Tato práce navrhuje techniku separace a detekce na čipu pro kvantové tečky (QD, „quantum dots“) konjugované s různými proteiny, za účelem sledování vlivu vazebného činidla na potlačení intenzity uorescence QD způsobené konjugací s proteinem a za účelem provedení multianalytické imunoanalýzy k identifikaci malých množství daného proteinu. Za optimálních podmínek byly biokonjugované QD úspěšně odděleny od těch nezkonjugovaných během 10 minut. Částice a cílové roztoky byly smíchány a detekce na čipu byla provedena za pomoci zařízení vyvinutého v naší laboratoři. Byl použit pouze jeden zdroj excitačního světla v kombinaci s několika filtry pro různé emisní vlnové délky. Fluorescence emitovaná dvěma typy konjugovaných QD mohla být poté zaznamenána současně, protože QD emitovaly světlo na různých vlnových délkách, ačkoliv byly excitovány při stejné vlnové délce. Smícháním dvou typů QD biokonjugovaných se dvěma druhy proteinů a protilátek jsme dokázali detekovat imunokomplexní píky s různými plochami. Plocha pod píkem závisela na koncentraci QD a antigenů, na postupu reakcí protilátka–antigen a ukázalo se, že je lineárně korelována s koncentrací antigenu. Ukázali jsme, že kapilární elektroforéza QD na čipu může být použita jako citlivá technika pro detekci biologických molekul. Hlavními výhodami této metody jsou jednoduchost, malé požadavky na objem vzorku i činidla a také vysoká účinnost separace.

Tomáš Křížek: Applications of Capillary Electrophoresis in Life Sciences (Dissertation thesis) ABSTRACT This thesis is focused on the applications of capillary electrophoresis in two important areas of life sciences, proteomics and enzyme assays. In the first part, Pluronic F-127 copolymer was studied as a sieving matrix for proteomic applications of capillary gel electrophoresis. The effect of thermoassociation of Pluronic F-127 on the separation selectivity was investigated and no difference in selectivity of the separation below, inside and above the thermoassociation temperature region was observed. The performance of Pluronic F-127 in capillary gel electrophoresis was compared with dextran as a commonly used sieving matrix. The results showed, that Pluronic F-127 offers superior performance for low-molecular-mass proteins because it provides higher separation power than dextran with significantly lower viscosity of the background electrolyte. The lower viscosity makes the polymer easier to replace after each analysis, which leads to remarkably higher repeatability of the experiments. On the other hand, dextran, due to its higher viscosity, was shown to be more convenient for separations of protein digests, where extremely high separation efficiency is required. The second part focuses on...

This diploma thesis deals with application of newly synthesized α-cyclodextrins derivatives, 2I -O-cinnamyl-α-cyclodextrin and 3I -O-cinnamyl-α-cyclodextrin, in capillary electrophoresis. Their unique feature lies in formation of cyclodextrin aggregates in an aqueous solution by inclusion of phenyl moiety of one molecule into cavity of another one. The influence of addition of 2I -O-cinnamyl-α-CD and 3I -O-cinnamyl-α-CD to background electrolyte (BGE) and its impact on effective mobilities of eighteen selected analytes were tested. Nine analytes were measured in the form of cations (aniline, antipyrine, L-histidine, D,L-tyrosine, D,L- phenylalanine, N-(1-naphtyl)ethylenediamine, 4-nitroaniline, p-aminoaceto-phenon and tyramine) and nine in the form of anions (N-acetyl-D,L-phenylalanine, N-acetyl-D,L-tryptophan, N-benzoyl-D,L-phenylalanine, N-boc-D,L-tryptophan, N-FMOC-D,L-valine, N-FMOC-alanine, N-FMOC-D,L-leucine, D,L-3-phenyllactic acid and (R)-(-)-mandelic acid). Electrophoretic mobilities of cations were tested in BGE at pH 2.2 and anions at pH 8.0. The measurements were conducted at 25 and 50 řC. At the beginning the buffer containing 2.5 mM TRIS was adjusted with phosphoric acid to pH 2.2. However, it was found, that phosphate anions might enter cyclodextrin cavity and disable potential...

