Master of Science / Department of Chemistry / Christopher T. Culbertson / The field of microfluidic devices has being developed quickly. It is aimed at integration of many chemical functions in a single chip, such as sample pretreatment, preconcentration, separation and detection, which provides a number of advantages including simplicity, automation, reduced analysis time, decrease in amount of samples and reduced formation of waste. Its potential applications have been conducted in the fields such as biotechnology, pharmaceuticals, life sciences, public health, agriculture and related areas. Membrane technology has been applied in analytical chemistry for many years and has won substantial growth in microfluidics over the past 10 years. Membrane is used to control transfer of some kinds of species, which can be employed for sample concentration, sample preparation, sample filtration and microreactors and so on. Sol-gel process, which usually involves catalytic hydrolysis of sol-gel precursor(s) and catalytic polycondensation of the hydrolyzed products and other sol-gel-active components present in the reaction medium to form a macromolecular network structure, is one of the most suitable methods for membrane fabrication. In this work, titanium membrane was fabricated inside glass microchips using the precursor of titanium isopropoxide. The resulted membranes demonstrated the excellent preconcentration effect. Followed separation and detection were also achieved. CE has been highly accepted as an efficient separation technique for qualitative and quantitative determination, which is performed using a narrow-bore capillary tube. It offers advantages of simplicity, high resolution separation, and minimal cost in terms of analysis time and sample consumption. In this work, protein separations were carried out by CE. Laser-induced fluorescence was used as detection. On-column fluorescence labeling using a fluorogenic labeling reagent was made. Under suitable experimental conditions, an excellent separation performance with about 1.4 million theoretical plate numbers was achieved.

MicroRNAs (miRNAs) are a class of small, single-stranded, non-protein coding RNA molecules that regulate cellular messenger RNA (mRNA) and protein levels by binding to specific mRNAs. Aberrant miRNA expression has been shown to be implicated in several diseases, including cancer. Extracellular miRNAs have been found to circulate in the bloodstream and some of their levels have been associated with different diseases. Furthermore, they hold promise as tissue- and blood-based biomarkers for cancer classification and prognostication. Blood-based biomarkers are attractive for cancer screening due to their minimal invasiveness, relatively low cost and ease of reproducibility. New miRNA analysis techniques will add toward the understanding of their biological functions. In this thesis, I investigate the utility of capillary electrophoresis (CE) and mass spectrometry (MS) for analysis of miRNAs through proof-of-concept experiments. In the fi rst part of this work, we developed a Protein-Facilitated Affinity Capillary Electrophoresis (ProFACE) assay for rapid quantification of miRNA levels in blood serum (see List of publications (6)). We also implemented a capillary electrophoresis with laser induced fluorescence detection (CE-LIF) method with online sample pre-concentration for detection of endogenous microRNAs in human serum and cancer cells. 3' modification of miRNA is a physiological and common post-transcriptional event that shows selectivity for specific miRNAs and is observed across species. Recent studies have shown that post-transcriptional addition of nucleotides to the 3' end of miRNAs is a mechanism for miRNA activity regulation. For example, such modifications in plants and C. elegans influence miRNA stability. In humans, effects on miRNA stability and on mRNA target repression have both been observed. Thus, there is a need for miRNA detection techniques which are direct and multiplexed, require minimal sample preparation and provide qualitative information regarding these modifications. We developed a multiplexed miRNA detection technique based on capillary electrophoresis coupled on line with electrospray ionization mass spectrometry (CE-ESI-MS). This method allowed a label-free, direct detection of multiple miRNAs extracted from cancer serum as well as their post-transcriptional modifications with a high mass accuracy.

