Analytische Chemie
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A novel synthetic strategy toward highly fluorinated BODIPY dyes with exceptional photostabilities relying on sustainable gold catalysis has been developed. A key to the tailored pyrrole precursors is the gold catalysis performed in ionic liquids as the reaction medium, allowing a facile recycling of the catalysts. The dyes prepared are well-matching with the spectral windows of popular rhodamine dyes and possess high brightness while showing a distinctly higher photostability than the rhodamines especially in aprotic solvents.
Immunoassays are in the majority of formats heterogeneous assays with one of the commodities – antibody or antigen – immobilized on a solid substrate or sometimes to nanoparticles or beads. All these formats require a washing step in order to separate bound from non-bound species before generating the measurement signal.
Measuring fluorescence polarization is a method to distinguish between a fluorescent macromolecule and a low-molecular weight fluorophore. After irradiation of the sample with polarized light, a macromolecule like e.g. an antibody, will emit still highly polarized light (high polarization, resp. small difference in degree of polarization to the background). In a fluorescence polarization immunoassay, the fluorescence of an analyte surrogate (a fluorescence “tracer”) is followed. Depolarization is high and so the measured polarization is low. When antibody is added, the tracer is bound, depolarization decreases and the in polarization is high. Increasing amounts of analyte more and more impede the tracer being bound by the tracer and thus the decreases, the typical sigmoidal relationship is obtained (Fig. 1). We dispose of 5 polarization platforms reading in plate mode, strip mode and cuvette mode. One is able to register polarization changes with time and so allows for kinetic measurements another one is hand-held. We have been comparing FPIA formats before [1-4] and now a comprehensive view on sensitivities, the importance of fast vs. slow binding kinetics as well as the choice of format can be presented.
Dieses Buch ist definitiv eine Empfehlung an alle, die mit RFA arbeiten müssen. Außerdem bietet es nützliche und praktische Informationen für Forscher, die an einem bestimmten Thema arbeiten, das im Antragsteil erwähnt wird. Dies wird ihnen während ihrer Forschung helfen, Proben vorzubereiten sowie Fehler in der RFA-Analyse zu erkennen und somit zu vermeiden.
Die Stärken des Buches liegen im verständlichen Text und darin, dass alle notwendigen Schritte der Analysenverfahren Punkt für Punkt aufgeführt sind. Obwohl die Autoren eine unglaubliche Arbeit geleistet haben, um den Leser auf den neuesten Stand zu bringen, sind einige Teile ziemlich kurz gehalten. Die Auflistung von weiterführender Literatur, einschließlich wichtiger Normen und Gesetze, verringert dieses Manko etwas und verweist die interessierten Leser auf ergänzende Quellen.
Chlorination of pool water and wastewater, in food and pharmaceutical production, as well as in pesticide and paper manufacturing is a routinely used technique. However, the amount of chlorine in water must be strictly adjusted, to ensure enough concentration to kill pathogenic bacteria and viruses, while preventing too high concentrations inducing negative effects on human health. As an indicator, a molecular fluorescent probe based on a BODIPY structure was designed. This indicator exhibits a sensitive and selective fluorescence response upon increasing concentrations of hypochlorite in aqueous solvent mixtures. Real-time analyses became possible after the integration of this fluorescent indicator into newly designed 2D & 3D microfluidic chips incorporating a passive sinusoidal mixer and a micro-hydrocyclone, respectively. A comparison of the two microfluidic systems, including their ability to prevent accumulation or circulation of microbubbles, has shown excellent fluidic behaviour for the micro-hydrocyclone device. This system was distinctly more robust against gas bubbles, showed a higher signal gain and allowed to halve the limit of detection to 0.02 mg L–1. The use of the 3D system to quantify the chlorine content of pool water samples for sensitive and quantitative chlorine monitoring has been demonstrated.
