4 resultados para simultaneous monitoring of process mean and variance

em ArchiMeD - Elektronische Publikationen der Universität Mainz - Alemanha


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Während in den letzten Jahren zahlreiche Biosensoren zum spezifischen Nachweis von DNA entwickelt wurden, ist die Anwendung oberflächen-sensitiver Methoden auf enzymatische Reaktionen ein vergleichsweise neues Forschungsgebiet. Trotz der hohen Empfindlichkeit und der Möglichkeit zur Echtzeit-Beobachtung molekularer Prozesse, ist die Anwendung dieser Methoden nicht etabliert, da die Enzymaktivität durch die Nähe zur Oberfläche beeinträchtigt sein kann. Im Rahmen dieser Arbeit wurde die enzymatische Verlängerung immobilisierter DNA durch eine DNA Polymerase mit Hilfe von Oberflächenplasmonen-Fluoreszenzspektroskopie (SPFS) und einer Quarzkristall-Mikrowaage (QCM) untersucht. Die Synthese von DNA wurde im Fall der QCM als Massenzuwachs detektiert, der sich im Abfall der Resonanzfrequenz des Schwingquarzes und einem Anstieg seiner Dissipationsenergie ausdrückte. Die viskoelastischen Eigenschaften der DNA-Schichten wurden bestimmt, indem die erhaltenen Daten mit einem auf Voigt basierenden Modell ausgewertet wurden. SPFS nutzt das evaneszente elektromagnetische Feld, das mit Oberflächenplasmonen einhergeht, zur oberflächen-sensitiven Anregung von Chromophoren. Auf diese Weise wurde der Einbau von Farbstoff-markierten Nukleotiden in die entstehende DNA-Sequenz als Indikator für das Voranschreiten der Reaktion ausgenutzt. Beide Meßtechniken konnten erfolgreich zum Nachweis der DNA-Synthese herangezogen werden, wobei die katalytische Aktivität des Enzyms vergleichbar zu der in Lösung gemessenen war.

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An accurate and sensitive species-specific GC-ICP-IDMS (gas chromatography inductively coupled plasma isotope dilution mass spectrometry) method for the determination of trimethyllead and a multi-species-specific GC-ICP-IDMS method for the simultaneous determination of trimethyllead, methylmercury, and butyltins in biological and environmental samples were developed. They allow the determination of corresponding elemental species down to the low ng g-1 range. The developed synthesis scheme for the formation of isotopically labeled Me3206Pb+ can be used for future production of this spike. The novel extraction technique, stir bar sorptive extraction (SBSE), was applied for the first time in connection with species-specific isotope dilution GC-ICP-MS for the determination of trimethyllead, methylmercury and butyltins. The results were compared with liquid-liquid extraction. The developed methods were validated by the analysis of certified reference materials. The liquid-liquid extraction GC-ICP-IDMS method was applied to seafood samples purchased from a supermarket. The methylated lead fraction in these samples, correlated to total lead, varied in a broad range of 0.01-7.6 %. On the contrary, the fraction of methylmercury is much higher, normally in the range of 80-98 %. The highest methylmercury content of up to 12 µg g-1 has been determined in shark samples, an animal which is at the end of the marine food chain, whereas in other seafood samples a MeHg+ content of less than 0.2 µg g-1 was found. Butyltin species could only be determined in samples, where anthropogenic contaminations must be assumed. This explains the observed broad variation of the butylated tin fraction in the range of <0.3-49 % in different seafood samples. Because all isotope-labelled spike compounds, except trimethyllead, are commercially available, the developed multi-species-specific GC-ICP-IDMS method has a high potential in future for routine analysis.