La mise au point de nouveaux matériaux possédant des propriétés antibactériennes est un sujet intéressant le monde médical et environnemental. Pour cela, les polymères commerciaux de Merrifield et de Wang-benzaldéhyde ont été modifiés par greffage de motifs calixarèniques polycationiques antibactériens inspirés des travaux du laboratoire et désignés pour interagir avec la surface bactérienne chargée négativement. Ces calixarènes ont été modifiés sur la partie basse, de façon contrôlée, par incorporation d'un groupe espaceur fonctionnel permettant d'accéder à un greffage ciblé du polymère. Nous avons en premier lieu évalué plusieurs types de fonctionnalités introduites sur le calixarène, permettant la fixation de ces motifs sur le support polymérique. Ainsi une amination réductrice a été choisie pour l’ancrage sur la résine de Wang-benzaldéhyde, tandis qu’un point d’ancrage de type pyridinium, pour la résine de Merrifield, a retenu notre attention et s'est avéré être un excellent candidat pour le greffage des calixarènes. La validité de ce point d’ancrage pyridinium a pu être vérifiée par l’incorporation d’une sonde fluorescente (pyrène) et caractérisée par fluorescence à l’état solide, par spectroscopie infrarouge et analyse élémentaire, ces deux dernières étant appliquées à l'ensemble des polymères préparés par la suite. Au travers d'une étude de capture–relargage en milieu aqueux de deux antibiotiques carboxyliques (type quinolone et ß-lactame) le polymère pyridinium modèle, sans calixarène, a montré tout son intérêt, face à la cholestyramine (Questran®) ou à l'Amberlite IRA-400, en tant que résine échangeuse d'anions et pouvant mener à une utilisation en tant que dépolluant/décontaminant. En amont des études antibactériennes de ces nouveaux matériaux nous avons cherché à quantifier la capacité du matériau à retenir/séquestrer des bactéries. L’électrophorèse capillaire, méthode analytique rapide et sensible, s’est présentée comme une solution adéquate. Sur le modèle E. coli., les résines polycationiques synthétisées ont été évaluées en tant que séquestrants en milieu aqueux. Les résultats obtenus prouvent l’efficacité de certaines d’entre elles; la capture a finalement été confirmée par microscopie de fluorescence confocale. Le nombre de bactéries fixées à la surface du matériau a pu être visuellement évalué / Development of new materials with antibacterial properties is a major concern in medical and environmental world. It’s for that reason that, Merrifield and Wang commercial polymers were modified by grafting polycationic calixarenic sub-units inspired by laboratory work and designed to interact with negatively charged bacterial surface. Those calixarenes were modified on the lower part, in a controlled manner, by the incorporation of a functional spacer group leading to a targeted grafting of the polymer. We have, at first, evaluated several kinds of functionalities introduced on the calixarene, giving us the opportunity to graft them on the polymeric support. Like this, a reductive amination was chosen to anchor the Wang-benzaldehyde resin, whereas a pyridinium anchoring point was pointed out as a very good candidate for the grafting of calixarenes. The validation of this pyridinium anchoring point was checked by incorporation of a fluorescent probe (pyrene) and characterized by solid state fluorescence, by infrared spectroscopy, those two lasts analysis were applied for all the other grafted polymers grafted after that. Through a capture-release study in aqueous media of two carboxylic antibiotics (quinolone and ß–lactame kind), the pyridinium polymer model, without calixarène, showed his interest faced to Cholestyramine (Questran®) or Amberlite IRA-400, as an anion exchange resin and leading to depoluting/decontamination applications. Before antibacterial studies of thoses new materials, we wanted to find a way to quantify the material capacity to catch/hold bacteria. Capillary electrophoresis, rapid and sensitive analytical method, appeared as a perfect solution. Using E. coli model, synthesized polycationic resins were evaluated as sequestering agent in aqueous media. Results obtained prove the efficiency of some of them; capture was finally confirmed by confocal fluorescent microscopy. The number of bacteria fixed by material surface could be visually evaluated