Luminescence techniques are amongst the most commonly used analytical methods in life and material sciences due to their sensitivity and their nondestructive and multiparametric character. Photoluminescence signals are, however, affected by wavelength, polarization and time dependent instrument specific effects, and provide only relative intensities. This hampers the comparability of fluorescence measure-ments and calls for simple tools for instrument characterization and the quantification of measured fluorescence intensities. Well characterized fluorescence standards for instrument calibration and performance validation (IPV) can be used as references for fluorescence signals. Of special importance is the correct determination of photoluminescence quantum yields (QF) (number of emitted per absorbed photons) that provides a direct comparison of the fluorescence efficiency of emitters. Such well characterized standards have been successfully developed by BAM for the relative determination of f values of transparent solutions of molecular and nanoscale emitters in the wavelength range from 350 and 1100 nm and will be soon certified. These standards can also be used to evaluate integrating sphere setups, which are increasingly being used for absolute measurements of QF values.
Gasoline adulteration is a frequent problem world-wide, because of the chance of quick, maximized profits. However, addition of cheaper ethanol or hydrocarbons like kerosene does not only result in economic damage but also poses problems for vehicles and the environment. To enable law enforcement forces, customers or enterprises to uncover such a fraudulent activity directly upon suspicion and without the need to organize for sampling and laboratory analysis, we developed a simple strip-based chemical test. Key to the favorable performance was the dedicated materials tailoring, which led to test strips that consisted of a cellulose support coated with silica, passivated with hexamethyldisilazane and functionalized covalently with a molecular probe. The probe fluoresces brightly across a broad solvent polarity range, enabling reliable quantitative measurements and data analysis with a conventional smartphone. The assays showed high reproducibility and accuracy, allowing not only for the detection of gasoline adulteration but also for the on-site monitoring of the quality of commercial E10 gasoline.
In der Masterarbeit wurde ein Screening gegen den CCMV mit einer linearen Peptidbibliothek entwickelt, wobei zwei lineare Binder identifiziert wurden. Das resynthetisierte Peptid wurde auf seine Bindungseigenschaften mittels ELISA und MST untersucht. Aufgrund eines Aminosäurefehlers in der selbst durchgeführten Resynthese des Peptids sind die Ergebnisse nicht vollständig übertragbar. Um eine umsetzbare Cyclusgröße für die Peptidbibliothek zu finden, wurden Ringgrößen mit sechs bis acht Aminosäuren untersucht. Da keiner der gewählten Ringgrößen bevorzugt war, wurde die Ringgröße aus acht Aminosäuren für die Peptidbibliothek gewählt. Der Ringschluss wurde durch die Bildung einer Disulfidbrücke erreicht. Dadurch war die Alkylierung der vorhandenen Thiolgruppe in den Abbruchsequenzen notwendig.
Neben den etablierten Alkylierungsreagenzien Iodessigsäure, Iodacetamid und Acrylamid wurden zwei Epoxide mituntersucht. Hierbei konnte nur bei Acrylamid und Propylenoxid eine vollständige Alkylierung beobachtet werden. Eine synthetische Peptidbibliothek aus zehn Aminosäuren pro Kopplungsschritt und einer Peptidlänge von acht Aminosäuren wurde erfolgreich nach der Split-and-Mix-Synthese hergestellt. Neben den kanonischen Aminosäuren wurde die synthetische Aminosäure 3-(3-Pyridyl)-alanin in die Peptidbibliothek mit eingebaut. Peptidsequenzen aus der cyclische Peptidbibliothek konnte mittels MALDI-TOF-MS identifiziert werden. Es konnte außerdem gezeigt werden, dass synthetische Aminosäuren mit proteinogenen Aminosäuren erfolgreich übersetzt werden. Anschließend wurde das entwickelte Screening auf eine cyclische Peptidbibliothek übertragen. Dabei konnte kein Binder identifiziert werden, da zu viele Nebenreaktionen auftraten. Ein alternativer Ringschluss über die Seitenketten von Lysin und Cystein wurden mit ortho-Phthaldialdehyd und 2,4,6-Trichloro-1,3,5-triazin (Cyanurchlorid) untersucht. Beide Bedingungen wiesen keinen erfolgreichen Ringschluss auf. Obwohl der Ringschluss über die Seitenketten von Lysin und Cystein nicht erfolgreich war, sollte ein Austausch von Cystein angestrebt werden, so dass kein freies Cystein in den Abbruchsequenzen vorhanden ist und die Alkylierung überflüssig wäre. Der Ringschluss durch Verwendung anderer Seitenketten bietet einen vielseitigen Ansatz.
