Analytische Chemie
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Eingeladener Vortrag
- nein (369)
In this thesis a protocol based on a direct competitive and non-competitive immunoassay using the microarray format was developed to screen emulated hybridoma supernatants against the drug carbamazepine (CBZ). As support, epoxy slides were manufactured by coating microscope glass slides with epoxy silanes. The manufactured epoxy slides showed similar capacity for protein immobilization as commercially available NEXTERION slides, but showed a higher autofluorescence background. On the epoxy slides, protein A, Cys-A, G and Cys-G were immobilized as antibody capturing coating. It could be shown that the Cys-tag considerably increased the immobilization of all compared proteins, especially of protein G. Protein Cys-G immobilized at pH 8-9 was considered the most suitable protein for antibody immobilization due to good spot uniformity and a high binding capacity for IgG. Additionally, in immobilization experiments it could be shown, that 10-500 fold excess of bovine IgG, introduced by two different fetal bovine serum (FBS) products, showed no significant competition on the immobilization of mice or goat IgG on protein Cys-G. To examine the captured antibodies for affinity to CBZ, the CBZ-TOTA-DY654 tracer was custom synthesized. It could be demonstrated, that the tracer antibody interaction was competitive with the tracer precursor, CBZ-TOTA-NH2 and CBZ. Furthermore, the synthesized tracer was also successfully used by externals to evaluate CBZ affine hybriodoma cells in flow cytometry. In the simulated screening, five antibodies with known affinity to CBZ were diluted in a cell culture medium. The screening method allowed to differentiate between affine antibodies (IC50 < 20 µg/ml) and unaffine antibodies (IC50 > 20 µg/ml), when concentrations of 0.1 µg/ml IgG or greater have been used. Of five applied antibodies, all highly affine clones (CE2 and B3212M) were reported as positives while no false-positive samples were observed. The screening included competitive, with around 89500 fold excess of CBZ, and non-competitive tracer incubation on different segments of the same chip, which resulted in total signal suppression on the competitive segment. The screening was performed in 20 hours, only a few nanoliters, of each simulated supernatant, were consumed in the screening process, furthermore in the method standard 96 well MTPs were used and no cleanroom facilities were required.
Ausgehend von dem in der Literatur beschriebenen Verfahren zur Bestimmung aromatischer Aminosäuren konnte eine Analysemethode zur Proteinbestimmung über die optische Detektion von Tyrosin und Phenylalanin nach chromatographischer Trennung der Protein-Hydrolysate entwickelt werden, die gegenüber dem Verfahren aus der Literatur erhebliche Vorteile besitzt. Der Einsatz von Rundbodenvials stellt eine Neuerung und kostengünstige Alternative zu der konventionellen Hydrolyse in Vakuumhydrolyseröhrchen wie auch zu den Mikrowellenvials der Mikrowellenhydrolyse dar, die das Potential für Miniaturisierungen der Proben hat und sich zudem für große Probendurchsätze eignet. Anstelle der Mikrowellenheizungen wurde ein konventionelles Ölbad als Heizquelle verwendet. Somit ergibt sich eine Verbesserung auch darin, dass diese Methode geringere Geräteinvestitionen erfordert. Wie die Ergebnisse nahelegen, sind sowohl Salzsäure als auch Bromwasserstoffsäure geeignete Reagenzien, die bei der sauren Hydrolyse von Proteinen verwendet werden können. Mit Hilfe antioxidativer Zusätze können Nebenreaktionen erfolgreich vermieden werden. Hier haben sich L-Cystein und Oxalsäure als wirkungsvolle Additive herausgestellt. Wichtige Verbesserungen betreffen vor allem die Zeitersparnis bei der Hydrolyse, die Aufarbeitung der Hydrolysate und die chromatographischen Separationen. Erstere konnte gegenüber der konventionellen Methode von 22 Stunden auf 45 Minuten reduziert werden. Die chromatographische Trennung wurde so weit optimiert, dass die Laufzeit von 64 auf 35 Minuten verkürzt werden konnte. Zudem konnte auf das zeitraubende und kontaminationsanfällige Eindampfen der Hydrolysate verzichtet werden. Während der Aufarbeitung der Hydrolysate konnten die Menge eingesetzter Chemikalien reduziert und damit mögliche Störungen während der optischen Detektion verringert werden. Letztere wurde zum einen durch das Einbeziehen der UV-Absorption bei 260 nm erweitert, sodass eine Alternative zur Absorption bei 215 nm vorliegt, die ebenere Basislinien im Chromatogramm ermöglicht. Zum anderen wurde gezeigt, dass mit Hilfe der Fluoreszenz die Empfindlichkeit der Methode gegenüber der UV-Absorption erheblich verbessert werden kann. Anhand der Probe eines Birkenpollenextraktes ließ sich die Anwendbarkeit der optimierten Methode auf Realproben demonstrieren.
We present special applications of electron backscatter diffraction (EBSD) which aim to overcome some of the limitations of this technique as it is currently applied in the scanning electron microscope. We stress that the raw EBSD signal carries additional information which is useful beyond the conventional orientation determination. The background signal underlying the backscattered Kikuchi diffraction (BKD) patterns reflects the chemical composition and surface topography but also contains channeling-in information which is used for qualitative real-time orientation imaging using various backscattered electron signals. A significantly improved orientation precision can be achieved when dynamically simulated pattern are matched to the experimental BKD patterns. The breaking of Friedel’s rule makes it possible to obtain orientation mappings with respect to the point-group symmetries. Finally, we discuss the
determination of lattice parameters from individual BKD
patterns. Subgrain structure in a single quartz grain. The increased noise level in the left map reflects the lower precision of a standard orientation determination using band detection by the Hough transform. The right map results from the same experimental raw data after orientation refinement using a pattern matching approach. The colors correspond an adapted inverse pole figure color key with a maximum angular deviation of about 2° from the mean orientation.
