L’émergence de nouvelles technologies basées sur la microfluidique de gouttes offre l’espoir de développer des immuno-essais complexes consommant peu d’échantillon. Cette thèse est dédiée d’une part au développement d’une plateforme d’analyse qui consiste au fractionnement d’un mélange de peptides amyloïdes par focalisation isoélectrique (IEF) pour ensuite procéder à l’immuno-détection en gouttes des différentes fractions. Une méthode d’IEF en capillaire a été développée et a permis la séparation des peptides Aβ 1-40, Aβ 2-40 et Aβ 5-40 ainsi que leur collecte dans des solutions individuelles. Ces solutions de collectes ont été analysées par Elisa sur particules magnétiques. L’immuno-essais a permis de déterminer la distribution des peptides dans les différentes collectes. Ces analyses ont d’abord été réalisées en « batch » où les particules magnétiques sont en suspension dans une solution non-confinée. Ensuite, un dispositif permettant de réaliser des immuno-essais en gouttes a été utilisé. Cette plateforme a été adaptée et optimisée pour le dosage des peptides Aβ 1-40 et Aβ 1-42 standard en vue de doser ces peptides dans le LCR et d’étendre l’analyse aux autres peptides (Aβ 2-40 et Aβ 5-40). D’autre part, une partie de la thèse a été dédiée à un travail sur micro-puce en vue de la conception d’un microsystème d’analyse totale intégrant en ligne les trois étapes majeures (séparation, compartimentalisation, immuno-détection en gouttes) de la plateforme d’analyse recherchée. Pour cette partie nous avons choisi le THV Dyneon qui a déjà servi au transport de bio-molécules en gouttes. Une première étude a consisté pour la première fois à réaliser des séparations électrocinétiques de protéines et de fluorophores hydrophobes dans les puces en THV Dyneon en utilisant deux modes de séparation (ECZ et NACE, respectivement). Ceci a permis de démontrer que ce nouveau matériau constitue un candidat prometteur pour le futur microsystème couplant séparation et compartimentalisation en gouttes. Une dernière étude a consisté au développement d’une méthode de marquage des peptides amyloïdes par un nouvel agent fluorescent, le Chromeo P540, particulièrement adapté pour des analyses IEF. Cet agent de marquage permettra de détecter les peptides durant la conception de l’interface de couplage (IEF/gouttes). / Emerging droplet-based microfluidic platform provide new opportunities to develop complex immunoassay with low sample consumption. This manuscript is dedicated on the development of an analytical platform for isoelectric focusing of amyloid peptides followed by their detection by immunoassay droplet-based microfluidic platform. A capillary isoeletric focusing method was developed first and allowed separation and collection of Aβ 1-40, Aβ 2-40 and Aβ 5-40 peptides in individual solutions. The fractions collected were analyzed off-line by Elisa on magnetic beads in batch-wise. This immune-detection allows the determination of peptide distribution in the different fractions collected. Then, we used a droplet-based microfluidic platform for immunoassay on magnetic beads. This platform was adapted and optimized to quantify standard Aβ 1-40 and Aβ 1-42 peptides. The next step will rely on the quantification of amyloid peptides on cerebrospinal fluid. In parallel, a study dedicated on microchip analyses to design a total-analysis-system has been performed. The final purpose is to integrate on-line three main steps; separation, compartmentalization and immune-detection in droplet. For this study, THV Dyneon has been chosen since this material was also used for the droplet-based microfluidic platform. A first study demonstrated for the first time electrokinetic separation, of biomolecules (proteins) and hydrophobic dyes in Dyneon THV chip using two separations modes (CZE and NACE respectively). This work highlighted the THV Dyneon micro-chip versatility. We can concluded that this new material is promising for further development dedicated to micro system combining separation and compartmentalization in droplet. A last work aimed to develop a labeling method for amyloid peptides with a new dye, the Chromeo P540, which is adapted for isoeletric focusing (IEF) analysis. This dye will allow peptides monitoring during the development of IEF/droplet interface.

