for universal amplification-free molecular diagnostics (Chapter 5). Finally, we interfaced the engineered electrokinetic concentrator with standard analytics to enhance their analysis sensitivity and greatly simplify their workflows (Chapter 6). At the end of the thesis, we conclude this thesis and present our outlooks on the future directions (Chapter 7).
by Wei Ouyang.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第19037号
農博第2115号
新制||農||1031(附属図書館)
学位論文||H27||N4919(農学部図書室)
31988
京都大学大学院農学研究科森林科学専攻
(主査)教授 神﨑 護, 教授 北島 薫, 教授 北山 兼弘
学位規則第4条第1項該当

Challenges encountered in pathogen identification and detection include the genetic heterogeneity of strains within species of some foodborne pathogens, isolation of injured cells, mixed strains or mixed species contamination of foods, and differentiation between viable and dead cells. The first objective of this research was to evaluate an isolation medium that was based on time-delayed release (5 to 6 h) of selective agents in tablet format to a modified Listeria recovery enrichment broth (mLRB) medium for enhanced and rapid recovery of injured Listeria. The second objective involved the use of Fourier transform infrared (FT-IR) spectroscopy and chemometric analysis for the differentiation of: Listeria monocytogenes epidemic clones (ECs); viable versus heat-killed populations; different mixed strains and mixed species of Listeria; and different injury treatments and repair in Listeria populations. Nitrite- or acid-injured Listeria at approximately 10 CFU/ml were recovered in mLRB medium, and cell populations enumerated at various times (12 to 48 h) of incubation at 37oC. Analysis of variance revealed that acid-injured Listeria populations in mLRBS6 (mLRB plus the selective agents at 6 h) were significantly higher (P < 0.05) than those in mLRBS0 (mLRB plus the selective agents at 0 h) at 24 h; however, the differences in populations on these two media were not significant for nitrite-injured Listeria. Cell populations of four strains of Listeria recovered in mLRBTD (mLRB plus the time-delayed release tablets of the selective agents) were significantly higher than when those strains were enriched in the U.S. Food and Drug Administration (FDA), International Organization for Standardization (ISO), and U.S. Department of Agriculture (USDA) broths at 24 h. Comparison between artificially contaminated milk and meat samples with a four-strain cocktail of Listeria resulted in cell populations that were significantly higher (P < 0.05) on mLRBTD for contaminated meat than on mLRBTD for contaminated milk at 24 h. FT-IR spectroscopy in the mid-infrared region (4000 to 600 cm-1) and chemometrics was successfully applied to discriminate L. monocytogenes strains belonging to the same EC (ECII or ECIV) (100% accurate spectral classification), intact and heat-killed populations of each EC strain (100% accurate spectral classification), and spectral wavenumbers 1650 to 1390 cm-1 were used to differentiate heat-killed from intact populations. FT-IR spectroscopy and chemometrics in the wavelength region 1800 to 900 cm-1 could successfully discriminate different mixed strains of L. monocytogenes (98.15% accurate spectral classification) and different mixed species of L. monocytogenes and L. innocua (92.06% accurate spectral classification) from individual strains; Wavelength range 1800 to 900 cm-1 was successfully used to discriminate between intact, acid-injured, and heat-injured Listeria, with repaired cells from acid and heat treatments clustering closer to intact cells (93.33% of spectra accurately classified). Delayed-addition of selective agents to broth medium improves recovery of injured Listeria by allowing repair time, could minimize contamination through manual addition of selective agents, and saves analyst time; FT-IR spectroscopy is a highly discriminatory and reproducible technique that can be used for the differentiation of strains and various physiological states of Listeria.

O Mycobacterium bovis causa a tuberculose bovina, doença zoonótica que propaga, provavelmente com baixa prevalência, em todo o território nacional e pode ser transmitida pelo leite. A adoção sistemática da pasteurização do leite contribuiu para a redução dos casos humanos da doença, mas em alguns países, como o Brasil, é comum o consumo de leite cru e de seus derivados, o que pode contribuir para ocorrência de casos humanos. A participação desse agente nos atuais índices de tuberculose humana, cujo principal agente é o M. tuberculosis, é pouco investigada, mas acredita-se que seja maior do que parece. As razões que contribuem para isso incluem o fato do tratamento humano ser o mesmo independentemente do agente,e as dificuldades e alto custo de distinguir as espécies. O método de diagnóstico \"gold standard\" em laboratório para Mycobacterium bovis em amostras clínicas é o isolamento do agente em meios como Stonebrink-Leslie ou similares. A riqueza em nutrientes dos meios, mais o lento metabolismo deste agente e o alto grau de contaminantes das amostras torna imprescindível o uso de descontaminantes que, via de regra, são tóxicos para o agente, dificultando o isolamento em amostras com níveis baixos do patógeno; cenário provável no caso do leite devido à mistura com o leite de animais sadios. Mas, a despeito de constituir-se uma importante zoonose transmitida pelo leite, não existe uma metodologia oficial para detecção desse agente em alimentos lácteos. Esses fatos justificam um estudo para avaliar o desempenho de meios líquidos, já utilizados em caros sistemas automatizados para detecção de micobactérias, como alternativa para um enriquecimento seletivo do M. bovis em amostras lácteas. Assim, amostras de leite integral esterilizado e de queijo tipo parmesão foram contaminadas com 10 a 100 UFC/ml ou g de M. bovis AN5 e enriquecidas em dois meios líquidos seletivos (MGIT e MGIT modificado), foram analisados nos dias 0, 7, 14, 21, 24, 28 e 32, mantidas em estufa a 37°C. Nessas datas, uma alíquota foi semeada em meio Stonebrink-Leslie e incubada a 37°C por 60 dias. No leite, houve crescimento exacerbado do M. bovis principalmente no MGIT modificado. No queijo, não foi possível isolar M. bovis de nenhuma amostra em MGIT modificado, devido à contaminação excessiva que deteriorou o meio de cultura. O MGIT modificado foi mais eficaz como enriquecimento para o Mycobacterium bovis, mas foi menos seletivo que o MGIT. Estudos futuros devem concentrar a investigação da curva de crescimento do agente na primeira semana de enriquecimento.
Mycobacterium bovis causes bovine tuberculosis, zoonotic disease raging, probably with low prevalence, throughout the national territory and can be transmitted through the milk. The systematic adoption of milk pasteurization helped reduce human cases of the disease, but in some countries, like Brazil, is the common consumption of raw milk and its derivatives, which may contribute to the occurrence of human cases. The participation of this agent in the current rates of human tuberculosis, the principal agent is M. tuberculosis, it is not investigated, but it is believed to be larger than it looks, the reasons contributing to this include the fact that human treatment is similar regardless of the agent and the difficulty / cost to distinguish between species. The diagnostic method \"gold standard\" for Mycobacterium bovis in clinical samples is the isolation of the agent means, Leslie as Stonebrink or the like. The species richness in nutrient media, over the slow metabolism this agent and the high degree of contamination of the samples makes indispensable using decontaminant that, as a rule, are toxic to the agent, making isolation in samples with low levels of the pathogen; scenario likely due to the milk mixture with the milk of healthy animals. But, despite constitute an important zoonosis transmitted by milk, there is no official method for detection of this agent in dairy foods. These facts justify a study to evaluate the performance of liquid media, as used in expensive automated systems for detection of mycobacteria, as alternative to a selective enrichment of M. bovis in milk samples. Thus, samples of sterilized milk and Parmesan cheese type were infected with 10 to 100 CFU / ml or g of M bovis AN5 and enriched in two selective liquid media (MGIT and modified MGIT) were analyzed on days 0, 7, 14, 21, 24, 28 and 32, maintaining at 37 ° C. On that date, an aliquot was plated on Stonebrink half- Leslie and incubated at 37 ° C for 60 days. In milk, there was overgrowth of M. bovis mainly in MGIT modified. Cheese, it was not possible to isolate M. bovis no sample in MGIT modified due to excessive contamination it deteriorated the culture medium. The modified MGIT was more effective as enrichment for Mycobacterium bovis, but was less selective than the MGIT. Future studies should focus on investigating the growth curve of the agent in the first week of enrichment.