During the processing of superconducting Nb3Sn wire, several intermediate intermetallic phases including a previously encountered Cu-Nb-Sn phase show up. The yet unknown crystal structure of this phase is now identified by a combination of different experimental techniques and database search to be of the hexagonal NiMg2 type with a proposed composition of about (Nb0.75Cu0.25)Sn2. The structure determination started from an evaluation of the lattice parameters from EBSD Kikuchi patterns from quenched material suggesting hexagonal or orthorhombic symmetry. A database search then led to the hexagonal NiMg2 type structure, the presence of which was confirmed by a Rietveld analysis on the basis of high energy synchrotron X-ray powder diffraction data. Assuming a partial substitution of Nb in orthorhombic NbSn2 by Cu, the change of the valence electron concentration provokes a structural transformation from the CuMg2 type for NbSn2 to the NiMg2 type for (Nb0.75Cu0.25)Sn2. In the previous literature the (Nb0.75Cu0.25)Sn2 phase described here has occasionally been referred to as Nausite.
We report on a study in which plasmid DNA in water was irradiated with 30 keV electrons generated by a scanning electron microscope and passed through a 100 nm thick Si3N4 membrane. The corresponding Monte Carlo simulations suggest that the kinetic energy spectrum of the electrons throughout the water is dominated by low energy electrons (<100 eV). The DNA radiation damage, single-strand breaks (SSB) and double-strand breaks (DSB), was determined by electrophoresis. The median lethal dose of D1/2 = 1.7 ± 0.3 Gy was found to be much smaller compared to partially or fully hydrated DNA irradiated under vacuum conditions. The ratio of DSB to SSB was found to be (1:12) as compared to 1:88) found for hydrated DNA. Our method enables quantitative measurements of radiation damage to biomolecules (DNA, proteins) in solutions under varying conditions (pH, salinity, cosolutes) for an electron energy range which is difficult to probe by standard methods.
An ideal sensor system is a combination of a selective receptor, an effective transducer, and a sensitive detector. To utilize molecularly imprinted polymers (MIPs) as responsive recognition phases in sensors, the employment of fluorescent molecules or nanoparticles (NPs) that show prominent changes in their spectroscopic properties after binding of the target molecule in the MIP’s cavity is particularly attractive. Such fluorescent MIPs (fMIPs) act through target-induced quenching, enhancement, or spectral shifts of the fluorescence. This contribution introduces different strategies of incorporation of fluorescent dyes, probes, and NPs into fMIPs. In addition, various sensing mechanisms are reviewed, and depending on the application of the sensor, the different deployable formats, their advantages, drawbacks, and impact will be presented and discussed.
The investigation of physical properties and chemical composition generates data important for answering art-historical questions. Due to technological developments, technical diagnostics in art and culture are in ever-greater demand in such fields of transdisciplinary research. Based on one example, a collar drawing from the Berlin Diez albums, the present paper aims to give some insights into the multi-instrumental approach in the material analysis of cultural artefacts. The same methods were also applied to other drawings from the Diez Albums.
Semicrystalline polymers have to be described by a three phase model consisting of a mobile amorphous (MAF), a crystalline (CF), and a rigid amorphous fraction (RAF). For nanocomposites based on a semicrystalline polymer the RAF is due to both the crystallites (RAFcrystal) and the filler (RAFfiller). Polymer nanocomposite based on poly(L-lactide) and MgAl layered double hydroxide nanofiller were prepared.
Due to the low crystallization rate of PLA ist crystallization can be suppressed by a high enough cooling rate, and the RAF is due only to the nanofiller. The MAF, CF, and RAF were estimated by Temperature Modulated DSC. For the first time CF, MAF, RAFcrystal, and RAFfiller could be estimated. It was found, that RAFfiller increases linearly with the concentration of the nanofiller for this system. Furthermore, RAFcrystal is only slightly influenced by the presence of the nanofiller.
This work presents an investigation of the accuracy of a radiographic simulation model applied to subsea pipeline inspections. Experimental measurements of a sample in a water tank are used to develop a set of calibrated simulation parameters for the modelling software aRTist. Image quality parameters such as signal-tonoise ratio, contrast and basic spatial resolution are compared with the aim of matching simulated values to experimental results. With this method signal-to-noise ratio was successfully matched while differences were still found in contrast-to-noise ratio comparisons. This means that measurements depending on absolute intensity are not accurate enough, however wall thickness measurements in tangential images, which are not based on absolute intensity, were found to produce similar results in simulated and experimental cases. The differences in contrast and intensity are thought to be due to detector backscatter and additional scatter from out-of-setup objects within the exposure bay, due to a lack of source collimation. These would affect the experimental results but were not included in the simulated setup. This was investigated by including different proportions of peripheral water and other objects in the modelled setup and examining the effect on Image quality parameters. Results show that this additional scatter has a significant impact on the radiograph, particularly on image contrast, and is therefore the likely cause of differences between experimental and simulated images. This implies that it will be very difficult to completely match simulated to experimental results, as including all possible scattering objects in the model would be very complex. An improvement could be made by using real subsea data to estimate this additional scattering, which could then be used to calibrate the model. However there would still be significant uncertainty in the ability of the model to accurately produce realistic intensity and contrast.