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Eingeladener Vortrag
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Fluorescent derivatives of the 129Xe NMR contrast agent cryptophane-A were obtained by functionalization with near infrared fluorescent dyes DY680 and DY682. The resulting conjugates were spectrally characterized, and their interaction with giant and large unilamellar vesicles of varying phospholipid composition was analyzed by fluorescence and NMR spectroscopy. In the latter, a chemical exchange saturation transfer with hyperpolarized 129Xe (Hyper-CEST) was used to obtain sufficient sensitivity. To determine the partitioning coefficients, we developed a method based on fluorescence resonance energy transfer from Nile Red to the membrane-bound conjugates. This indicated that not only the hydrophobicity of the conjugates, but also the phospholipid composition, largely determines the membrane incorporation. Thereby, partitioning into the liquid-crystalline phase of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine was most efficient. Fluorescence depth quenching and flip-flop assays suggest a perpendicular orientation of the conjugates to the membrane surface with negligible transversal diffusion, and that the fluorescent dyes reside in the interfacial area. The results serve as a basis to differentiate biomembranes by analyzing the Hyper-CEST signatures that are related to membrane fluidity, and pave the way for dissecting different contributions to the Hyper-CEST signal.
The fluorine content of polymer particles labelled with 2,2,2-trifluoroethylamine was reliably quantified with overlapping sensitivity ranges by XPS and solid-state NMR. This provides a first step towards reference materials for the metrological traceability of surface group quantifications. The extension of this concept to fluorescence spectroscopy is illustrated.
The surface of poly(methyl methacrylate) particles with different amounts of a grafted layer of poly(acrylic acid) was labeled with varying degrees of an amino derivative of fluorescein isothiocyanate. The resulting fluorescent polymer particles were analyzed by absorption spectroscopy and by steady-state and time-resolved fluorescence spectroscopy including measurements of the fluorescence anisotropy. The combined results indicate that the overall decrease in fluorescence intensity with increasing surface concentrations of the fluorophore can be traced back to the formation of non-fluorescent aggregates. A mechanism is proposed, in which the excitation energy migrates between identical fluorophores until it is transferred to non-fluorescent aggregates acting as an energy trap. Increases in the surface fluorophore concentration increase both the probability for energy transfer between identical fluorophores and the probability for energy transfer to non-fluorescent aggregates. Furthermore, we suggest that this mechanism also applies to fluorescent protein conjugates and rationalizes the nonlinear dependence of the fluorescence emission on the labeling density.
Unter dem Motto „Innovation und Qualitätssicherung in der (Bio)Analytik“ werden in der Arbeitsgruppe Fluoreszenzspektroskopie der BAM, Bundesanstalt für Materialforschung und -prüfung, funktionelle Chromophor-Systeme, einfache Signalverstärkungs- und Multiplexingstrategien sowie innovative Validierungs- und rückführbare Standardisierungskonzepte für verschiedene fluorometrische Messgrößen und Methoden entwickelt. Im Mittelpunkt stehen dabei molekulare Fluorophore, Nanokristalle mit größenabhängigen optischen Eigenschaften (sogenannte Quantenpunkte, QDs) und fluoreszierende Partikel variabler Größe sowie Sonden und Sensormoleküle für neutrale und ionische Analyte und für die Charakterisierung von funktionellen Gruppen. Dabei erfolgen auch methodische Entwicklungen für die Fluoreszenzspektroskopie, die Fluoreszenzmikroskopie, die Milcrofluorometrie, die Sensorik und die Mikroarraytechnologie. Ziele sind u. a. das Design und die Untersuchung von multiplexfähigen selektiven und sensitiven Sonden für die Biomarkeranalytik, die Entwicklung von Methoden zur Charakterisierung der signalrelevanten Eigenschaften dieser Chromophor-Systeme und zur Charakterisierung von funktionellen Gruppen an Oberflächen und ihre Validierung sowie die Entwicklung und Bereitstellung von formatadaptierbaren, flexibel ersetzbaren
Standards für die fluoreszenzbasierte Multiparameteranalytik.
The objective of this study was to introduce differential sensing techniques to synthetic systems that act, like olfactory receptors, as transporters in lipid bilayer membranes. Routine with most alternative chemosensing ensembles, pattern generation has, quite ironically, remained inaccessible in lipid bilayers because the number of available crossresponsive sensor components has been insufficient. To address this challenge, we here report on the use of cationic hydrazides that can react in situ with hydrophobic analytes to produce cationic amphiphiles which in turn can act as countercation activators for polyanionic transporters in fluorogenic vesicles. To expand the dimension of signals generated by this system, a small collection of small peptides containing a positive charge (guanidinium, ammonium) and one to three reactive hydrazides are prepared. Odorants are used as examples for hydrophobic analytes, perfumes to probe compatibility with complex matrices, and counterion-activated calf-thymus DNA as representative polyion–counterion transport system. Principal component and hierarchical cluster analysis of the obtained multidimensional patterns are shown to differentiate at least 21 analytes in a single score plot, discriminating also closely related structures such as enantiomers, cis–trans isomers, single-atom homologs, as well as all tested perfumes. Inverse detection provides access to analytes as small as acetone. The general nature of the introduced methodology promises to find diverse applications in current topics in biomembrane research.
A series of activatable optical probes for the model enzyme penicillin G amidase based on intramolecular formation of non-fluorescent H-dimer between two identical dyes were synthesized. The probes are based on a self-immolative linker, which allows positioning both dyes in close spatial proximity to ensure efficient quenching of probes with absorption and fluorescence emission in the near-infrared (NIR) range. A detailed photophysical investigation of the novel optical probes led to a revision of a previously anticipated quenching mechanism and revealed their potential for optimizing the performance of activatable probes based on H-dimer formation. A kinetic analysis indicated that the fluorescence progress curves can be used to qualitatively extract enzyme kinetic parameters.