Clostridium difficile is a pathogen for both humans and animals and is often associated with antibiotic-associated diarrhea. Recently, several human cases of C. difficile-infection with increased mortality and morbidity have been reported. In studies performed in different countries C. difficile has been found in meat. Therefore the question whether C. difficile can be a zoonotic agent has been raised. The aim of this study was to optimize a method for isolation of C. difficile from faeces. When C. difficile is isolated from animals that do not have diarrhea the sample must be cultivated in an enrichment broth. Parameters influencing the enrichment were tested such as enrichment before and after spore selection, enrichment time, alcohol and heat chock for spore selection and if the samples had to be centrifuged or not before cultivation on agar plates. Enrichment in broth before spore selection was better than after. Heat and alcohol chock showed similar results, therefore you can chose which method you want. Cultivation from the pellet after centrifugation of the sample was better than cultivating directly from the inoculated broth. When the sample had low concentration of bacteria long enrichment time, 7 days or more, was best. The next step will be isolation of C. difficile from food-producing animals and humans and the strains will then be compared to se if the same strain is found in humans and in animals, to se if C. difficile-infection can be a zoonoz.

Genetic regulatory networks are of great importance in terms of scientific interests and practical medical importance. Since a number of high-throughput measurement devices are available, such as microarrays and sequencing techniques, regulatory networks have been intensively studied over the last decade. Based on these high-throughput data sets, statistical interpretations of these billions of bits are crucial for biologist to extract meaningful results. In this thesis, we compare a variety of existing regression models and apply them to construct regulatory networks which span trancription factors and microRNAs. We also propose an extended algorithm to address the local optimum issue in finding the Maximum A Posterjorj estimator. An E. coli mRNA expression microarray data set with known bona fide interactions is used to evaluate our models and we show that our regression networks with a properly chosen prior can perform comparably to the state-of-the-art regulatory network construction algorithm. Finally, we apply our models on a p53-related data set, NCI-60 data. By further incorporating available prior structural information from sequencing data, we identify several significantly enriched interactions with cell proliferation function. In both of the two data sets, we select specific examples to show that many regulatory interactions can be confirmed by previous studies or functional enrichment analysis. Through comparing statistical models, we conclude from the project that combining different models with over-representation analysis and prior structural information can improve the quality of prediction and facilitate biological interpretation. Keywords: regulatory network, variable selection, penalized maximum likelihood estimation, optimization, functional enrichment analysis.

Availability of genome sequences for numerous bacterial species comprising of different bacterial strains allows elucidation of species and strain specific adaptations that facilitate their survival in widely fluctuating micro-environments and enhance their pathogenic potential. Different bacterial species use different strategies in their pathogenesis and the pathogenic potential of a bacterial species is dependent on its genomic complement of virulence factors. A bacterial virulence factor, within the context of this study, is defined as any endogenous protein product encoded by a gene that aids in the adhesion, invasion, colonization, persistence and pathogenesis of a bacterium within a host. Anecdotal evidence suggests that bacterial virulence genes are undergoing diversifying evolution to counteract the rapid adaptability of its host&rsquo
s immune defences. Genome sequences of pathogenic bacterial species and strains provide unique opportunities to study the action of diversifying selection operating on different classes of bacterial genes.

À l’heure où l’intégration des enjeux environnementaux dans le développement de procédés éco-efficients joue un rôle essentiel dans le moteur de l’innovation responsable, la chimie verte est devenue l’un des sujets de préoccupation majeure. Ainsi, le développement de nouveaux procédés éco-respectueux pour la production d’ingrédients naturels issus de matières premières végétales renouvelables est devenu une démarche incontournable dans le modèle de recherche. L’objectif de cette thèse a consisté au développement d’une stratégie d’éco-valorisation innovante employant les fluides sub/supercritiques pour l’extraction, la caractérisation, la production et l’imprégnation sur support cosmétique de produits naturels d’origine végétale. Pour cela, nous avons utilisé comme modèle végétal : les graines oléagineuses de la plante Kniphofia uvaria, sélectionnée pour des applications cosmétiques grâce à ses propriétés bioactives antioxydantes et anti-âge. Dans un premier temps, le développement de méthodes complémentaires en SFC ainsi que le développement du couplage SFC-MS a été réalisé à l’aide de la source APCI afin d’identifier les molécules responsables des activités bioactives des graines de Kniphofia uvaria. Ainsi, le développement d’un système hybride (U)HPLC/SFC-HRMS a été réalisé afin de mettre en place ce couplage. Des optimisations en termes de proportion et nature de solvant make-up ainsi qu’un travail au niveau des paramètres SFC et MS ont été faits afin de d’améliorer la sensibilité et la spécificité des analyses lipidiques. Dans un second temps, nous nous sommes attachés au développement d’une stratégie d’enrichissement en composés bioactifs à l’aide des méthodes : SFE et CPC. Ainsi, en SFE, des optimisations en termes de température, pression, nature/proportions de co-solvant dans le fluide ont été réalisées alors qu’en CPC, des optimisations au niveau de l’injection ont été faites. Des conditions optimales pour le fractionnement sélectif des anthraquinones et des triglycérides ont été déterminées en SFE et CPC. Dans un dernier temps, ce travail a consisté à développer un couplage en-ligne pour extraire et imprégner sélectivement sur silice cosmétique : les anthraquinones. Le développement et l’optimisation de ce procédé en-ligne ont été réalisés à l’échelle du laboratoire et ont démontré la faisabilité de ce couplage ainsi qu’un intérêt certain pour l’obtention de produits naturels sous une première forme galénique, destinée à une future incorporation dans la formulation de cosmétiques
Nowadays, green chemistry is a great challenge. It seeks innovation in the development of eco-efficient processes. The production of natural products from renewable materials by these new environmentally friendly processes is more and more used. The aim of this Ph.D thesis is to develop an eco-valuation strategy to extract, characterize, produce and impregnate natural products onto a cosmetic support using sub/supercritical fluids. Consequently, we used oleaginous plant seeds from Kniphofia uvaria as a plant model, which was selected for its interesting cosmetic properties such as antioxidant or anti-ageing. Firstly, the SFC-MS hyphenation with the APCI as an ionization source was developed to screen bioactive molecules; responsible of cosmetic properties. This coupling was performed by the hybrid combination of (U)HPLC/SFC-HRMS. Various optimizations in terms of the solvent make-up (nature and proportion), modulation with SFC and MS parameters were carried out in order to improve sensitivity and selectivity of lipid analysis. Secondly, an enrichment strategy to concentrate bioactive compounds in the final extract was developed by SFE and CPC. Thus, in SFE, experimental parameters (temperature, pressure, nature/proportion of the modifier in the CO2 fluid) were optimized while in CPC, the injection optimization was realized. Methods for the selective fractionation of anthraquinones and triglycerides were obtained in CPC and SFE. Finally, an on-line sub/supercritical extraction-impregnation process was developed to extract and for simultaneously impregnating anthraquinones onto a cosmetic silica. Development and optimization of this process was realized on a laboratory scale. Consequently, this study demonstrated the feasibility of this concept and it presents a great interest to provide natural products as a galenic form, which could be used in the cosmetic formulation

