Monitoring the Collapse of pH-Sensitive Liposomal Nanocarriers and Environmental pH Simultaneously: A Fluorescence-Based Approach

Nowadays, the encapsulation of therapeutic compounds in so-called carrier systems is a very smart method to achieve protection as well as an improvement of their temporal and spatial distribution. After the successful transport to the point of care, the delivery has to be released under controlled conditions. To monitor the triggered release from the carrier, we investigated different fluorescent probes regarding their response to the pH-induced collapse of pH-sensitive liposomes (pHSLip), which occurs when the environmental pH falls below a critical value. Depending on the probe, the fluorescence decay time as well as fluorescence anisotropy can be used equally as key parameters for monitoring the collapse. Especially the application of a fluorescein labeled fatty acid (fPA) enabled the monitoring of the pHSLips collapse and the pH of its microenvironment simultaneously without interference. Varying the pH in the range of 3 < pH < 9, anisotropy data revealed the critical pH value at which the collapse of the pHSLips occurs. Complementary methods, e.g., fluorescence correlation spectroscopy and dynamic light scattering, supported the analysis based on the decay time and anisotropy. Additional experiments with varying incubation times yielded information on the kinetics of the liposomal collapse

Flash Photolysis Study of Complexes between Salicylic Acid and Lanthanide Ions in Water

In the natural environment humic substances (HS) represent a major factor determining the speciation of metal ions, e.g., in the context of radionuclide migration. Here, due to their intrinsic sensitivity and selectivity, spectroscopic methods are often applied, requiring a fundamental understanding of the photophysical processes present in such HS–metal complexes. Complexes with different metal ions were studied using 2-hydroxybenzoic acid (2HB) as a model compound representing an important part of the chelating substructures in HS. In flash photolysis experiments under direct excitation of 2HB in the absence and the presence of different lanthanide ions, the generation and the decay of the 2HB triplet state, of the phenoxy radical, and of the solvated electron were monitored. Depending on the lanthanide ion different intracomplex processes were observed for these transient species including energy migration to and photoreduction of the lanthanide ion. The complexity of the intracomplex photophysical processes even for small molecules such as 2HB underlines the necessity to step-by-step approach the photochemical reactivity of HS by using suitable model compounds

Upconversion Luminescence Properties of NaYF₄:Yb:Er Nanoparticles Codoped with Gd³⁺

The temperature-dependent upconversion luminescence of NaYF₄:Yb:Er nanoparticles (UCNP) containing different contents of Gd³⁺ as additional dopant was characterized. The UCNP were synthesized in a hydrothermal synthesis and stabilized with citrate in order to transfer them to the water phase. Basic characterization was carried out using TEM and DLS to determine the average size of the UCNP. The XRD technique was used to investigate the crystal lattice of the UCNP. It was found that due to the presence of Gd³⁺, an alteration of the lattice phase from α to β was induced which was also reflected in the observed upconversion luminescence properties of the UCNP. A detailed analysis of the upconversion luminescence spectraespecially at ultralow temperaturesrevealed the different effects of phonon coupling between the host lattice and the sensitizer (Yb³⁺) as well as the activator (Er³⁺). Furthermore, the upconversion luminescence intensity reached a maximum between 15 and 250 K depending on Gd³⁺ content. In comparison to the very complex temperature behavior of the upconversion luminescence in the temperature range <273 K, the luminescence intensity ratio of ²H_11/2→⁴I_15/2 to ⁴S_3/2→⁴I_15/2 (<i>R</i> = G1/G2) in a higher temperature range can be described by an Arrhenius-type equation

Oxazine Dye-Conjugated DNA Oligonucleotides: Förster Resonance Energy Transfer in View of Molecular Dye–DNA Interactions