This diploma thesis deals with the determination of glucosinolates in plant material by capillary electrophoresis and high performance liquid chromatography. The chemical structure, biosynthesis, degradation, and also biological effects of glucosinolates are described. One part of this work also deals with the methods, which glucosinolates in plant materials were determined by. The experimental part describes the separation of intact glucosinolates by capillary electrophoresis and high performance liquid chromatography. Two plant materials were available for the determination of glucosinolates, namely lyophilized rapeseed leaves and broccoli juice. Micellar electrokinetic chromatography using a cationic surfactant was used to determine intact glucosinolates by capillary electrophoresis. After finding the optimal conditions for the separation of intact glucosinolates, it was found that it is impossible to determine these substances in plant samples. The reason was interference from the matrix, which interfered with this determination. While using high performance liquid chromatography under optimal conditions, some of the intact glucosinolates were identified in a rapeseed plant sample. Furthermore, the calibration dependencies of individual glucosinolates were obtained and the recovery and...

<p>U radu je: 1. Određena efikasnost elektrokinetičkih tretmana (konvencionalni tretman, tretman sa izmenom polariteta, tretman sa dve anode i heksagonalni dvodimenzionalni elektrokinetički tretman) koji izuzimaju dodatak agenasa u tretirani sediment, za uklanjanje metala; 2. Ispitan je uticaj diskontinuiteta napajanja strujom (tokom noći), kao i uticaj oscilacija jačine električne energije pri primeni solarnih panela na efikasnost tretmana; 3. Ispitan je uticaj geohemije sedimenta, fizičko-hemijskih promena u sistemu i karakteristika prisutnog zagađenja (oblici pojavljivanja metala, koncentracije) na efikasnost elektrokinetičkog procesa. 4. Izvr&scaron;ena je procena rizika u sistemu sediment/voda pre, tokom i nakon tretmana; 5. Primenjen je matematički model na dobijene rezultate. Na osnovu određene efikasnosti i procene rizika tretmana elektrokinetičke remedijacije na realan problem Velikog Bačkog kanala zaključeno je, da je najbolja opcija dvoanodni tretman u kome se primenjuju solarne panele (e9). Dobijeni rezultati tretmana ukazuju da diskontinuiteti napajanja strujom (tokom noći), kao i uticaj oscilacija jačine električne energije pri primeni solarnih panela nemaju značajnog uticaja na efikasnost procesa. &Scaron;to se tiče, geohemije sedimenta, pokazalo se da ona ima snažan uticaj i da upravo ona određuje fizičko-hemijske promene koje se događaju u sedimentu tokom sprovođenja tretmana elektrokinetičke remedijacije. Ključ uspeha ovih tretmana sedimenta jeste i zadržavanje metala u frakcijama (karbonatna i reducibilna) koje lak&scaron;e migriraju. Primenom matematičkog modela zaključeno je da se mogu predvideti promene koncentracija metala bez sprovođenja eksperimenata, &scaron;to je od krucijalnog značaja za buduća istraživanja.</p> / <p>This work includes: 1. An investigation into efficiency of various electrokinetic treatments (a conventional treatment, and treatments with changing polarity, two anodes and two-dimensional hexagonal electrodes) without the addition of agents to the treated sediment, for the removal of metals; 2. An investigation of the influence of discontinuous (off during the night) and oscillating power supplies on the application of solar panels and their effect on the efficacy of the treatment; 3. A study on the influence of sediment geochemistry, physico-chemical changes in the system and the characteristics of the pollution present (metals speciation and concentration) on the efficiency of the electrokinetic process; 4. A risk assessment of the sediment/water system before, during and after the treatment; 5. Mathematical modelling of the results obtained. Based on the risk assessment and the efficiency of the electrokinetic treatments in the Veliki Backa canal sediment, it was concluded that the best remediation option was a two anode treatment using solar panels (e9). The investigation into the influence of treatments using discontinuous (off during the night) and oscillating power supplies showed they did not have a significant impact on the efficiency of the process. Sediment geochemistry was shown to have a strong influence, determining the physical and chemical changes that occur in the sediment during the implementation of the electrokinetic remediation. The key to success for these treatments is the retention of metals in the fractions (carbonate and reducible) which more easily migrate. It is concluded that the mathematical model can be applied to successfully predict thechanges in the concentration of metals without experimentation, which is of crucial importance for future studies.</p>