This diploma thesis deals with the application of two permanently positively charged monosubstituted β-cyclodextrin derivatives (PEMEDA-β-CD, PEMPDA-β-CD) as chiral selectors in capillary electrophoresis. Use of PEMPDA-β-cyclodextrin in capillary electrophoresis has not been reported in literature. Properties of PEMEDA-β- cyclodextrin are already known, but its application for separation of amino acid enantiomers has not been published yet. Cyclodextrin derivatives were tested as additives in different buffers of different pH and with eventual addition of organic modifier. As suitable background electrolyte 15 mmol·l-1 borate buffer, pH = 9.5 without organic modifier was chosen. Furthermore the influence of chiral selector on separation and eventual enantioseparation of chosen analytes was evaluated. Addition of cyclodextrin derivatives in concentration range 0.0 - 5.0 was tested. Fourteen anionogenic analytes, including native amino acids, N- blocked amino acids and profens, were detected with UV-VIS detector at optimal wavelength 214, 254 or 280 nm. Both chiral selectors were suitable for enantioseparation of N-boc-D,L-tryptophan, which was baseline separated at concentration of selector as low as 0.5 mmol·l-1 . Tested amino acids blocked with terc-butoxycarbonyl and D,L-ketoprofen were partially...

Capillary zone electrophoresis was used for chiral separation of eleven diquat derivatives. These N-heteroaromatic dications containing structural motif of 2,2'-bipyridine have recently been studied for their interesting electrochemical properties as well as for the axial chirality of their molecules. The combination of these properties could potencially lead to interesting applications in the future. For enantioseparation of diquats (DQ) commercially available randomly sulfated α-, β-, and γ-cyclodextrins with high degree of substitution were used. A succesfull chiral separation was achieved using all of the three sulfated cyclodextrins as chiral selectors (CS). Baseline enantioseparation was achieved for 82 %, 91 % respectively 100 % of the analyzed DQ in the presence of HS-α-CD, HS-β-CD, HS-γ-CD respectively. The highest separation efficiency and resolution were obtained in the backround electrolyte containing 22 mmol/L NaOH, 35 mmol/L H3PO4 (pH2,5) and 6 mmol/L HS-β-CD. Using three available nonracemic DQ an identification of the particular M- and P-enantiomers was done for the three corresponding DQ structures. Apparent stabillity constants of complexes of the DQ derivatives with above mentioned cyclodextrins as CS were determined by means of capillary affinity electrophoresis. The stability...

Capillary electrophoresis (CE) is highly efficient separation method based on the different migration velocity of ions in liquid media in electric field. It is commonly used in analytical laboratories and due to the different separation principle it is applied as a complementary method to the chromatographic methods (HPLC and UHPLC). Beside the applicability of CE for quantitative/qualitative analysis, the method can be used also for physico-chemical characterization of compounds (e.g. determination of acid dissociation constants of weak electrolytes or stability constants of complexes). This work is focused on the applicability of CE methods for determination of physico- chemical characteristic of compounds (acid dissociation constants of triazole fungicides and stability constants of dibenzo-18-crown-6, benzo-18-crown-6 and 18-crown-6 ether complexes with metal ions in hydro-organic solvent mixtures) and on the possibility to use affinity CE (ACE) with electrospray ionization-mass spectrometric detection (ACE- ESI/MS) for the study of non-covalent interactions of compounds. For the online hyphenation of CE and ESI/MS, two highly sensitive CE-ESI/MS interfaces were tested: i) porous tip and ii) nano-sheath liquid flow. The ability of the CE-ESI/MS interfaces to effectively decouple spray and...