This thesis focuses on Human herpesvirus 4 and 6, two ubiquitous viruses with a long list of putative disease associations, ranging from malignancies such as lymphomas and carcinomas to multiple sclerosis. To date, the relatively limited genetic data on these organisms hinders the understanding of their variability and their genetic structure at a population level. We here explore wet lab techniques for the production of genetic data in a cost-effective manner in order to reach the order of magnitude that is required to unravel the genetics these viruses. After successfully identifying individuals affected by integrated chromosomally inherited human herpesvirus 6 from public datasets of sequencing data, the sequences of the infecting virus were produced by target enrichment means from the source biological sample. The testing of an in-house target enrichment protocol followed, aiming to target latently infecting virus in human saliva. While the protocol still needs optimization and the aid of alternative techniques to be suitable for cost-effective, large-scale studies, the results were very satisfactory (up to >800-fold enrichment). In parallel, long range PCRs were used to produce human herpesvirus 4 latency genes sequences from one large human healthy saliva panel including populations previously unexplored in terms of human herpesvirus 4 isolates. Thanks to the combination of wet lab techniques and data analysis, the presence of genetic patterns in the two studied viruses is emerging, with human herpesvirus 6 presenting differences in diversity between its two species, as well as signs of geographical patterns possibly in part hidden by recombination events. Different bioinformatics approaches showed instead a stronger geographical stratification in human herpesvirus 4, with regional-driven clades. This information would allow us for a correct study design when addressing the relationship between virus and disease, taking into account the natural variation of the virus, as well as help to pinpoint genetic features that might be determinant for disease triggering or development. The strong geographical patterns presented by the diseases associated to these viruses strengthen the notion of the importance of this investigation and opens an avenue of research focused on disclosing the putative relationship between viruses strain variation and the risk for these virus–associated diseases.

碩士
國立暨南國際大學
應用化學系
100
Abstract Titanium dioxide (TiO2) affinity enrichment has been widely used in phosphoproteomic studies. Although many attempts have been made to fabricate TiO2-based materials for selective enrichment of phosphopeptides, the effects of structural properties, such as crytsallinity, pore diameter, porosity, and specific surface area of TiO2, on selectivity has not been widely explored. Herein, we synthesized TiO2 nanowires with well-defined nanopores and high specific surface area for selective and reproducible enrichment of phosphopeptides from complex peptide mixture. TiO2 nanowires showed improved performance than TiO2 beads for selective and reproducible detection of phosphopeptides from tryptic digests of ß-casein/BSA mixture (1:1500 molar ratio) and HeLa cell lysates (200 μg). We attributed the high selectivity and reproducibility of TiO2 nanowires for phosphopeptide enrichment to the narrow pore diameter distribution and not to the crytsallinity. The average coefficient of variation of phosphopeptide peak intensity values between three replicates was 20.9 %. Venn diagrams showed extensive overlap of identified phosphopeptides (63.3 %) and phosphoproteins (71.1 %) between three replicates. Quantitative phosphopeptide analysis of HeLa cell lysate digest (from 100 μg to 10 μg ) externally spiked α-casein as internal standard reveal good linear relationship with an average correlation coefficient greater than 0.95. Based on these results, we expect that TiO2 nanowires will be of great interest for quantitative phosphoproteome analysis.

博士
國立暨南國際大學
應用化學系
105
In this work, we have developed a novel synthesis route for the production of titanium (IV) sulfonate-functionalized nanodiamonds (Ti4+-SND) as Ti4+-IMAC (Immobilized metal-ion affinity chromatography)adsorbents for selective enrichment of phosphopeptides. Nanodiamonds were first functionalized with polyarginine via EDC-mediated coupling reaction and then the titanium-sulfonate functionalization was performed by mixing with polystyrene sulfate and TiCl4 solutions. The obtained Ti4+-SNDs were used to specific capture of phosphopeptides from standard protein digests and nonfat milk digests. The results demonstrated that by taking advantage of the strong Ti4+-sulfate chelating effect and high Ti4+ loading amount, the Ti4+-SND show remarkable selectivity (β-casein/BSA = 1:1000), good sensitivity (10 fmol), and high recovery in phosphopeptide analysis. This novel approach for developing and producing IMAC adsorbents was further validated using different metal ions including Zr4+ and Fe3+. Both of the synthesized Zr4+ and Fe3+ IMAC adsorbents show excellent performance in selective enrichment of phosphopetides from complex peptide mixtures. Keywords: Polyarginine、Polystyrene sulfonate、Ti-IMAC、Phosphopeptides

Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a disease with a mortality rate of over 40%, and is a major public health concern in the endemic regions of Thailand and northern Australia. Bp is a resilient pathogen capable of surviving in diverse environments including soil, fresh and seawater, and plant and animal tissues for extended lengths of time. Bp is intrinsically resistant to antibiotics, which contributes to persistence and relapse in over 25% of melioidosis patients, and there is currently no vaccine. Our lab has previously shown that immunization with Bp outer membrane vesicles (OMVs) derived from Bp grown in Luria Burtani broth provides significant protection against melioidosis in mice. However, this protection was limited to the acute phase of infection and animals immunized with OMVs were unable to clear the bacteria. In this work, we show that by manipulating the growth media to simulate various bacterial niches, including the natural hypertonic soil environment (NaCl-supplemented), the limited-nutrient host macrophage intracellular environment (M9CG minimal media), and quorum sensing conditions (QS-molecule supplemented), OMV protein content can be modified to include those proteins potentially important for Bp survival and may contribute to protection against chronic or persistent infection. Here, we characterize the composition of selectively enriched Bp OMVs and demonstrate that enriched OMVs are non-toxic and well-tolerated both in vitro and in vivo. Immunization of BALB/c mice with QS OMVs elicits significantly greater OMV-, CPS-, and LPS- serum IgG along with cell-mediated immune responses compared to mice immunized with LB OMVs. LB, M9CG, and QS OMV immunization provided equal protection against aerosolized Bp through the acute phase of infection, and M9CG OMV-immunized mice demonstrated fewer signs of morbidity and less weight-loss over the course of infection, indicating potential control of the bacteria. These results suggest that immunizing with OMVs selectively enriched with intracellular proteins may elicit the necessary immune responses to protect against persistent melioidosis.
Nicole L Kikendall

碩士
國立暨南國際大學
應用化學系
100
Reversible oxidative cysteine modifications play a critical role in the redox-based signaling mechanism. Dynamic profiling oxidation state of the cysteine residues in protein such as Peroxiredoxin (Prx) presents a formidable challenge. Here, we present a novel approach to selectively isolate sulfopeptides (peptides containing cysteine sulfonic acid and sulfinic acid) from complex digests using TiO2-coated nanodiamonds (TiO2-coated NDs) prior to MS analysis. The method was applied to selectively concentrate sulfopeptides from either a highly dilute solution or a highly complex peptide mixture. This method allowed us to identify the 22 distinct cysteine oxidation status out of a total 35 present in performic-acid-oxidized BSA by MALDI-TOF MS and all the distinct cysteine oxidation status by LC-ESI-MS/MS. Finally, we applied the new approach to identify the cysteine oxidation status of hydrogen peroxide–treated Prx and BSA by LC-ESI-MS/MS. Cysteine residues were found to display in either cysteine sulfonic acid or cysteine sulfinic acid status after hydrogen peroxide treatment. Enhanced detection of low abundance sulfopeptides containing active cysteine positions (C52, C71, C83, and C173) was achieved due to the highly selective enrichment using TiO2-coate NDs.

碩士
國立暨南國際大學
應用化學系
101
Selective isolation and concentration of phosphopeptides is a prerequisite step prior to MS analysis. Currently titanium oxide affinity chromatography has gained more and more popularity among the common phosphopeptide isolation methods. Here, we present a new hybrid material, TiO2-coated nanodiamonds (TiO2-NDs), for selective enrichment of phosphopeptides from complex peptide mixtures. The results showed that TiO2-NDs outperformed the commercial TiO2 beads for higher enrichment capacity and specificity toward phosphopeptides. We identified 932 unique phosphopeptides and 643 phosphorylation site from 100 μg of HeLa cells, which represented a 1.5-fold increase in phosphopeptide number and a 1.5-fold increase in phosphosites in comparison with the results obtained using commercial TiO2 beads. This new hybrid material (TiO2-NDs) can be easily incorporated as an effective alternative for TiO2 beads into existing protocols for phosphopeptide isolation, and can facilitate more comprehensive profiling of phosphoproteome.