Contamination of natural bodies of water or soil with oils and lubricants (or generally, hydrocarbon derivatives such as petrol, fuels, and others) is a commonly found phenomenon around the world due to the extensive production, transfer, and use of fossil fuels. In this work, we develop a simple system for the on-field detection of total petroleum hydrocarbons (TPHs) in water and soil. The test is based on the measurement of the fluorescence signal emitted by the molecular rotor 2-[ethyl[4-[2-(4-nitrophenyl)ethenyl]phenyl]amino]ethanol (4-DNS-OH). This dye is embedded in a hydrophobic polymeric matrix (polyvinylidene fluoride), avoiding interactions with water and providing a robust support for use in a test strip fashion. Together with the strips, an embedded optical system was designed for fluorescence signal read-out, featuring a Bluetooth low-energy connection to a commercial tablet device for data processing and analysis. This system works for the detection and quantification of TPHs in water and soil through a simple extraction protocol using a cycloalkane solvent with a limit of detection of 6 ppm. Assays in surface and sea waters were conclusive, proving the feasibility of the method for in-the-field operation.
In vitro cultured 3D models of CRC have been demonstrated to hold considerable worth in drug discovery, drug resistance analysis, and in studying cell-cell and cell-matrix interactions that occur in the tumor microenvironment. The 3D models resemble the in vivo physiological microenvironment by replicating the cell type composition and tissue architecture. Molecularly imprinted polymers (MIPs) have been investigated for use instead of antibodies against small nonimmunogenic structures, such as sialic acid (SA). Glyco-conjugates including SA are present on all cells, and often deregulated on cancer cells. Here, we present a novel approach for targeting and detecting colorectal cancer cells (CRC) by using in vitro cultured HT29 3D spheroids co-cultured in vitro with either fluorescent MIPs targeting SA, SA-MIPs, or the two lectins targeting SA, MAL I, and SNA. Both formaldehyde-fixed and viable HT29 3D spheroids with or without SA-MIPs were imaged in 3D by confocal microscopy. The results revealed a preserved cell morphology and viability of the HT29 3D spheroids co-cultured in vitro with SA-MIPs. However, the lectins MAL I and SNA targeting the alpha-2,3 or alpha-2,6 SA glycosidic linkages, respectively, affected the cell viability when co-cultured with the viable HT29 3D spheroids, and no living cells could be detected. Here, we have shown that the SA-MIPs could be used as a safe and low-cost diagnostic tool for targeting and detecting cancer cells in a physiologically relevant 3D cancer model in vitro.
Rapid tests and assays to be used outside of a laboratory for non-trained personal and also at a point of need are becoming increasingly important in areas such as health, food, security, or the environment. Specially on that regard, paper-based sensors are emerging as a new class of devices because they fulfil the requisites of the "World Health Organization" to be ASSURED: affordable, sensitive, specific, user-friendly, rapid and robust, equipment free and deliverable to end-users. The physical, chemical and mechanical properties of cellulose or glass fiber paper in combination with the facility of preparation are making these materials of great interest while looking for cost-efficient and green alternatives for device production technologies. To improve the sensitivity of these systems, a particularly promising approach is the employment of gated indicator delivery systems using preorganized nanoscopic solid structures incorporated on paper strips to produce an exponential amplification of the detectable signal. Having in mind these concepts, several examples of (bio)gated materials incorporated into sensing membranes will be presented for the detection of small organic molecules, having fluorescence or electrochemiluminescence signal as output signal. Compared with fluorescence, it has been demonstrated that the non-optical excitation has significantly reduced the background signal, and with the help of a portable potentiostat in combination with a home-made 3D-printed case fitted onto a smartphone, the sensitivity of the sensing system has been improved tremendously, from the lower ppb range (fluorescence) to the lower ppt range. With this study, the applicability of ECL detection on paper strips in combination with gated indicator-releasing materials has been demonstrated for the first time, presenting a novel synergistic match. Considering the modularity of the system developed, the platform technology potential is obvious, promising expansion of the general concept to many other analytes, applications and scenarios.