Orientador: Ana Valéria Colnaghi Simionato / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-24T11:07:57Z (GMT). No. of bitstreams: 1 Buzatto_AdrianaZardini_M.pdf: 6706551 bytes, checksum: 371c3400224fcd4031972f241185d555 (MD5) Previous issue date: 2014 / Resumo: O câncer está entre as maiores causas de óbitos no mundo, porém, se a enfermidade for detectada no início, grande parte dos casos pode atingir a cura. Os nucleosídeos são moléculas originadas da degradação do RNA e suas concentrações em fluidos biológicos são alteradas na presença de tumores devido à maior taxa de replicação celular, portanto são potenciais biomarcadores tumorais. A investigação do perfil de tais metabólitos pode auxiliar no diagnóstico e no tratamento do câncer. Neste trabalho, um novo método para a análise de nucleosídeos em soro sanguíneo por eletroforese capilar (CE-UV) no modo cromatografia micelar eletrocinética capilar (MECK) foi desenvolvido e validado. O surfactante catiônico brometo de cetiltrimetilamônio (CTAB) foi empregado, com consequente inversão do fluxo eletrosmótico. Os analitos foram extraídos da matriz de soro sanguíneo através de extração em fase sólida (SPE) e um amplo estudo sobre estratégias de pré-concentração foi realizado para obtenção de limites de quantificação mais baixos e adequados. Sete padrões de nucleosídeos mais o padrão interno foram separados em c.a. 25 min sob as condições de análise otimizadas, i.e. eletrólito de corrida composto por ácido bórico 30 mmol/L, CTAB 50 mmol/L e metanol 10% (v/v) em pH 9,90; tensão de -10 kV; temperatura de 20°C; injeção hidrodinâmica a 50 mbar por 120 s seguida por aplicação 25 kV entre vials de eletrólito por 15 s; e capilar de sílica fundida com 50 µm de diâmetro interno e 56 cm de comprimento efetivo. Foram atingidas eficiências entre 4,3 ? 104 e 1,0 ? 105 e resolução mínima de 1,5. A metodologia foi validada em um pool de soro sanguíneo de indivíduos sadios e foram obtidos limites de quantificação entre 1,0 e 4,0 µmol/L, coeficientes de correlação linear entre 0,9801 e 0,9969 e precisões e exatidões menores que 20%. O método foi aplicado a amostras de 60 indivíduos sadios e 66 portadores de câncer de próstata e um estudo quimiométrico foi realizado para avaliação da potencialidade dos nucleosídeos como biomarcadores tumorais, com obtenção de especificidade acima de 90%. Tal metodologia, portanto, pode ser empregada em conjunto com as já bem estabelecidas para o diagnóstico do câncer de próstata / Abstract: Cancer is amongst the major causes of death worldwide, but, if the disease is detected early, most cases can be cured. Nucleosides are molecules originated from RNA degradation and their concentrations in biological fluids are altered during tumor development due to a higher rate of cell replication; therefore, they are putative tumor biomarkers. The investigation of the profile of such metabolites may aid in the diagnosis and treatment of cancer. In this work, a novel method for the analysis of nucleosides in blood serum by capillary electrophoresis (CE-UV) under micellar electrokinetic capillary chromatography (MECK) mode was developed and validated. The cationic surfactant cetyltrimethyl ammonium bromide (CTAB) was employed, with consequent reversal of the electrosmotic flow. The analytes were extracted from blood serum samples by solid phase extraction (SPE) and a wide study on pre-concentration strategies was accomplished in order to obtain lower and more adequate limits of quantification. Seven nucleoside standards plus the internal standard were separated within c.a. 25 min under the optimized conditions, i.e. electrolyte composed by 30 mmol/L boric acid, 50 mmol/L CTAB and 10% methanol (v/v) with pH 9,90; voltage of -10 kV; temperature of 20°C; hydrodynamic injection under 50 mbar for 120 s followed by 25 kV for 15 s between electrolyte vials; and fused silica capillary with 50 µm internal diameter and 56 cm of effective length. Efficiencies between 4.3 ? 104 and 1.0 ? 105 and a minimum resolution of 1.5 were achieved. The methodology was validated in a pool of blood serum samples from healthy individuals and limits of quantification between 1,0 and 4,0 µmol/L were obtained, as well as linear correlation coefficients between 0,9801 and 0,9969 and precision and accuracy lower than 20%. The method was applied to samples from 60 healthy individuals and 66 prostate cancer patients and a chemometric study was employed in order to evaluate the potentiality of the nucleosides as tumor biomarkers, with specificity higher than 90%. Therefore, this methodology may be applied in conjunction with those already established for prostate cancer diagnosis / Mestrado / Quimica Analitica / Mestra em Química

Exposomics is an emerging area of study that looks at how the environment around a person or persons affects their overall health. Biomarkers have emerged as useful tools to better understand how the exposome affects a person. In this work, we investigate two potential endogenous biomarkers, homocysteine and glutathione that have been previously implicated in a number of diseases that have been linked to environmental causes. We also investigated an environmental exposure, the fungicide Ziram, which epidemiologically has been linked to diseases. In our investigation, we utilized capillary electrophoresis and capillary electrophoresis-mass spectrometry to develop a method for homocysteine and identify a derivative to keep it from auto-oxidizing. We utilized mass spectrometry to identify the best ionization technique to detect Ziram and confirmation of where it binds on thiol-containing molecules. We also developed a method to extract glutathione-Ziram from serum and utilized liquid chromatography-mass spectrometry to begin a validation of glutathione-Ziram. Although future work is needed, we believe that this work was the beginning steps to understand biomarkers and their role in the exposome.