碩士
國立暨南國際大學
應用化學系
102
A facile and effective approach to synthesize chitosan-immobilized magnetic nanoparticles was proposed using carbon-encapsulated iron nanoparticles (Fe@CNPs) as the magnetic core.Poly(acrylic acid)-coated Fe@CNPs were prepared by free radical polymerization of acrylic acid and used as magnetic substrates for subsequent Chitosan immobilization.The chitosan-immobilized magnetic probes were applied for rapid and selective identification of phosphopeptides from complex peptide mixtures by MALDI-TOF MS.

碩士
大葉大學
生物產業科技學系碩士在職專班
93
Salmonellae is one of the most common causes of human gastroenteritis. This study compared the performance of three selective media namely CAS (CHROMagar Salmonella medium), HE (Hektoen enteric agar) and XLD (Xylose lysine desoxycholate agar) and three enrichment broths namely GN (Gram-negative broth), SB (Selenite broth) and SBG (Selenite brilliant green sulfa enrichment broth) to optimize the use of plating media and enrichment broths for isolation of Salmonella spp. from human stools. The 304 stools were cultured onto the above three selective media by direct inoculation and after enrichment in GN and SB. The other 155 stools were tested for SBG and SB enrichment experiments. The standard biochemical identification tests and the serogrouping test were also used to identify Salmonella spp. The 109 Salmonella belong to 20 serotypes. The isolation rate of Salmonella is higher when stools were suspended in saline than plated on HE and XLD directly (42 isolates and 29 isolates, respectively). The sensitivity and specificity for direct plating were 53.5% and 87.6%, respectively, for XLD agar, and for CAS these values were 35.2% and 83.9%, respectively, and for HE these values were 40.9% and 81.5%, respectively. The sensitivities of XLD for direct plating was statistically significantly higher than CAS and HE The sensitivities for the detection of Salmonellae after GN enrichment were 45.1% and 52.1% for HE and XLD was statistically significantly higher than CAS 28.1%. The specificity for the detection of Salmonellae after GN enrichment on CAS, HE, XLD were not significantly different. XLD medium can be recommended for use for the isolation of Salmonella spp. with SB enrichment (sensitivity, 86.0%, specificity, 75.7%). The SB (66 isolates) enrichment procedure increased the number of Salmonella spp. isolates was significantly different from GN (45 isolates) and without enrichment (45 isolates) (p<0.005). Althought SB and SBG were not significantly different from each other in sensitivity, but the specificity of SBG (85.2%) was better than SB (69.6%) (p<0.005). There were more strains Citrobacter spp. on HE medium then CAS and XLD medium (p<0.005) and more strains Pseudomonas aeruginosa on CAS medium then HE (p<0.01) and XLD medium (p<0.005). There were more Citrobacter spp.and Pseudomonas aeruginosa after SB enrichment than SBG (p<0.05). The use of plating on CAS, HE, XLD after SBG enrichment demonstrated high levels of sensitivity and specificity were not significantly different from each other. It can be recommended for the use for the isolation of Salmonella spp. from human stools. The specificity of the CAS was 88.0% after slective enrichment in SBG, while the specificity after oxidase test ruled out false positive result was 94.0%. The higher specificity reduces the need for confirmatory test, thereby cutting technical time and reagent requirements. The CAS with slective enrichment in SBG can be recommended as best choice for Salmonella isolation from human stools.

碩士
國立中山大學
化學系研究所
100
This study examines the use of unmodified magnetite nanoparticles (Fe3O4 NPs) for selective extraction and enrichment of the catecholamines dopamine (DA), noradrenaline (NE), and adrenaline (E), prior to analysis using capillary electrophoresis with UV detection. Coordination between Fe3+ on-the-surface Fe3O4 NPs and the catechol moiety of catecholamines enables Fe3O4 NPs to capture catecholamines from an aqueous solution. We obtained maximum loading of catecholamines on the NP surface by adjusting the pH of the solution to 7.0. In addition, catecholamine loading on the Fe3O4 NPs increased in conjunction with NP concentrations. Ligand exchange found H3PO4 to be efficient in the removal of adsorbed catecholamines on the NP surface. Adding 1.2% poly(diallyldimethylammonium chloride) to the background electrolyte caused efficient separation of the liberated catecholamines with baseline resolution within 20 min. Under optimal extraction and separation conditions, the limit of detections at a signal-to-noise ratio of 3 for E, NE, and DA were 9 nM, 8 nM, and 10 nM, respectively. Significantly, we successfully used the combination of a phenylboronate-containing spin column and the proposed method to determine the concentrations of NE and DA in urine and the content of NE in Portulaca oleracea L. leaves.

碩士
國立暨南國際大學
應用化學系
96
Mass spectrometric identification of phosphoproteins and their phosphorylation sites is challenging , due to their low abundance in nature. Therefore, it is essential to perform an enrichment strategy prior to MS analysis. In this work, we employed polyarginine (PA) - coated diamond nanoparticles (nominal size 100 nm) as high affinity probes to selectivity capture phosphoproteins / phosphopeptides from a complex solution of tryptic digested proteins. Compared to IMAC and MOAC methods for phosphopeptide enrichment, The polyarginine-coated nanodiamonds show an exceptionally high affinity for multiphosphorylated peptides due to multiple arginine-phosphate interactions. The efficacy of this method was demonstrated by analyzing a small volume (50 μL) of tryptic digests of proteins such as α-casein、β-casein and non-fat milk at a concentration as low as 1 × 10-9 M. The concentration is much lower than that can be achieved by using other commercial technologies kits. This work presents a new strategy that not only effectively extract phosphorylated peptides from complex samples but also can selectively enrich multiphosphorylated peptides for direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. The new affinity-based protocol is expected to find useful applications in characterizing multiple phosphorylation sites on proteins of interest in both complex and diluted environments.

abstract: Microbial electrochemical cells (MXCs) are promising platforms for bioenergy production from renewable resources. In these systems, specialized anode-respiring bacteria (ARB) deliver electrons from oxidation of organic substrates to the anode of an MXC. While much progress has been made in understanding the microbiology, physiology, and electrochemistry of well-studied model ARB such as Geobacter and Shewanella, tremendous potential exists for MXCs as microbiological platforms for exploring novel ARB. This dissertation introduces approaches for selective enrichment and characterization of phototrophic, halophilic, and alkaliphilic ARB. An enrichment scheme based on manipulation of poised anode potential, light, and nutrient availability led to current generation that responded negatively to light. Analysis of phototrophically enriched communities suggested essential roles for green sulfur bacteria and halophilic ARB in electricity generation. Reconstruction of light-responsive current generation could be successfully achieved using cocultures of anode-respiring Geobacter and phototrophic Chlorobium isolated from the MXC enrichments. Experiments lacking exogenously supplied organic electron donors indicated that Geobacter could produce a measurable current from stored photosynthate in the dark. Community analysis of phototrophic enrichments also identified members of the novel genus Geoalkalibacter as potential ARB. Electrochemical characterization of two haloalkaliphilic, non-phototrophic Geoalkalibacter spp. showed that these bacteria were in fact capable of producing high current densities (4-8 A/m2) and using higher organic substrates under saline or alkaline conditions. The success of these selective enrichment approaches and community analyses in identifying and understanding novel ARB capabilities invites further use of MXCs as robust platforms for fundamental microbiological investigations.
Dissertation/Thesis
Ph.D. Microbiology 2013

A method for the selective concentrating of DNA targets using capillary affinity gel electrophoresis is presented. Complementary ssDNA targets are retained through hybridization with oligonucleotide probes immobilized within polyacrylamide gels while non-complementary targets are removed. The captured DNA targets were concentrated by step elution, where a localized thermal zone was applied in small steps along the capillary. Evaluation of the selective capture of a 150 nt DNA target in a complicated mixture was carried out by factorial analysis. Gels with a smaller average pore size were found to retain a higher amount of complementary targets. This was thought to be due to the ssDNA target migrating through the gel by reptation, eliminating hairpin structures, making the complementary region of the target available for hybridization. This method was applied to a series of DNA targets of different lengths, 19 nt, 150 nt, 250 nt and 400 nt. The recovery of the method ranged from 0.5 to 4% for the PCR targets, and 13 to 18% for the 19 nt oligonucleotide target. The purity was calculated to be up to 44% for the PCR targets and up to 86% for the 19 nt target. This was an improvement in purity of up to 15 times and 1100 times in comparison to the original samples for the PCR targets and 19 nt oligonucleotide, respectively. The 19 nt targets were selective concentrated and delivered into a microfluidic based DNA biosensing platform. The purity of the sample improved from 0.01% to 50% while recovery decreased from 100% to 20% for a sample with 0.5 nM complementary and 1 μM non-complementary targets. An improvement in the response of the sensing platform was demonstrated on 19 nt oligonucleotide targets delivered by selective concentration versus concentration alone into the microfluidic biosensing system.