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The role of biological markers (biomarkers) in public health, pediatric medicine and clinical toxicology cannot be understated. Clinically validated biomarkers used in newborn screening (NBS) serve to detect individuals at risk for a disease in the population, pre-symptomatically diagnose affected neonates early in life and/or accurately predict disease progression and treatment responses to therapy. However, there is urgent need for the discovery of more specific biomarkers that can improve screening accuracy in a high throughput, cost-effective yet ethical manner. The major objectives of this thesis were to develop innovative nontargeted metabolite profiling methodologies based on multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS) for early detection of treatable genetic diseases, as well as comprehensive surveillance of drugs of abuse (DoA) in high risk subjects. Chapter II introduces a multiplexed MSI-CE-MS strategy for confirmatory testing of known biomarkers for various inborn errors of metabolism from a dried blood spot (DBS) that was rigorously validated using proficiency test specimens from Centres for Disease Control and Prevention (CDC) and authentic neonatal samples from Newborn Screening Ontario (NSO) with quality assurance. Additionally, MSI-CE-MS together with temporal signal pattern recognition revealed for the first time a novel class of pathognomonic marker elevated in galactosemia, namely N-galactated amino acids. Chapter III describes an untargeted metabolomic study to discover biomarkers of cystic fibrosis (CF) to reduce the high false positive rate and widespread carrier identification by current two-tiered NBS algorithms that rely on genetic testing. A panel of metabolites from retrospective DBS specimens, including several amino acids, ophthalmic acid and an unknown peptide, allowed for differentiation of asymptomatic CF neonates from screen-positive yet unaffected carriers and transient hypertrypsinogenemic cases. Chapter IV develops and validates a high throughput MSI-CE-MS assay for rapid screening for DoA and their metabolites in human urine with improved specificity and broad spectrum coverage as compared to classic targeted immunoassays. This method can also applied to ensure compliance, drug efficacy and patient safety while detecting for potential substitution or adulteration when using high resolution MS/MS. In summary, this thesis contributes an innovative methodology and data workflow for biomarker discovery for improved neonatal screening of rare genetic diseases in the population, which was also applied for more effective drug surveillance strategies in public health given the alarming worldwide opioid crisis. / Thesis / Doctor of Philosophy (PhD)