The purpose of this study is the characterization of the membrane-water interfaces of both sarcoplasmic reticulum membrane (SR) and charged lipid bilayers under varied properties of the surrounding aqueous solution. In this work we studied the electrokinetic properties of liposomes and SR vesicles as well as the interaction of lipophilic ions with these membranes. The study of electrokinetic properties is based on the measurements of electrophoretic mobility of SR membrane vesicles and PC/PG liposomes. Electrophoretic mobility of SR vesicles was measured as a function of ionic strength for six pH values (pH 4.0, 4.7, 5.0, 6.0, 7.5, and 9.0). Electrophoretic mobility of single-layered and multi-layered PC/PG liposomes was measured at neutral pH as a function of ionic strength. For interpretation of electrophoretic mobility studies, SR vesicles (at pH 4, 7, and 9) and multi-layered and single-layered liposome sizes were determined using photoelectron microscopy. The study of the interaction of lipophilic ions with these membranes is based on (1) measurements of their partition coefficients described in terms of an ion partition model based on the Langmuir adsorption model and (2) electrophoretic mobility measurements of SR vesicles and PC liposomes in suspension with varied concentration of lipophilic ions. SR-water and PC-water partition coefficients were measured as a function of concentration for two anions tetraphenylborate (TePB-) and pentabromophenol (PBP-) and two cations (Imipramine+, and Clomipramine+). The anions belong to a class of pesticides and the cations are drugs once prescribed as anti-depressants. Partition into the SR membrane was shown to be significantly greater for all lipophilic ions except TePB-, which only showed this effect at the higher lipophilic ion range of the data. The PC-water partition coefficient was also measured for TePP+. Since the lipid bilayer of SR is not significantly different than that of PC liposomes, we believe the differences in partition are due to excess lipophilic ions being absorbed to the proteins of SR. The electrokinetics of charged PCPG liposomes, and PC liposomes with absorbed lipophilic ions could be understood in terms of the charge being located below their surface and screened by counter-ions inside the polar head-group region. We call this model the "permeable surface model". The assumptions of this model are that (1) the charge exists on a plane at a depth, d, below the surface of the liposome within the lipid head-group region and (2) small ions (Na+, K+, Cl-) are able to penetrate the lipid head-group region with a molar membrane-water partition coefficient of 0.4. Using this model we were able to obtain the depth of sorption of lipophilic ions in PC liposomes. We found values of 0.13 nm for TePB-, 0.5 nm for PBP-, 0.12 nm for Imipramine+, 0.17 nm for Clomipramine and 0.25 nm for TePP+. The depth of lipophilic ions in PC is a valuable quantity for the study of the effect of lipophilic ions on membrane function. For PCPG mobility we found the charged plane due to PG lipids was 0.2 nm for single-layered liposomes and 0.1 nm for multi-layered liposomes. This is consistent with the relative size of PC and PG head groups The dependence of SR mobility on pH was found to be directly correlated with the total charge of the A, P, and N domains of the Ca2+-ATPase as determined by the amino acid residues and their corresponding pKa values in water. We found that detached charged plane model, a new model developed in our group, could be fit to the mobility of SR as a function of ionic strength while other soft particle models failed. The assumptions of this model are that (1) the friction caused by protruding proteins on the surface of SR can be represented by a homogeneous retardation layer of thickness D and softness parameter λRL, and (2) the charge of the APN domain can be represented as a plane of charge embedded in the retardation layer at a distance s from the membrane surface. The best-fit values for λRL, and s were not consistent for different pH value studies. The detached charged plane model was unable to predict the mobility of SR vesicles in the presence of lipophilic ions if we assumed that the lipophilic ions were sorbing to the detached charged plane that represents the native charge of the APN domains of SR. At high lipophilic ion concentration the experimental mobilities consistently were greater in magnitude than the values predicted by the model. We concluded that there is significant absorption of lipophilic ions to the proteins in SR membrane, and that the lipophilic ion sorption sites are not the same as the detached plane of charge that represents the native charge of the APN domain.