碩士
國立暨南國際大學
應用化學系
106
In this work, we have developed a novel synthesis strategy for the production of TiO2 hollow nanocage for highly efficient enrichment of phosphopeptides via a Nanodiamond(ND)-assisted template method. We reduced the ND surface to OH groups and then coated the silica dioxide and titanium dioxide on the ND by sol-gel method, and the ND template was removed by calcination at 650 degrees for eight hours. The nanostructure of the obtained TiO2 hollow nanocage was characterized by nitrogen adsorption-desorption isotherms, X-ray powder diffraction (XRD), transmission electron microscopy (TEM), and the corresponding selected area electron diffraction (SAED). We can see that the metal oxide particles are coated on the surface of ND. The obtained TiO2 hollow nanocages exhibited high adsorption capacity, and excellent enrichment specificity (tryptic digest of β-casein/BSA at a molar ratio of 1:4000) and sensitivity (tryptic digest of 10 fmol of β-casein). Moreover, the hollow TiO2 nanocages provided effective enrichment efficiency of low-abundance phosphopeptides from HeLa cell digests. This ND-assisted template method represents an efficient avenue for the preparation of irregular shape hollow TiO2 cages in the nanoscale range, and serves as a new, scalable, and broadly applicable hard-template strategy for material science.

碩士
國立臺灣大學
化學研究所
98
Identification of multi-phosphorylated peptides by liquid chromatography-tandem mass spectrometry（LC-MS/MS）remains a challenging task due to the poor ionization of the species. For this reason, various kits to selectively enrich phosphopeptides prior to MS analysis have been developed and commercialized, but most of them are selective only for singly phosphorylated peptides. Interestingly, poly-arginine-coated nanodiamond（PA-ND）shows a high selectivity for multi-phosphorylated peptides and this has been demonstrated by Chang et al. For further applications in real sample analysis with LC-MS/MS, an effective elution buffer to separate the target molecules from the PA-ND substrate has yet to be found. In this work, we have successfully synthesized a much highly selective probe by coating PA on ND（average diameter: 86.9 nm → 140.6 nm）in 0.1 M MES buffer. The zeta potential in deionized water increased from -27.6 mV to +43.29 mV, better than the previous one, ~0 mV. Furthermore, we found a better enrichment condition, with 40 % acetonitrile containing 1 % trifluoroacetic acid, that allows us to selectively probe multi-phosphorylated peptides in very complex samples（with impurities ≥5000 higher than of the target analyte）. Finally, we also found that 12.5 % ammonium water containing 0.5 M potassium phosphate can easily elute the enriched substance from the probe. LC-MS/MS can therefore successfully identify a triply phosphorylated peptide extracted from a highly mixture by the newly synthesized PA-ND particles and with the newly developed protocols.

碩士
國立暨南國際大學
應用化學系
97
Abstract Despite recent technical advances, comprehensive characterization of protein phosphorylation remains a challenge. We explored the possibility using a two–step enrichment procedure for fractionation and selective identification of multiply and singly phosphorylated segments from enzymatic digestion of complex proteins. The procedure involved isolation of multiply phosphorylated peptides from solution using polyarginine-coated nonadiamond and subsequent isolation of singly phosphorylated peptides from the same pool of solution using TiO2-coated magnetic probes. The two types of probes can be independently characterized by MALDI mass spectrometry and provide complementary datasets for phosphoproteomic analysis. In addition, the total number of identified phosphopeptides obtained by the two-step enrichment procedure was almost doubled than that obtained by IMAC, TiO2, or PA affinity-based methods without the use of additional biological material. Improvements to comprehensive phosphopeptide identification via the two-step enrichment procedure are discussed. To our knowledge, this is the first demonstration of the two-step enrichment procedure for selective isolation and fractionation of multiply and singly phosphorylated peptides from the same pool of peptides solution.

A new system which offers a low-cost and high performance means for detecting four bacterial pathogens Aeromonas salmonicida, Tenacibaculum maritimum, Lactococcus garvieae and Yersinia ruckeri in covertly infected farmed salmonid fish has been developed. The system couples Selective Enrichment Culture (SEC), Polymerase Chain Reaction (PCR) and Enzyme Hybridization Assay (EHA) technologies to provide streamlined high-throughput sample processing suitable for large scale surveillance and monitoring programs. The SEC media used for the technology were those developed by T.Wilson and J.Carson for the Cooperative Research Centre (CRC) for Aquaculture Ltd, except for the A. salmonicida medium which was developed using information obtained during the CRC project and by performing Minimum Inhibitory Concentration (MIG) assays with 32 antimicrobial agents that were not previously tested. Laboratory sensitivity of the A. salmonicida medium was 100% as determined by Most Probable Number (MPN) analysis and specificity of the medium was 97%. Bacterial DNA and RNA were extracted from the SEC media using guanidinium isothiocyanate (GuSCN) and Whatman Polyfiltronics GF/B 96-well Uni-filter plates. Sensitivity of the system as determined by PCR was between 1 and 16 CFU per 200 μI of selective-enrichment medium for DNA and between 1 and 9 CFU per 200 μI of selective-enrichment medium for RNA. The use of vacuum filtration and the 96-well glass microfibre filter plate allowed for rapid high-throughput and inexpensive extraction. Due to the high sensitivity of PCR, the technology is prone to false-positive results due to amplicon carry-over. To help prevent the occurrence of these erroneous results the photochemical IP-10 was added to the procedure. IP-10 inactivates PCR amplicons rendering them incapable of re-amplification in subsequent PCR reactions. Nucleolink ™ PCR-enzyme hybridization strips were used to perform sensitive streamlined (RT)PCR-EHA. For each bacterium 4 fg of pure target DNA or RNA was detected. With this system only one tube per sample from cDNA to EHA was required, decreasing the cost and time involved in sample transfer and decreasing the risk of cross-contamination between samples. In laboratory trials the final SEC-(RT-)PCR-EHA system proved to be very sensitive with 1-16 CFU from selective-enrichment culture detected. Specificity of the system was >99%. The system was also rapid with the 96-well nucleic acid extraction to EHA results achieved in as little as 8 hours. Test validation with field samples was achieved by determining test specificity and sensitivity from an epidemiological perspective. During this testing the sensitivity and specificity of the system matched that obtained using purified nucleic acids in the laboratory. Validation was conducted using a total of 10 fish trials using fish from different environments and from different sample sources. The system described here uses two types of technology, PCR and RT-PCR. The SEC-PCR-EHA system detects genomic DNA from the target pathogen, demonstrating evidence of infection, past or present and is ideal for use in surveillance programs and for quarantine. The SEC-RT-PCR-EHA system detects ribosomal RNA from the target pathogens. This system gives a more accurate indication of the presence of live bacteria and therefore of live covert infection and is useful when monitoring changes in the disease status of a population of fish over a short period of time. The system was developed using inexpensive materials instead of proprietary products, and disposable equipment usage was minimised in an effort to keep the system low-cost. The sensitivity of the system was maximised to ensure the detection of covertly infected fish and specificity of the system was as good as the PCR primers would allow. The system utilises 96-well technology to minimise the volume of reagents and to enable streamlined sample processing using a multichannel pipette. In summary, a low-cost, high-performance and streamlined highthroughput sampling system has been developed.