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What is the intracellular fate of nanoparticles (NPs) taken up by the cells? This question has been investigated for polystyrene NPs of different sizes with a set of molecular biological and biophysical techniques.rnTwo sets of fluorescent NPs, cationic and non-ionic, were synthesized with three different polymerization techniques. Non-ionic particles (132 – 846 nm) were synthesized with dispersion polymerization in an ethanol/water solution. Cationic NPs with 120 nm were synthesized by miniemulsion polymerization Particles with 208, 267 and 603 nm were produced by seeding the 120 nm particle obtained by miniemulsion polymerization with drop-wise added monomer and polymerization of such. The colloidal characterization of all particles showed a comparable amount of the surface groups. In addition, particles were characterized with regard to their size, morphology, solid content, amount of incorporated fluorescent dye and zeta potential. The fluorescent intensities of all particles were measured by fluorescence spectroscopy for calibration in further cellular experiments. rnThe uptake of the NPs to HeLa cells after 1 – 24 h revealed a much higher uptake of cationic NPs in comparison to non-ionic NPs. If the same amount of NPs with different sizes is introduced to the cell, a different amount of particles is present in the cell medium, which complicates a comparison of the uptake. The same conclusion is valid for the particles’ overall surface area. Therefore, HeLa cells were incubated with the same concentration, amount and surface area of NPs. It was found that with the same concentration always the same polymer amount is taking up by cells. However, the amount of particles taken up decreases for the biggest. A correlation to the surface area could not be found. We conclude that particles are endocytosed by an excavator-shovel like mechanism, which does not distinguish between different sizes, but is only dependent on the volume that is taken up. For the decreased amount of large particles, an overload of this mechanism was assumed, which leads to a decrease in the uptake. rnThe participation of specific endocytotic processes has been determined by the use of pharmacological inhibitors, immunocytological staining and immunofluorescence. The uptake of NPs into the endo-lysosomal machinery is dominated by a caveolin-mediated endocytosis. Other pathways, which include macropinocytosis and a dynamin-dependent mechanism but exclude clathrin mediated endocytosis, also occur as competing processes. All particles can be found to some extent in early endosomes, but only bigger particles were proven to localize in late endosomes. No particles were found in lysosomes; at least not in lysosomes that are labeled with Lamp1 and cathepsin D. However, based on the character of the performed experiment, a localization of particles in lysosomes cannot be excluded.rnDuring their ripening process, vesicles undergo a gradual acidification from early over late endosomes to lysosomes. It is hypothesized that NPs in endo-lysosomal compartments experience the same change in pH value. To probe the environmental pH of NPs after endocytosis, the pH-sensitive dye SNARF-4F was grafted onto amino functionalized polystyrene NPs. The pH value is a ratio function of the two emission wavelengths of the protonated and deprotonated form of the dye and is hence independent of concentration changes. The particles were synthesized by the aforementioned miniemulsion polymerization with the addition of the amino functionalized copolymer AEMH. The immobilization of SNARF-4F was performed by an EDC-coupling reaction. The amount of physically adsorbed dye in comparison to covalently bonded dye was 15% as determined by precipitation of the NPs in methanol, which is a very good solvent for SNARF-4F. To determine influences of cellular proteins on the fluorescence properties, a intracellular calibration fit was established with platereader measurements and cLSM imaging by the cell-penetrable SNARF-4F AM ester. Ionophores equilibrated the extracellular and intracellular pH.rnSNARF-4F NPs were taken up well by HeLa cells and showed no toxic effects. The pH environment of SNARF-4F NPs has been qualitatively imaged as a movie over a time period up to 1 h in pseudo-colors by a self-written automated batch program. Quantification revealed an acidification process until pH value of 4.5 over 24 h, which is much slower than the transport of nutrients to lysosomes. NPs are present in early endosomes after min. 1 h, in late endosomes at approx. 8 h and end up in vesicles with a pH value typical for lysosomes after > 24 h. We therefore assume that NPs bear a unique endocytotic mechanism, at least with regards to the kinetic involvedrn

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In this thesis, interactions of folic acid with tea and tea components at the level of intestinal absorption have been investigated. Firstly, the interaction between folic acid and tea as well as tea catechins was studied in vitro, using Caco-2 cell monolayers and secondly, a clinical trial was designed and carried out. In addition, targeting of folic acid conjugated nanoparticles to FR expressing Caco-2 cells was studied in order to evaluate the principle of nutrient-receptor-coupled transport for drug targeting. In the first part of this work, it was shown that EGCG and ECG (gallated catechins) inhibit folic acid uptake (IC50 of 34.8 and 30.8 µmol/L) comparable to MTX (methotrexate) under these experimental conditions. Moreover, commercial green and black tea extracts inhibited folic acid uptake with IC50 values of approximately 7.5 and 3.6 mg/mL, respectively. These results clearly indicate an interaction between folic acid and green tea catechins at the level of intestinal uptake. The mechanism responsible for the inhibition process might be the inhibition of the influx transport routes for folates such as via RFC and/or PCFT. For understanding the in vivo relevance of this in vitro interaction, a phase one, open-labeled, randomized, cross-over clinical study in seven healthy volunteers was designed. For the 0.4 mg folic acid dose, the mean Cmax decreased by 39.2% and 38.6% and the mean AUC0 decreased by 26.6% and 17.9% by green tea and black tea, respectively. For the 5 mg folic acid dose, the mean Cmax decreased by 27.4% and mean AUC0 decreased by 39.9% when taken with green tea. The results of the clinical study confirm the interaction between tea and folic acid in vivo leading to lower bioavailabilities of folic acid. In the second part of the thesis, targeting studies using folic acid conjugated nanoparticles were conducted. Folic acid conjugated nanoparticles were shown to be internalized by the cell via FR (folate receptor) mediated endocytosis. DNA block copolymer micelles equipped with 2, 11 and 28 folic acid units respectively were applied on FR expressing Caco-2 cells. There was a direct proportion in the amount of internalized nanoparticle and the number of folic acid units on the periphery of the nanoparticle. To sum up, throughout this thesis, the importance of folic acid for nutrition and nutrient and drug related interactions of folic acid at intestinal level was shown. Furthermore, significance of FRs in targeting for cancer chemotherapy was demonstrated in in vitro cell culture experiments. Folic acid conjugated DNA block copolymer micelles were suggested as efficient nanoparticles for targeted drug delivery.