Micellar electrokinetic capillary chromatography (MECC) is a form of capillary zone electrophoresis. Addition of a surfactant produces micelles in an aqueous/organic buffer. Separation of drugs is obtained via differences in the electrophoretic mobilities of the analytes within the capillary, resulting from their electrophoretic velocity and the electroosmotic flow of the buffer in a given electric field. The migration order is determined by the differential partitioning of the drugs between the micelles and the aqueous/organic phase. Barbiturates were extracted from various biological fluids at pH 4.5 with TOXI-TUBES B. MECC analyses were performed using a Waters Quanta 4000 Capillary Electrophoretic System with a 745 Data Module with a 75 μ x 60 cm capillary and an aqueous/organic buffer of 85% 10 mM borate, 10 mM phosphate, 100 mM sodium dodecyl sulfate and 15% acetonitrile at a pH of 8.5 with a voltage of 20 kV using ultraviolet absorption detection at 214 nm. Migration times were: phenobarbital, 7.78 min.; butalbital, 8.01 min.; butabarbital, 8.23 min.; mephobarbital (internal standard), 8.88 min.; amobarbital, 9.41 min.; pentobarbital, 10.03 min. and secobarbital, 10.79 min. Correlation coefficients (r) between peak areas and concentration ranges of 3 to 60 μg/mL were from 0.964 to 0.999. Coefficients of variation (CV) raged from 2.6 to 8.6% between days and 2.3 to 9.8% within day. Application of this methodology to four forensic cases of butalbital intoxication detected concentrations of 0.7 to 12.7 μg/mL in blood; 0.8 to 1.9 μg/mL in vitreous humor and 1.5 to 7.6 μg/mL in urine. MECC is applicable to forensic analysis of barbiturates extracted from biological fluids.

Sites contaminated with polycyclic aromatic hydrocarbons can be simultaneously contaminated with trace elements. Co-contaminated soil is considered a complex problem since inorganic and organic contaminants behave differently and thereby often require different remediation strategies. Despite the fact that co-contaminated soils are a common problem, existing research on remediation of contaminated soil mostly focuses on either organic or inorganic contaminants. In the present study, the possibilities of electrokinetic remediation as an alternative to commonly used remediation technologies was investigated. An experiment was setup to evaluate the effects of electrokinetic remediation on both polycyclic aromatic hydrocarbons and trace elements, such as arsenic, cadmium, chromium, copper, lead and zinc, simultaneously. The experiment was performed in 12 litre large plexiglass cells. Two cells with electrokinetic treatment, equipped with iron electrodes, to amend the soil with iron via intentional corrosion of the electrodes, and one control. The cells were filled with contaminated soil and deionized water was pumped through the cells to simulate a groundwater flow. The experiment ran for two months, during which pore water was sampled weekly and simulated groundwater monthly, to monitor changes in contaminant concentrations. Soil samples were taken at the start and at the end of the experiment. Results showed that the concentration of polycyclic aromatic hydrocarbons in the simulated groundwater decreased from 0.39 ± 0.15 μg L-1 to 0.12 ± 0.064 μg L-1 during the experiment. However, at the point of writing, samples of soil at the end of the experiment has not yet been analysed for concentration of polycyclic aromatic hydrocarbons, something that needs to be done to validate previously described results. In terms of the trace elements, the applied method did not increase their mobility, nor did it decrease it. Moreover, the trace elements behaved similarly in the control cell as in the treatment cells. This was observed for all the tested trace elements, indicating that electrokinetic remediation in this particulate case may be a suitable remediation technology for organic, but not that successful for inorganic contaminants. In regard to this, further research is required to establish whether electrokinetic remediation is a promising remediation technology for co- contaminated soil and how it can be optimized to ensure remediation success for both organic and inorganic contaminants.

Capillary electrophoresis (CE) is an analytical chemistry approach that allows for the efficient separation by charge of diverse classes of compounds for analysis, including secondary metabolites. The goal of this work was to optimize a buffer system for plant tissue analysis using micellar electrokinetic chromatography (MEKC), and by doing so to understand the role of buffer components in the performance of this form of capillary electrophoresis. In this experiment we implemented a factorial design to optimize buffer composition for separating plant tissue and secondary metabolites. The results of this experiment will be used to optimize a universal buffer for MEKC analysis that can be used on any variety of plant tissues. To determine the feasibility of this, a diverse set of plant secondary metabolite chemical standards in solution were tested as well as Helianthus annuus tissue to confirm the separation in a real biological sample. The results of this optimization yield insights into the utility of buffer components like electrolyte and pH for MEKC separation.

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