Excellent electronic properties of semiconducting single-walled carbon nanotubes (sc-SWCNTs) motivated the investigation for using them in different application areas such as microelectronics, sensorics, MEMS and MOEMS. However, challenges arise from the lack of selectivity with respect to electronic type and chirality as well as ensuring high quality, high purity and well-aligned SWCNTs during fabrication process. Catalytic chemical vapour deposition (CCVD) has shown great potential in direct synthesis of high quality SWCNTs with chiral or type selectivity. This thesis addresses three important aspects for growth of sc-SWCNT covering method development for fast screening for complex catalyst systems, process development for type-selective growth of SWCNTs and transfer of processes to a specific CVD reactor capable to scale the processes up to 8-inches wafer embedded in the microtechnologic process line. Multi-wavelengths Raman spectroscopy is applied to analyze type and chiral compositions of SWCNTs. In addition, different microscopic techniques of SEM, TEM and AFM are utilized to analyze surface morphology of catalyst layers and size of the nanoparticles as well as structure-related properties of SWCNTs. Initially, systematic studies on monometallic Co and bimetallic Co-Mo systems with different bilayer thickness configurations and their influences on the properties of grown SWCNTs are conducted on chip level. It is shown by adjusting the catalyst deposition conditions of bilayer catalyst as well as optimization of gas environments in CCVD process, structure-related properties of SWCNTs are dramatically enhanced. Furthermore, by utilizing shutter-assisted sputter deposition of gradient layer catalyst, a fast and efficient method for screening different bilayer configurations of Co-Mo, Co-Ru and Ni-Ru has been developed. By utilizing gradient layer deposition with finely resolved catalyst thicknesses, random network SWCNT is grown on bimetallic Co-Mo system under certain process condition with 45% (at 633 nm) and 75% (at 785 nm) semiconducting enrichment of long and high quality SWCNT. In contrast, bimetallic Co-Ru system under certain process condition is developed to grow in-plane SWCNT with 85% (at 633 nm) and 75% (at 785 nm) semiconducting enrichment of short and low quality SWCNT. In addition, different configurations of the bimetallic Co-Ru system are prepared from salt precursors by spin-coating technique. For a mixture of cobalt (II) chloride and ruthenium (III) nitrosylacetate, random network SWCNT with 70% (at 633 nm) and 95% (at 785 nm) semiconducting enrichment of long SWCNTs with high quality is obtained on wafer level. Random network SWCNT with high degree of semiconducting enrichment is used as channel material for thin-film transistors fabrication that results in CNTFET with on/off ratio in the order of 10*3:Bibliographic description 3 Vorwort 9 List of abbreviations and symbols 11 1 Introduction 15 2 Fundamentals of carbon nanotubes 21 2.1 Chemical bonds in carbon structures 21 2.2 Different allotropes of carbon 22 2.3 History of carbon nanotubes research 23 2.4 Structure of carbon nanotubes 24 2.5 Electronic properties of carbon nanotubes 26 2.6 Synthesis of carbon nanotubes 27 2.7 Growth mechanism of carbon nanotubes by CCVD 29 2.8 Catalyst for CCVD synthesis of SWCNTs 31 2.8.1 Catalyst nanoparticle formation from thin film 32 2.8.2 Mechanism of solid state dewetting 33 2.9 CCVD synthesis of SWCNT 35 2.10 Selective synthesis of SWCNT 37 3 Experimental 39 3.1 Preparation of different catalyst/support systems 39 3.1.1 Homogenous layer of catalyst prepared by PVD 39 3.1.2 Gradient layer deposition of catalyst by IBSD 41 3.1.3 Homogenous layer of catalyst prepared by spin coating 45 3.2 CVD reactors for synthesis of SWCNT 46 3.2.1 R&D vertical flow CVD reactor with showerhead 46 3.2.2 Industrial vertical flow CVD reactor with showerhead 47 3.2.3 Horizontal flow tube CVD reactor 49 3.3 Methods for characterization 50 3.3.1 Atomic force microscopy 50 3.3.2 Raman spectroscopy 50 3.3.3 Spectroscopic ellipsometry 56 3.3.4 X-ray reflection 56 3.3.5 Scanning electron microscopy 56 3.3.6 Transmission electron microscopy 56 4 Growth of SWCNT using PVD catalyst layer in vertical CVD reactor A 57 4.1 Monometallic Co catalyst supported on SiO2 57 4.1.1 Surface and morphological analysis of SiO2/Co 57 4.1.2 Analysis of CCVD grown SWCNT on SiO2/Co 59 4.1.3 Chirality and diameter analysis of SWCNTs on SiO2/Co 61 4.2 Monometallic Co catalyst supported on Al2O3 62 4.2.1 Surface and morphological analysis of Al2O3/Co 62 4.2.2 Analysis of CCVD grown SWCNT on Al2O3/Co 63 4.2.3 Chirality and diameter analysis of SWCNTs on Al2O3/Co 67 4.3 Bimetallic Co-Mo catalyst supported on Al2O3 68 4.3.1 Surface and Morphological analysis of Al2O3/Co-Mo 68 4.3.2 Effect of IBSD deposition parameters on NP formation 71 4.3.3 Analysis of CCVD grown SWCNT on Al2O3/Co-Mo 72 4.3.4 Chirality and diameter analysis of SWCNTs on Al2O3/Co-Mo 76 4.4 Comparison of SWCNT from different catalyst configurations 77 5 Growth of SWCNT using gradient layer of catalyst 79 5.1 Analysis of grown SWCNT on Co-Mo using step gradient A 79 5.2 Analysis of grown SWCNT on Co-Mo using step gradient B 80 5.2.1 Growth of SWCNT by utilizing shutter at position I 80 5.2.2 Growth of SWCNT by utilizing shutter at position II 82 5.2.3 Effect of vacuum breaking on CCVD growth of SWCNT 83 6 Growth of SWCNT using gradient layer catalyst in vertical CVD reactor B 87 6.1 SWCNT growth on gradient layer of monometallic catalyst 87 6.1.1 Analysis of CCVD grown SWCNT on gradient layer of Co 87 6.1.2 Analysis of CCVD grown SWCNT on gradient layer of Ni 89 6.1.3 Comparison of SWCNT properties for monometallic of Ni and Co 90 6.2 SWCNT growth on gradient layer of bimetallic catalyst 92 6.2.1 Analysis of CCVD grown SWCNT on gradient layer of Co-Mo 92 6.2.2 Analysis of CCVD grown SWCNT on gradient layer of Co-Ru 95 6.2.3 Comparison of SWCNTs on Co-Mo and Co-Ru catalyst systems 98 6.2.4 Analysis of CCVD grown SWCNTs on gradient layer of Ni-Ru 100 7 Growth of SWCNT using spin-coated catalyst precursor in horizontal CVD reactor 103 7.1 Effect of CCVD growth temperature on SWCNT properties 103 7.2 Effect of catalyst calcination temperature on SWCNT properties 103 7.3 Analysis of CCVD grown SWCNT on Co and Co-Ru 105 7.3.1 Monolayer configuration of different Co precursors 105 7.3.2 Bilayer configuration of Co and Ru precursors 106 7.3.3 Trilayer configuration of Co and Ru precursors 107 7.3.4 Monolayer configuration of Mixture Co and Ru precursors 109 7.3.5 Comparison of SWCNTs on different catalyst configurations 110 8 Growth of SWCNT using spin-coated catalyst precursor in vertical CVD reactor B 113 8.1 Growth of SWCNT on Mixture of Co and Ru precursors 113 8.2 Effect of CVD reactor geometry on SWCNT properties 115 8.3 Effect of catalyst preparation technique on SWCNT properties 116 8.4 Wafer-level growth of SWCNT on bimetallic Co-Ru 117 9 SWCNT-based device fabrication 119 9.1 Different approaches for SWCNT-based device fabrication 119 9.2 Growth-based technique for SWCNT-based device fabrication 121 9.2.1 FET fabrication on in-plane random network SWCNT 121 9.2.2 FET fabrication on out-of-plane random network SWCNT 123 10 Summary and outlook 127 Appendix 131 Bibliography 171 List of tables 183 List of figures 185 Versicherung 197 Theses 199 Curriculum vitae 201 List of publications 203
Die hervorragenden elektronischen Eigenschaften von halbleitenden, einwandigen Kohlenstoff-Nanoröhren (sc-SWCNTs haben die Untersuchung dazu veranlasst, sie in verschiedenen Anwendungsbereichen wie der Mikroelektronik, Sensorik, MEMS und MOEMS einzusetzen. Herausforderungen ergeben sich jedoch aus dem Mangel an Selektivität bezüglich elektronischer Bauart und Chiralität sowie der Sicherstellung hoher Qualität, hoher Reinheit und gut aufeinander abgestimmter SWCNTs während des Herstellungsprozesses. Die Katalytische chemische Gasphasenabscheidung (CCVD) zeigt ein großes Potenzial bei der direkten Synthese von hochqualitativen SWCNTs mit Chiraler- oder Typenselektivität. Diese Dissertation behandelt drei wichtige Aspekte für das Wachstum von sc-SWCNT und deckt die Methodenentwicklung des schnellen Screenings für komplexe Katalysatorsysteme, die Prozessentwicklung für das typselektive Wachstum von SWCNTs und die Übertragung von Prozessen in einen spezifischen CVD-Reaktor ab. Der Reaktor, welcher eingebettet in die mikrotechnologische Prozesslinie ist, kann Wafer bis zu 8- Zoll verarbeiten. Raman-Spektroskopie mit mehreren Wellenlängen wird verwendet, um die Zusammensetzung von SWCNTs zu analysieren. Darüber hinaus werden verschiedene mikroskopische Techniken von REM, TEM und AFM verwendet, um die Oberflächenmorphologie von Katalysatorschichten und die Größe der Nanopartikel sowie die strukturbezogenen Eigenschaften von SWCNTs zu analysieren. Zunächst werden systematische Untersuchungen an monometallischen Co- und Bimetall-Co-Mo-Systemen mit unterschiedlichen Doppelschichtdickenkonfigurationen durchgeführt und deren Einfluss auf die Eigenschaften gewachsener SWCNTs auf Chipebene untersucht. Es wird gezeigt, dass durch Einstellung der Katalysatorabscheidungsbedingungen des Doppelschichtkatalysators sowie durch Optimierung der Gasumgebung im CCVD-Prozess die strukturbezogenen Eigenschaften von SWCNTs drastisch verbessert werden können. Darüber hinaus wurde durch die Verwendung eines Gradientenschichtkatalysators, welcher mittels einer Shutter-unterstützten Zerstäubungsabscheidung hergestellt wurde, ein schnelles und effizientes Verfahren zum Untersuchen verschiedener Doppelschichtkonfigurationen von Co-Mo, Co-Ru und Ni-Ru entwickelt. Unter Verwendung der Abscheidung einer Gradientenschicht mit einer fein aufgelösten Katalysatordicke wurden ungerichtete SWCNTs auf einem bimetallischen Co-Mo-System unter definierten Prozessbedingungen mit 45% (bei 633 nm) und 75% (bei 785 nm) halbleitender Anreicherung von langem und hochwertigem SWCNT gezüchtet. Im Gegensatz dazu wird das bimetallische Co-Ru-System unter definierten Prozessbedingungen entwickelt, um SWCNT in der Ebene mit 85% (bei 633 nm) und 75% (bei 785 nm) halbleitender Anreicherung von kurzer und geringer Qualität von SWCNT zu wachsen. Außerdem werden verschiedene Konfigurationen des Bimetall-Co-Ru-Systems aus Salzvorläufern durch Spin-Coating-Technik hergestellt. Es zeigt sich für die Bimetallkonfiguration, die durch Mischung von Cobalt (II) -chlorid und Ruthenium (III) -nitrosylacetat, ein zufälliges Netzwerk SWCNT zu 70% (bei 633 nm) und 95% (bei 785 nm) halbleitender Anreicherung langer SWCNTs mit hohem Anteil hergestellt wurde Qualität wird auf Waferebene gewachsen. Ein zufälliges Netzwerk-SWCNT mit einem hohen Grad an halbleitender Anreicherung wird als Kanalmaterial für die Herstellung von Dünnschichttransistoren verwendet, was zu einem CNTFET mit einem Ein / Aus-Verhältnis um 10*3 führte.:Bibliographic description 3 Vorwort 9 List of abbreviations and symbols 11 1 Introduction 15 2 Fundamentals of carbon nanotubes 21 2.1 Chemical bonds in carbon structures 21 2.2 Different allotropes of carbon 22 2.3 History of carbon nanotubes research 23 2.4 Structure of carbon nanotubes 24 2.5 Electronic properties of carbon nanotubes 26 2.6 Synthesis of carbon nanotubes 27 2.7 Growth mechanism of carbon nanotubes by CCVD 29 2.8 Catalyst for CCVD synthesis of SWCNTs 31 2.8.1 Catalyst nanoparticle formation from thin film 32 2.8.2 Mechanism of solid state dewetting 33 2.9 CCVD synthesis of SWCNT 35 2.10 Selective synthesis of SWCNT 37 3 Experimental 39 3.1 Preparation of different catalyst/support systems 39 3.1.1 Homogenous layer of catalyst prepared by PVD 39 3.1.2 Gradient layer deposition of catalyst by IBSD 41 3.1.3 Homogenous layer of catalyst prepared by spin coating 45 3.2 CVD reactors for synthesis of SWCNT 46 3.2.1 R&D vertical flow CVD reactor with showerhead 46 3.2.2 Industrial vertical flow CVD reactor with showerhead 47 3.2.3 Horizontal flow tube CVD reactor 49 3.3 Methods for characterization 50 3.3.1 Atomic force microscopy 50 3.3.2 Raman spectroscopy 50 3.3.3 Spectroscopic ellipsometry 56 3.3.4 X-ray reflection 56 3.3.5 Scanning electron microscopy 56 3.3.6 Transmission electron microscopy 56 4 Growth of SWCNT using PVD catalyst layer in vertical CVD reactor A 57 4.1 Monometallic Co catalyst supported on SiO2 57 4.1.1 Surface and morphological analysis of SiO2/Co 57 4.1.2 Analysis of CCVD grown SWCNT on SiO2/Co 59 4.1.3 Chirality and diameter analysis of SWCNTs on SiO2/Co 61 4.2 Monometallic Co catalyst supported on Al2O3 62 4.2.1 Surface and morphological analysis of Al2O3/Co 62 4.2.2 Analysis of CCVD grown SWCNT on Al2O3/Co 63 4.2.3 Chirality and diameter analysis of SWCNTs on Al2O3/Co 67 4.3 Bimetallic Co-Mo catalyst supported on Al2O3 68 4.3.1 Surface and Morphological analysis of Al2O3/Co-Mo 68 4.3.2 Effect of IBSD deposition parameters on NP formation 71 4.3.3 Analysis of CCVD grown SWCNT on Al2O3/Co-Mo 72 4.3.4 Chirality and diameter analysis of SWCNTs on Al2O3/Co-Mo 76 4.4 Comparison of SWCNT from different catalyst configurations 77 5 Growth of SWCNT using gradient layer of catalyst 79 5.1 Analysis of grown SWCNT on Co-Mo using step gradient A 79 5.2 Analysis of grown SWCNT on Co-Mo using step gradient B 80 5.2.1 Growth of SWCNT by utilizing shutter at position I 80 5.2.2 Growth of SWCNT by utilizing shutter at position II 82 5.2.3 Effect of vacuum breaking on CCVD growth of SWCNT 83 6 Growth of SWCNT using gradient layer catalyst in vertical CVD reactor B 87 6.1 SWCNT growth on gradient layer of monometallic catalyst 87 6.1.1 Analysis of CCVD grown SWCNT on gradient layer of Co 87 6.1.2 Analysis of CCVD grown SWCNT on gradient layer of Ni 89 6.1.3 Comparison of SWCNT properties for monometallic of Ni and Co 90 6.2 SWCNT growth on gradient layer of bimetallic catalyst 92 6.2.1 Analysis of CCVD grown SWCNT on gradient layer of Co-Mo 92 6.2.2 Analysis of CCVD grown SWCNT on gradient layer of Co-Ru 95 6.2.3 Comparison of SWCNTs on Co-Mo and Co-Ru catalyst systems 98 6.2.4 Analysis of CCVD grown SWCNTs on gradient layer of Ni-Ru 100 7 Growth of SWCNT using spin-coated catalyst precursor in horizontal CVD reactor 103 7.1 Effect of CCVD growth temperature on SWCNT properties 103 7.2 Effect of catalyst calcination temperature on SWCNT properties 103 7.3 Analysis of CCVD grown SWCNT on Co and Co-Ru 105 7.3.1 Monolayer configuration of different Co precursors 105 7.3.2 Bilayer configuration of Co and Ru precursors 106 7.3.3 Trilayer configuration of Co and Ru precursors 107 7.3.4 Monolayer configuration of Mixture Co and Ru precursors 109 7.3.5 Comparison of SWCNTs on different catalyst configurations 110 8 Growth of SWCNT using spin-coated catalyst precursor in vertical CVD reactor B 113 8.1 Growth of SWCNT on Mixture of Co and Ru precursors 113 8.2 Effect of CVD reactor geometry on SWCNT properties 115 8.3 Effect of catalyst preparation technique on SWCNT properties 116 8.4 Wafer-level growth of SWCNT on bimetallic Co-Ru 117 9 SWCNT-based device fabrication 119 9.1 Different approaches for SWCNT-based device fabrication 119 9.2 Growth-based technique for SWCNT-based device fabrication 121 9.2.1 FET fabrication on in-plane random network SWCNT 121 9.2.2 FET fabrication on out-of-plane random network SWCNT 123 10 Summary and outlook 127 Appendix 131 Bibliography 171 List of tables 183 List of figures 185 Versicherung 197 Theses 199 Curriculum vitae 201 List of publications 203