Tous les modèles théoriques, mathématiques et empiriques utilisés pour l’évaluation de la mobilité électrophorétique de peptides regroupent trois paramètres de base : la masse du peptide, son nombre de résidus (sa longueur) et sa charge. Or, ces modèles ont une faiblesse commune qui est le paramètre de charge (q). Lors de l’estimation de cette variable, l’usage d’approximation pour les constantes de dissociation acide (pKa) des groupes ionisables des peptides entraîne une erreur sur ce calcul. Une solution utilisée par plusieurs auteurs est de développer les modèles pour la mobilité à bas pH seulement en assumant la protonation complète de tous les groupes acides et basiques. Afin de comprendre l’effet du résidu acide aminé (a.a.) voisin sur l’ionisation des groupes carboxyles et amines terminaux (Cʹ et Nʹ) d’un peptide, les valeurs de pKa de plusieurs séries de peptides ont été déterminés par CE suite à l’optimisation d’une méthode. Les peptides étudiés étaient de type Z-X et X-Z où X est un ou plusieurs résidus voisins variable tandis que Z est un résidu invariable tel la phénylalanine, la glycine et l’alanine. L’objectif primaire de ce projet consistait à déterminer les valeurs réelles du pKa des groupes Cʹ et Nʹ de 69 peptides afin d’élucider l’effet de leur environnement immédiat, i.e., le résidu voisin. Ce travail systématique de compilation des pKa est une approche rarement utilisée. En ayant accès à ces valeurs, la source d’incertitude sur les constantes d’ionisation des peptides auquel font face beaucoup d’auteurs est éliminée. Ceci permet une analyse plus approfondie de leur comportement électrophorétique. Pour la série de dipeptides étudiés, nous avons observé que plus l’hydrophobicité du résidu X augmentait, plus les pKa Cʹ et Nʹ baissait selon le résidu Z. Les résidus X hydrophiles possèdent des chaines latérales chargées ou très polaires qui influencent de façon plus importante le processus d’ionisation que les résidus X hydrophobes. Les données analysées ont aussi permis de déterminer que plus la distance du résidu X au Cʹ ou Nʹ est grande, plus son influence sur le pKa diminue. Également, le prolongement d’un peptide de 1 à 6 résidus, tel les oligoglycines, fait augmenter le pKa-Cʹ et diminuer le pKa-Nʹ via un processus expliqué par la réduction des interactions électrostatiques entre les fonctions Cʹ et Nʹ. Ces résultats illustrent que les pKa Cʹ et Nʹ de peptides ne sont pas exacts lorsqu’ils sont basés uniquement sur l’identité de l’acide aminé terminal, comme c’est le cas pour l’approche classique de modélisation. Les valeurs de pKa de dipepides obtenus par titrages potentiométriques ont été comparés aux pKa déterminés par CE. Des écarts importants ont été obtenus ce qui a mis en lumière la nécessité de contrôler les protocoles expérimentaux de manière rigoureuse pour mieux comparer les résultats provenant de deux techniques. Par la suite, à l’aide des mobilités de peptides obtenus expérimentalement à haut et bas pH, nous avons comparé les résultats de modélisations calculés pour les pKa expérimentaux à ceux de la littéraire afin d’évaluer les avantages du modèle d’Offord. Enfin, des simulations de séparations par CE de 9 peptides obtenues avec les programme informatique PeakMaster ont été comparées avec les résultats expérimentaux. Ceci a démontré l’utilité et la supériorité des pKa expérimentaux comparativement aux pKa les plus communément utilisés avec les approches traditionnelles. / All theoretical, mathematical and empirical models used to evaluate the electrophoretic mobilities of peptides use three basic parameters: the peptide mass, its number of residues (its length) and its charge. However, all models have a common flaw: the charge parameter (q). When estimating this variable, the use of approximations for the ionization constants (pKa) leads to imprecise values for the calculation. One solution is to limit the development of mobility models to only low pH and assume full protonation of all acidic and basic groups. In order to understand the effect of the neighboring amino acid (a.a.) residue on the terminal carboxylate and amine functions of a peptide (Cʹ et Nʹ), the pKa values of multiple series of peptides were determined by capillary electrophoresis (CE) using an optimized method. The peptides studied were of the form Z-X and X-Z, where X was one or multiple variable residues and Z was an invariable residue such as phenylalanine, glycine or alanine. The core of this project consisted of determining the actual Cʹ and Nʹ pKa values for 69 peptides to elucidate the effect of their immediate microenvironment. i.e., the effect of their nearest neighbor. This systematic compilation of pKa’s is an approach that is rarely used. With these values on hand, the uncertainty of the ionisation constant that most authors face is almost eliminated and the analysis of the electrophoretic behavior of peptides can be enhanced. For the peptides studied, we observed that increasing the hydrophobicity of residue X lead to a decrease in Cʹ and Nʹ pKa values, but to different degrees depending on the terminal residue (Z) studied. Hydrophilic X residues possess charged or highly polar side-chains, which influenced Cʹ and Nʹ ionisations to a greater degree than hydrophobic X residues. Additionally, the elongation of a peptide from one to six residues, such as the oligoglycine series studied, lead to an increase in pKa-Cʹ and a decrease in pKa-Nʹ, which might be explained by a diminishing electrostatic interaction between the Cʹ and Nʹ functional groups. These results illustrate that the Cʹ and Nʹ pKa values for peptides are not accurate when based solely on the identity of the Cʹ and Nʹ a.a. residues, which is the classical way peptide charge is estimated for modeling not only mobilities but also chemical and biological reactivity. Potentiometric titration of dipeptides produced pKa values that were compared to pKa’s determined by CE. Significant discrepancies were obtained which brought to light the necessity of rigorously controlling experimental protocols when comparing results obtained with two techniques. Mobility data for high and low pHs were calculated using experimental and literature tabulated pKa values to estimate charge and to therefore understand the benefit gained when applying the Offord mobility model. Finally, simulations of separating 9 peptides by CE were carried out with the computer program PeakMaster and compared with experimental results. This showed the utility and superiority of our cumulated pKa values versus the most widely cited approximate pKa values from the literature.