La glycosylation est l’une des principales modifications post-traductionnelles des protéines. La glycosylation des protéines est fortement modifiée lors de diverses pathologies comme le cancer, la polyarthrite rhumatoïde ou les troubles congénitaux de la glycosylation («Congenital disorders of glycosylation», CDGs). Ainsi, la nature et les proportions relatives des oligosaccharides liés aux protéines peuvent être utilisées pour dépister, pronostiquer voire suivre l’évolution de pathologies. Les travaux de cette thèse se sont focalisés sur l’étude de la glycosylation pour permettre le dépistage de certaines pathologies : les CDGs, la maladie d’Alzheimer (MA) et la dégénérescence rétinienne. Plusieurs stratégies fondées sur l’électrophorèse capillaire (EC) ont été envisagées pour répondre à cet objectif. Tout d’abord, le développement d’une méthode par EC de zone (ECZ) l’apolipoprotéine C-III (ApoC-III) intacte, une O-glycoprotéine impliquée dans le dépistage des troubles de la O-glycosylation, a permis de séparer les différentes glycoformes selon leur degré de sialylation. L’analyse d’échantillons d’ApoC-III standard provenant de différents fournisseurs par spectrométrie de masse (MS) MALDI -TOF a mis en évidence une hétérogénéité liée à la présence (inattendue) de formes carbamylées. Un traitement du plasma basé sur une immunocapture de l’ApoC-III suivie d’une dérivation sur billes magnétiques de la protéine par un fluorophore a permis de séparer ses différentes glycoformes en ECZ. Ensuite, une analyse N-glycomique de fluides biologiques employant une nouvelle technique de préparation d’échantillon que nous avons adaptée au plasma et au liquide céphalorachidien, a été réalisée par EC en gel couplée à une détection par fluorescence induite par laser (ECG-LIF) sur des patients sains et atteints de la MA. Cette étude a permis de mettre en évidence quelques modifications des N-glycanes chez ces patients. Enfin la combinaison des deux stratégies d’analyse de la glycosylation (glycoprotéine intacte et glycanes libérés), a permis de détecter la transferrine intacte présente dans le liquide vitré à des concentrations faibles ainsi que ses N-glycanes libérés, dans le cadre du dépistage d’une maladie oculaire. L’EC-QTOF-MS a également été explorée pour l’analyse de N-glycanes dérivés avec un nouveau fluorophore permettant d’améliorer la sensibilité en MS. / Glycosylation is one of the most main types of post-translational modifications of proteins. Disease-associated modifications in protein glycosylation have been observed for various pathologies such as cancer, rheumatoid arthritis, or «Congenital disorders of glycosylation» (CDGs). They are often exploited for diagnosis, prognosis and monitoring of these diseases. This thesis work focused on the glycosylation study with the aim to allow the screening of different pathologies: CDGs, Alzheimer’s disease and retinal degeneration diseases. Different strategies based on capillary electrophoresis (CE) were considered to fulfil this goal. First, the developement of a CZE analysis of intact apolipoprotein C-III (ApoC-III), an O-glycoprotein implied in the screening of O-glycosylation disorders, allowed the separation of its glycoforms according to their sialylation degree. The MALDI-TOF mass spectrometry (MS) analysis of standard ApoC-III batches from different standard ApoC-III batches from different suppliers highlighted an additonnal heterogeneity due to (unexpected) carbamylated species. A plasma pretreatment based on an immunocapture of apoC-III followed by protein derivatization on magnetic beads using a fluorophore allowed to separate its glycoforms by CZE. Second, a glycomic analysis of biologicial fluids using a new sample treatment method that we adjusted to plasma and cerebrospinal fluid samples was performed by CGE-LIF on controls and Alzheimer’s patients. It allowed to highlight some modifications of N-glycans for this disease. Finally, the combination of both strategies of glycosylation analysis (intact glycoprotein and released N-glycans) allowed the detection of intact transferrin present in vitrous humor but also of its released N-glycans for the screening of retinal degeneration disease. CE-QTOF-MS was also investigated for the analysis of released N-glycans derivatized by a new fluorophore which increases MS sensitivity.

