We implemented an assay combining fluorescence and bright‐field microscopy to measure ligand binding to dopamine D3 receptors in live mammalian cells. For fluorescence intensity quantification from microscopy images, we developed a machine learning‐based user‐friendly software membrane tools. For the experiments, a novel fluorescent ligand NAPS‐Cy3B was synthesized. The subnanomolar affinity of NAPS‐Cy3B makes it a suitable ligand for the characterization of D3 receptors and its ligands in live HEK293 cells.
Carboxylate sensing solid-contact ion-selective electrodes (ISEs) were created to provide a proof-of-concept ISE development process covering all aspects from in silico ionophore design to functional sensor characterization. The biscarbazolylurea moiety was used to synthesize methylene-bridged macrocycles of different ring size aiming to fine tune selectivity towards different carboxylates. Cyclization was achieved with two separate strategies, using either amide synthesis to access up to –[CH2]10– macrocycles or acyl halides to access up to –[CH2]14– macrocycles. Seventy-five receptor–anion complexes were modelled and studied with COSMO-RS, in addition to all free host molecules. In order to predict initial selectivity towards carboxylates, 1H NMR relative titrations were used to quantify binding in DMSO-d 6/H2O solvent systems of two proportions – 99.5%:0.5% m/m and 90.0%:10.0% m/m, suggesting initial selectivity towards acetate. Three ionophores were selected for successful sensor prototype development and characterization. The constructed ion-selective electrodes showed higher selectivity towards benzoate than acetate, i.e., the selectivity patterns of the final sensors deviated from that predicted by the classic titration experiments. While the binding constants obtained by NMR titration in DMSO-d 6/H2O solvent systems provided important guidance for sensor development, the results obtained in this work emphasize the importance of evaluating the binding behavior of receptors in real sensor membranes.
Solid-contact ion-selective electrodes with carbazole-derived ionophores were prepared. They were characterized as acetate sensors, but can be used to determine a number of carboxylates. The potentiometric response characteristics (slope, detection limit, selectivity, and pH sensitivity) of sensors prepared with different membrane compositions (ionophore, ionophore concentration, anion exchanger concentration, and plasticizer) were evaluated. The results show that for the macrocyclic ionophores, a larger cavity provided better selectivity. The sensors exhibited modest selectivity for acetate but good selectivity for benzoate. The carbazole-derived ionophores effectively decreased the interference from lipophilic anions, such as bromide, nitrate, iodide, and thiocyanate. The selectivity, detection limit, and linear range were improved by choosing a suitable plasticizer and by reducing the ionophore and anion exchanger concentrations. The influence of the electrode body’s material upon the composition of the plasticized poly(vinyl chloride) membrane, and thus also upon the sensor characteristics, was also studied. The choice of materials for the electrode body significantly affected the characteristics of the sensors.
Monoaminoacridines (1-, 2-, 3-, 4-, and 9-aminoacridine) were studied for suitability as matrices in the negative ion mode matrix-assisted laser desorption/ionization mass spectrometry (MALDI(−)-MS) analysis of various samples. This is the first study to examine 1-, 2-, and 4-aminoacridine as potential matrix material candidates for MALDI(−)-MS. In addition, spectral (UV−Vis absorption and fluorescence), proton transfer-related (basicity and autoprotolysis), and crystallization properties of these compounds were characterized experimentally and/or computationally. For testing the capabilities of these aminoacridines as matrix materials, four samples related to cultural heritage materialsstearic acid, colophony resin, dyer's madder dye, and a resinous case-study sample from a shipwreckwere analyzed with MALDI(−)-MS. A novel algorithm (implemented as an executable Python script) for MS data analysis was developed to compare the five matrix materials and to help mass spectrometrists rapidly identify peaks originating from the sample and matrix material. It was determined that all five of the studied aminoacridines can successfully be used as matrix materials in MALDI(−)-MS analysis. As an interesting finding, in several cases, the best mass spectra were obtained by using a relatively small amount of matrix material mixed with an excess amount of sample. 3-and 4-aminoacridine outperformed the other aminoacridines in the ease of obtaining acceptable spectra, average number of ions identified in the mass spectra, and low dependence of the sample-to-matrix mass ratio on experimental results.
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