In this work, the photophysical properties of two oxazine dyes (ATTO 610 and ATTO 680) covalently attached via a C6-amino linker to the 5′-end of short single-stranded as well as double-stranded DNA (ssDNA and dsDNA, respectively) of different lengths were investigated. The two oxazine dyes were chosen because of the excellent spectral overlap, the high extinction coefficients, and the high fluorescence quantum yield of ATTO 610, making them an attractive Förster resonance energy transfer (FRET) pair for bioanalytical applications in the far-red spectral range. To identify possible molecular dye–DNA interactions that cause photophysical alterations, we performed a detailed spectroscopic study, including time-resolved fluorescence anisotropy and fluorescence correlation spectroscopy measurements. As an effect of the DNA conjugation, the absorption and fluorescence maxima of both dyes were bathochromically shifted and the fluorescence decay times were increased. Moreover, the absorption of conjugated ATTO 610 was spectrally broadened, and a dual fluorescence emission was observed. Steric interactions with ssDNA as well as dsDNA were found for both dyes. The dye–DNA interactions were strengthened from ssDNA to dsDNA conjugates, pointing toward interactions with specific dsDNA domains (such as the top of the double helix). Although these interactions partially blocked the dye-linker rotation, a free (unhindered) rotational mobility of at least one dye facilitated the appropriate alignment of the transition dipole moments in doubly labeled ATTO 610/ATTO 680–dsDNA conjugates for the performance of successful FRET. Considering the high linker flexibility for the determination of the donor–acceptor distances, good accordance between theoretical and experimental FRET parameters was obtained. The considerably large Förster distance of ∼7 nm recommends the application of this FRET pair not only for the detection of binding reactions between nucleic acids in living cells but also for monitoring interactions of larger biomolecules such as proteins

Fluorescence Line-Narrowing Spectroscopy as a Tool to Monitor Phase Transitions and Phase Separation in Efficient Nanocrystalline Ce_<i>x</i>Zr_1–<i>x</i>O₂:Eu³⁺ Catalyst Materials

Despite the wide range of industrial applications for ceria-zirconia mixed oxides (Ce_<i>x</i>Zr_1–<i>x</i>O₂), the complex correlation between their atomic structure and catalytic performance is still under debate. Catalytically interesting Ce_<i>x</i>Zr_1–<i>x</i>O₂ nanomaterials can form homogeneous solid solutions and, depending on the composition, show phase separation under the formation of small domains. The characterization of homogeneity and atomic structure of these materials remains a major challenge. High-resolution emission spectroscopy recorded under cryogenic conditions using Eu³⁺ as a structural probe in doped CeZrO₂ nanoparticles offers an effective way to identify the different atomic environments of the Eu³⁺ dopants and, subsequently, to monitor structural parameters of the ceria-zirconia mixed oxides. It is found that, in stoichiometric CeZrO₂:Eu³⁺, phase separation occurs at elevated temperatures beginning with the gradual formation of (pseudo)­cubic crystallites in the amorphous materials at 500 °C and a sudden phase separation into tetragonal, zirconia-rich and cubic, ceria-rich domains over 900 °C. The presented technique allows us to easily monitor subtle changes even in amorphous, high surface area samples, yielding structural information not accessible by conventional techniques such as X-ray diffraction (XRD) and Raman. Moreover, in reference experiments investigating the reducibility of largely unordered Ce_0.2Zr_0.8O₂:Eu³⁺, the main reduction peak in temperature-programmed reduction measurements appeared at exceptionally low temperatures below 200 °C, thus suggesting the outstanding potential of this oxide to activate catalytic oxidation reactions. This effect was found to be dependent on the amount of Eu³⁺ dopant introduced into the CeZrO₂ matrix as well as to be connected to the atomic structure of the catalyst material