碩士
長庚大學
醫學生物技術研究所
95
ABSTRACT ENRICHMENT OF STEM CELL DERIVED NEURON BY βIII-TUBULIN TARGETED LINEAGE SELECTION Neural stem cells are the prime candidate for studying the neuro-genesis and as a donor sources for the cell replacement therapy in several neurological diseases. Study indicated that neural stem cell has the potential to proliferate in vitro and differentiate into neuronal-like cells with biochemical and electro-physiological properties. Although the neural stem cell provided an unlimited source of neuron, the current differentiation protocol could be difficult to achieve the sufficient amount of homogenous neurons for the therapeutic cell transplantation. Therefore, we employed a hybrid approach of “lineage selection” and “forced differentiation” for enriching the stem cell derived neuron with the lineage restricted genes and promoters. To accomplish our goal, we studied on the possible use of the three difference approaches: (1) βIII-tubulin as a promoter based lineage selection, (2) Calbindin-1 as a neuronal inducer in forced differentiation and (3) βIII-tubulin and calbindin-1 as a hybrid approach for neuronal differentiation. Our results shown: first, the characterized the minimal promoter of βIII-tubulin was sufficient for up-regulating the reporter gene in the differentiated cells and was applied in the MEB5 differentiation and the GFP expressed cells were always recognized by the MAP2 antibody. Furthermore, we demonstrate the effectiveness of the both NRSE dsRNA and NRSF siRNA strategies in mediating the neuronal differentiation. Second, we employed the over-expression of calbindin-1 for promoting the neuronal differentiation of neural stem cell through tetracycline and βIII-tubulin regulated system. The exogenous overexpressed calbindin-1 was significantly increased the neuronal differentiation in MEB5 with more than 70% of transfected cells had been identified by MAP2 in compared with 30% in control group. These were more primary neuritic outgrowth and longer neuritic length in the calbindin-1 expressed cells, whereas it greatly reduced the glial differentiation from 30% to 9%. Finally, we employed the conditional inducible calbindin-1-GFP expression by βIII-tubulin promoter during neuronal differentiation; this novel approach was not only using calbindin-1 as a neuronal regulator and protector, but also using βIII-tubulin as neuronal specific lineage selection for enrichment of the neuronal differentiation. Our data shown that this approach was enriched the specificity of the neuronal differentiation, and a proposed positive feedback facilitates the neuronal differentiation.

