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Eingeladener Vortrag
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The unique optical properties, such as size-tunable absorption and emission, caused semiconductor nanocrystals to attract a great deal of interest for recent technological developments. For the evaluation of semiconductor nanocrystals as new materials for various applications like optoelectronic devices, knowledge of the structure–property relationships is indispensable, but still presents a challenge. Here, we address these challenges for thioglycolic acid-capped CdTe nanocrystals with a focus on the quantification of thiol ligands, identification of the ligand shell structure and their influence on the optical properties of these nanocrystals. We present the use of a simple analytical technique, the Ellman's test, and ICP-OES analysis for the study of the surface chemistry of these nanomaterials. Together with theoretical calculations, the results of these studies show the strong influence of the amount of Cd–thiolates present in the ligand shell on the concentration-dependent emission properties, thereby providing the basis for a better understanding of the chemical nature of the NC–ligand interface. In this context, the present work contributes to the establishment of a clearer picture and better control of the surface chemistry, which will provide the basis for the design of highly emitting nanocrystals and the prediction of their applicability.
The rational design of bright optical probes and dye–biomolecule conjugates in the NIR-region requires fluorescent labels that retain their high fluorescence quantum yields when bound to a recognition unit or upon interaction with a target. Because hydrophilicity-controlled dye aggregation in conjunction with homo-FRET presents one of the major fluorescence deactivation pathways in dye–protein conjugates, fluorescent labels are required that enable higher labeling degrees with minimum dye aggregation. Aiming at a better understanding of the factors governing dye–dye interactions, we systematically studied the signal-relevant spectroscopic properties, hydrophilicity, and aggregation behavior of the novel xS-IDCC series of symmetric pentamethines equipped with two, four, and six sulfonic acid groups and selected conjugates of these dyes with IgG and the antibody cetuximab (ctx) directed against the cancer-related epidermal growth factor (EGF) receptor in comparison to the gold standard Cy5.5. With 6S-IDCC, which displays a molar absorption coefficient of 190 000 M1 cm1 and a fluorescence quantum yield (Φf) of 0.18 in aqueous media like PBS and nearly no aggregation, we could identify a fluorophore with a similarly good performance as Cy5.5. Bioconjugation of 6S-IDCC and Cy5.5 yielded highly emissive targeted probes with comparable Φf values of 0.29 for a dye-to-protein (D/P) ratio <1 and a reduced number of protein-bound dye aggregates in the case of 6S-IDCC. Binding studies of the ctx conjugates of both dyes performed by fluorescence microscopy and FACS revealed that the binding strength between the targeted probes and the EGF receptor at the cell membrane is independent of D/P ratio. These results underline the importance of an application-specific tuning of dye hydrophilicity for the design of bright fluorescent reporters and efficient optical probes. Moreover, we could demonstrate the potential of fluorescence spectroscopy to predict the size of fluorescence signals resulting for other fluorescence techniques such as FACS.
The increase in information content from bioassays and bioimaging requires robust and efficient strategies for the detection of multiple analytes or targets in a single measurement, thereby addressing current health and security concerns. For fluorescence techniques, an attractive alternative to commonly performed spectral or color multiplexing presents lifetime multiplexing and the discrimination between different fluorophores based on their fluorescence decay kinetics. This strategy relies on fluorescent labels with sufficiently different lifetimes that are excitable at the same wavelength and detectable within the same spectral window. Here, we report on lifetime multiplexing and discrimination with a set of nanometer-sized particles loaded with near-infrared emissive organic fluorophores chosen to display very similar absorption and emission spectra, yet different fluorescence decay kinetics in suspension. Furthermore, as a first proof-of-concept, we describe bioimaging studies with 3T3 fibroblasts and J774 macrophages, incubated with mixtures of these reporters employing fluorescence lifetime imaging microscopy. These proof-of-concept measurements underline the potential of fluorescent nanoparticle reporters in fluorescence lifetime multiplexing, barcoding, and imaging for cellular studies, cell-based assays, and molecular imaging.
Water-dispersible and (bio)functionalizable nanoclays have a considerable potential as inexpensive carriers for organic molecules like drugs and fluorophores. Aiming at simple design strategies for red-emissive optical probes for the life sciences from commercial precursors with minimum synthetic effort, we systematically studied the dye loading behavior and stability of differently functionalized laponites. Here, we present a comprehensive study of the absorption and emission properties of the red emissive hydrophobic and neutral dye Nile Red, a well-known polarity probe, which is almost insoluble and nonemissive in water. Adsorption of this probe onto disk-shaped nanoclays was studied in aqueous dispersion as function of dye concentration, in the absence and presence of the cationic surfactant cetyltrimethylammonium bromide (CTAB) assisting dye loading, and as a function of pH. This laponite loading strategy yields strongly fluorescent nanoclay suspensions with a fluorescence quantum yield of 0.34 at low dye loading concentration. The dye concentration-, CTAB-, and pH-dependent absorption, fluorescence emission, and fluorescence excitation spectra of the Nile-Red6#8211;nanoclay suspensions suggest the formation of several Nile Red species including emissive Nile Red monomers facing a polar environment, nonemissive H-type dimers, and protonated Nile Red molecules that are also nonfluorescent. Formation of all nonemissive Nile Red species could be suppressed by modification of the laponite with CTAB. This underlines the great potential of properly modified and functionalized laponite nanodisks as platform for optical probes with drug delivery capacities, for example, for tumor and therapy imaging. Moreover, comparison of the Nile Red dimer absorption spectra with absorption spectra of previously studied Nile Red aggregates in dendrimer systems and micelles and other literature systems reveals a considerable dependence of the dimer absorption band on microenvironment polarity which has not yet been reported so far for H-type dye aggregates.
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.
Carbamazepine (CBZ) is one of the most frequently detected pharmaceuticals in water samples. For the determination of this anthropogenic marker, various immunoassay formats were tested and evaluated in order to identify the most suitable one. For these direct competitive assays, the analyte was labelled with the enzyme horseradish peroxidase (HRP) or alkaline phosphatase (AP), and seven substrates with specific detection properties were used. The quality criteria for the standard curves were fulfilled by all HRP assays and the chemiluminescence AP format. Furthermore, intra- and inter-plate coefficients of variation as a measure of the achievable precision were determined for the samples. The application of the AP assays to surface water was unfeasible due to CBZ concentrations below the quantifiable concentration range. Surface as well as waste water samples could be analyzed with the HRP assays. Here, the HRP assay employing the chromogenic substrate 3,3',5,5'-tetramethylbenzidine yielded the best results.
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.