Dye Dynamics in Three-Color FRET Samples

Time-resolved emission data (fluorescence decay and fluorescence depolarization) of two three-color Förster resonance energy transfer (tc-FRET) systems consisting of a carbostyril donor (D), a ruthenium complex (Ru) as relay dye, and a Cy5 derivative (Cy) or, optionally, an anthraquinone quencher (Q) were carefully analyzed using advanced distribution analysis models. Thereby, it is possible to get information on the flexibility and mobility of the chromophores which are bound to double stranded (ds) DNA. Especially the distance distribution based on the analysis of the fluorescence depolarization is an attractive approach to complement data of fluorescence decay time analysis. The distance distributions extracted from the experimental data were in excellent agreement with those determined from accessible volume (AV) simulations. Moreover, the study showed that for tc-FRET systems the combination of dyes emitting on different time scales (e.g., nanoseconds vs microseconds) is highly beneficial in the distribution analysis of time-resolved luminescence data in cases where macromolecules such as DNA are involved. Here, the short lifetimes can yield information on the rotation of the dye molecule itself and the long lifetime can give insight in the overall dynamics of the macromolecule

Ultrasonic Approach for Formation of Erbium Oxide Nanoparticles with Variable Geometries

Ultrasound (20 kHz, 29 W·cm^–2) is employed to form three types of erbium oxide nanoparticles in the presence of multiwalled carbon nanotubes as a template material in water. The nanoparticles are (i) erbium carboxioxide nanoparticles deposited on the external walls of multiwalled carbon nanotubes and Er₂O₃ in the bulk with (ii) hexagonal and (iii) spherical geometries. Each type of ultrasonically formed nanoparticle reveals Er³⁺ photoluminescence from crystal lattice. The main advantage of the erbium carboxioxide nanoparticles on the carbon nanotubes is the electromagnetic emission in the visible region, which is new and not examined up to the present date. On the other hand, the photoluminescence of hexagonal erbium oxide nanoparticles is long-lived (μs) and enables the higher energy transition (⁴S_3/2–⁴I_15/2), which is not observed for spherical nanoparticles. Our work is unique because it combines for the first time spectroscopy of Er³⁺ electronic transitions in the host crystal lattices of nanoparticles with the geometry established by ultrasound in aqueous solution of carbon nanotubes employed as a template material. The work can be of great interest for “green” chemistry synthesis of photoluminescent nanoparticles in water

Dye Tool Box for a Fluorescence Enhancement Immunoassay

Immunochemical analytical methods are very successful in clinical diagnostics and are nowadays also emerging in the control of food as well as monitoring of environmental issues. Among the different immunoassays, luminescence based formats are characterized by their outstanding sensitivity making this format especially attractive for future applications. The need for multiparameter detection capabilities calls for a tool box of dye labels in order to transduce the biochemical reaction into an optically detectable signal. Here, in a multiparameter approach each analyte may be detected by a different dye with a unique emission color (covering the blue to red spectral range) or a unique luminescence decay kinetics. In the case of a competitive immunoassay format for each of the different dye labels an individual antibody would be needed. In the present paper a slightly modified approach is presented using a 7-aminocoumarin unit as the basic antigen against which highly specific antibodies were generated. Leaving the epitope region in the dyes unchanged but introducing a side group in positon 3 of the coumarin system allowed us to tune the optical properties of the coumarin dyes without the necessity of new antibody generation. Upon modification of the parent coumarin unit the full spectral range from blue to deep red was accessed. In the manuscript the photophysical characterization of the coumarin derivatives and their corresponding immunocomplexes with two highly specific antibodies is presented. The coumarin dyes and their immunocomplexes were characterized by steady-state and time-resolved absorption as well as emission spectroscopy. Moreover, fluorescence depolarization measurements were carried out to complement the data stressing the different binding modes of the two antibodies. The binding modes were evaluated using the photophysics of 7-aminocoumarins and how it was affected in the respective immunocomplexes, namely, the formation of the intramolecular charge transfer (ICT) as well as the twisted intramolecular charge transfer (TICT). In contrast to other antibody–dye pairs reported a distinct fluorescence enhancement upon formation of the antibody–dye complex up to a factor of 50 was found. Because of the easy emission color tuning by tailoring the coumarin substitution for the antigen binding in nonrelevant position 3 of the parent molecule, a dye tool box is on hand which can be used in the construction of competitive multiparameter fluorescence enhancement immunoassays (FenIA)