This thesis presents microfluidic devices for selection and amplification of nucleic acids (aptamers) that bind to specific targets. Aptamers are very attractive molecules in many biological applications due to their interesting properties including high target binding affinities and stability. Using conventional platforms for aptamer generation (SELEX, systematic evolution of ligands by exponential enrichment) is labor-intensive and time consuming. Microfluidic devices have been developed to improve the aptamer enrichment efficiency. However, aptamer generation using these devices is still inefficient because they require complicated flow control components for sample and reagent handling and additional off-chip processes. We developed microfluidic SELEX platforms for rapid isolation of aptamers that possess greatly simplified designs which enable easy chip fabrication and operation. The simplicity of the devices is achieved by utilizing a combination of bead-based selection and amplification of target binding nucleic acids, and gel-based electrokinetic transfer of nucleic acids. In the devices, nucleic acids that bind to targets are isolated on target-functionalized microbeads or target cells in a microchamber and electrokinetically transported to another chamber through a gel-filled microchannel by an electric field. The strands are then hybridized onto reverse primers immobilized on microbeads and amplified via polymerase chain reaction (PCR) using on-chip temperature control. The amplified strands are separated from the beads and electrophoretically transferred back into the selection chamber for subsequent SELEX rounds. Using the devices, we demonstrated enrichment of target-binding nucleic acids against human immunoglobulin E (IgE), the glucose-boronic acid complex, and MCF-7 cancer cells. With the physical and functional integration allowed by the monolithic design realized in our devices, the total process time for selection of aptamers was drastically reduced compared with that required by conventional aptamer selection platforms. Moreover, the binding affinities of the selected strands using our devices are comparable to those of aptamers obtained using the conventional platforms.

碩士
國立暨南國際大學
應用化學系
104
In this work, we have developed a facile method for efficient production of metal-ion and polyarginine co-functionalized nanodiamonds （denotes as PA-ND@PSS-Mn+） as a novel IMAC platform for phosphoproteomic analysis. Polyarginine-coated nanodiamonds （PA-ND） were mixed with poly-styrenesulfonate（PSS） via multivalent electrostatic interactions. Immobilization of zirconium ion on the surface of poly-arginine/poly-styrenesulfonate（PSS） functionalized nanodiamonds was done by a 30-min incubation in ZrCl4 solution at room temperature. The PA-ND@PSS-Zr4+　was further characterized using particle size and zeta-potential analyzer, energy dispersive spectroscopy, and EDTA titration, and its performance for selective enrichment of phosphorpeptides was evaluated using the tryptic digests of a complex BSA/β-casein protein mixture in a molar ratio of 1000:1. Experimental results showed that PA-ND@PSS-Zr4+ can be applied for selective identification of singly phosphorylated peptides as well as multiply phosphorylated peptides via a two-step elution process.　 The obtained zirconium ion and polyarginine co-functionalized nanodiamonds were successfully applied to the selective enrichment of both standard protein digests and real samples.