In recent years, the use of natural medicinal properties of plants has again come to the forefront of interest of the public. Because of their therapeutic properties, these plants are commonly used in alternative treatments for minor ailments. This thesis deals with the determination of polyphenolic acids in extracts from aronia, echinacea, tetradenia and centella. One of the aims of the thesis was to compare the amount of polyphenolic acids in individual plants with their antioxidant properties. The theoretical part includes a general introduction to flavonoids, introduction to polyphenolic acids, introduction to the principles of capillary zone electrophoresis and a summary of selected foreign studies for the determination of flavonoids. An important chapter of the theoretical part is a description of individual plants with an explanation of their possible therapeutic use. The experimental part is devoted to the process of optimization of electrophoretic determination of selected polyphenolic acids and evaluation of their content. It was found that in the extracts six representatives of polyphenolic acids occur in minimal amounts, often below the limit of quantification or detection of the method.

The past few years have seen a rapid expansion in interest in the characterization of the entire complement of proteins, or proteome. Micro total analysis systems (μTAS) are an emerging promising method, offering rapid, sensitive and low sample consumption separations. I have demonstrated microchip capillary electrophoresis (CE) devices made of CaF2. New methods have been developed for micromachining enclosed capillaries in CaF2. CE analysis of fluorescently labeled amino acids was used to illustrate bioanalytical applications of these microdevices. Initial on-chip infrared spectroscopy results for qualitative analyte identification were achieved in microfluidic CaF2 channels. I have also shown the evaluation of poly(methylmethacrylate) (PMMA) and thermoset polyester (TPE) microchips for use in protein profiling. To improve separation efficiency and reduce protein adsorption, dynamic coating and poly(ethylene glycol) (PEG) grafting using atom transfer radical polymerization (ATRP) have been used in PMMA microdevices. Proteins, peptides and protein digests have been separated electrophoretically in these PMMA microchips. My results demonstrate that PMMA microdevices should be well suited as microfluidic systems for high performance separations of complex biological mixtures. In-channel ATRP has been developed for the surface modification of TPE microdevices. Characterization indicates that PEG-modified microchannels have much lower and more pH-stable electroosmotic flow, more hydrophilic surfaces and reduced nonspecific protein adsorption. CE of amino acid and peptide mixtures in these PEG-modified TPE microchips had good reproducibility. Phosducin-like protein and phosphorylated phosducin-like protein were also separated to measure the phosphorylation efficiency. My results show that PEG-grafted TPE microchips have broad potential application in biomolecular analysis. Cancer marker analysis is important for medical research and applications. I report a method that can covalently attach appropriately oriented antibodies of interest on monolith surfaces. To reduce nonspecific adsorption, protein solutions were used to effectively block the monolith surface. Selective preconcentration and elution of human chorionic gonadotropin have been performed in my affinity columns, demonstrating that this type of system should have promising applications in cancer marker detection.

Organotin compounds are one of the most produced and most used organometallic compounds. Some of these substances are endocrine disruptors, persistent organic polutants and their high toxic effects are observed. That’s why their presence in the environment caused by human activity could endanger many organisms. The aim of this thesis is summarize their properties and their occurrence in the environment. Then the quick, easy and relatively cheap method for determination of trialkyltin compounds in heavily poluted aquatic sediments using capillary zone electrophoresis is developed.

This thesis summarizes knowledge about mycotoxins, with focus to ochratoxin A. It also summarizes its tolerable levels of food intake, detoxification and analytical methods for mycotoxins. The work also includes a chapter describing liposomes that were used for the analysis of ochratoxin A by liposomal electrokinetic capillary electrophoresis (LECK). The practical part includes the analysis of ochratoxin A from Aspergillus ochraceus Wilhelm and Aspergillus melleus Yukawa fungi cultivated on a rye and optimization of the method for analysis of ochratoxin A based on liposomes of different compositions. By capillary zone electrophoresis, ochratoxin A is not sufficiently separated and detected in the extracted mixture; conversely, when liposome solutions are used, different migration behavior can be achieved while stabilizing ochratoxin A in solution due to amphiphilic interactions between mycotoxins and liposomes. Therefore, the LEKC method was used for this work. The most suitable liposome composition has been shown to be molar ratios of 25% cholesterol (membrane stabilization) / 50% 2-oleoyl-1-palmitoyl-sn-glycerol 3-phosphocholine (main zwitterionic lipid) / (25% 1,2-diacyl-sn-glycerol)-3-phospho-L-serine (introduction of negative charge).