Abstract P-39: Fluorescent Silver Nanoclusters with Immunoglobulins and Albumins

Background: Multiplex biomedical assays including molecular genetic tests and immunoanalysis require multiple fluorophores with a wide excitation range and different emission spectra. In comparison with organic fluorophores and quantum dots, the metal nanoclusters (NC) consisting of a few to hundred atoms have the following advantages: small size, large Stokes shift, prolonged fluorescence lifetime and biocompatibility. Our research was aimed at construction of fluorescent AgNC with the main blood proteins and transmission electron microscopy (TEM). Methods: AgNC were synthesized from AgNO3 in the presence of albumins and immunoglobulins (Ig) of different classes and origin at pH 9-11 with NaBH4 recovery. The resulting AgNC with proteins were loaded to "Formvar/Carbon 200 Mesh Copper" copper grids (Ted Pella, USA) and examined using TEM system JEM 2100 Plus (JEOL, Japan) without contrast. Fluorescence excitation/emission spectra were measured in quartz cuvette using the FluoroMax + spectrofluorometer (Horiba Scientific, Japan). Results: Recovery of Ag+ ions did not occur in the presence of IgG and albumins without NaBH4 at different temperatures, pH, and incubation time. Broad excitation spectra of AgNC were in a range 340-540 nm. Their emission spectra correlated with the original AgNO3 concentration and did not depend on protein and pH. NC stabilized with IgG or albumin with blue fluorescence and emission maximum at 420 nm contained NC from 0.6 nm and higher. Green AgNC with proteins had bright fluorescence at 430-470 nm and red NC showed emission maximum at 650 nm. TEM revealed discrete AgNC and their numerous aggregates in each sample of fluorescent NC in spite of different fluorescent emission spectra. According to the MTT test, AgNC with human IgG and BSA with protein concentrations up to 3 mg/ml were not toxic for human larynx carcinoma HEp-2 cells despite cytotoxicity of silver nanoparticles covered with IgG or albumin envelopes as well as Cd and AuNC with BSA. Conclusion: AgNC with antibodies and albumin with a broad size range and aggregation possess tunable fluorescence emission spectra with broad excitation at 340-540 nm. Different emission spectra permit AgNC to be used in multiplex assays. AgNC were not toxic for human tissue culture and may be applied for bioimaging.

Rapid Synthesis of Thiol-Co-Capped-CdTe/CdSe/ZnSe Core Shell-Shell Nanoparticles: Their Optical and Structural Morphology

CdTe QDs has been demonstrated in many studies to possess good outstanding optical and photo-physical properties. However, it has been established from literature that the toxic Cd2+ that tends to leak out into nearby solutions can be protected by less toxic ZnS or ZnSe shells leading to the synthesis of core-shells and multi-core-shells. Hence, this has allowed the synthesis of CdTe multi-core-shells to have gained much interest. The preparation of most CdTe multi-core-shells reported from various studies usually has a longer reaction time (6–24 h) in reaching their highest emission maxima. The synthesis of CdTe multi-core-shells in this study only took 35 min to obtain a highest emission maximum compared to what has been reported from the literature. CdTe multi-core-shells were synthesized by injecting 7, 14, and 21 mL each of Zn complex solution and Se ions into the reacting mixture containing CdTe core-shells (3 h) at 5 min intervals over a 35 min reaction time. The emission maxima of the MPA-TGA-CdTe multi-core-shells at 21 mL injection was recorded around 625 nm. Therefore, we are reporting the rapid synthesis of five different thiol co-capped CdTe/CdSe/ZnSe multi-core-shell QDs with the highest emission maxima obtained at 35 min reaction time.

Synthesis and optical properties of new 4-substituted pyrimidines - D-A type chromophores

The set of 4-aryl(hetaryl)pyrimidines, where aryl/hetaryl is a highly electron donating substituent was synthesized. Optical and electrochemical properties of the synthesized compounds were studied and the values of a forbidden band gap energy ( ) were determined. The narrowest bandgap was found to be inherent to 4-ferrocenylpyrimidine (1.8 eV) and the most long-wavelength emission maximum ‑ to para-substituted pyrimidine which structure embeds a thiophene moiety between N-hexylcarbazolyl fragment and the pyrimidine core (577 нм).

Sponge-derived Ageladine A affects the in vivo fluorescence emission spectra of microalgae

In several marine hosts of microalgae, fluorescent natural products may play an important role. While the ecological function of these compounds is not well understood, an interaction of these molecules with the photosynthesis of the symbionts has been suggested. In this study, the effect of Ageladine A (Ag A), a pH-dependent fluorophore found in sponges of the genus Agelas, on microalgal fluorescence was examined. The spectra showed an accumulation of Ag A within the cells, but with variable impacts on fluorescence. While in two Synechococcus strains, fluorescence of phycoerythrin increased significantly, the fluorescence of other Synechococcus strains was not affected. In four out of the five eukaryote species examined, chlorophyll a (Chl a) fluorescence intensity was modulated. In Tisochrysis lutea, for example, the position of the fluorescence emission maximum of Chl a was shifted. The variety of these effects of Ag A on microalgal fluorescence suggests that fluorophores derived from animals could play a crucial role in shaping the composition of marine host/symbiont systems.

Preparation and luminescent properties of multicolored SrAl2O4:Eu2+, Dy3+/SiO2-coated red-emitting coumarin color converter/polyamide 6 luminous fiber with warm-toned luminescence

Novel SrAl2O4:Eu2+, Dy3+/SiO2-coated red-emitting coumarin color converter (SiO2@RECC)/polyamide 6 (PA6) luminous fibers with warm-toned luminescence color were prepared on the basis of the energy transfer and color conversion from SrAl2O4:Eu2+, Dy3+ to RECC. The mass concentrations of RECC used in the fibers were adjusted to obtain different luminescence colors. Scanning electron microscopy images, photoluminescence (PL) emission spectra, RGB color coordinates, luminescence photos, and luminescence lifetimes were analyzed. Results demonstrated that the PL emission spectra of luminous fibers contain two emission peaks in the range of 475–700 nm. The increase in RECC concentration contributed to the gradual redshift of the second emission peak, and the ratio of the second emission maximum to the first emission maximum also increased gradually. The luminescence color of the fibers shifted gradually toward red in the dark. Moreover, the fibers showed light yellow, yellow, orange, and orange-reddish luminescence in the dark when the mass concentrations of RECC were fixed at 0.1%, 0.6%, 1.0%, and 1.4%, correspondingly. The luminescence lifetimes of the fibers can be sustained for at least 13,288 s. The prepared warm-toned SrAl2O4:Eu2+, Dy3+/SiO2@RECC/PA6 luminous fibers have considerable application prospects, given their excellent luminescence properties.

Development of a 1,3a,6a-triazapentalene derivative as a compact and thiol-specific fluorescent labeling reagent

For the fluorescence imaging of biologically active small compounds, the development of compact fluorophores that do not perturb bioactivity is required. Here we report a compact derivative of fluorescent 1,3a,6a-triazapentalenes, 2-isobutenylcarbonyl-1,3a,6a-triazapentalene (TAP-VK1), as a fluorescent labeling reagent. The reaction of TAP-VK1 with various aliphatic thiols proceeds smoothly to afford the corresponding 1,4-adducts in high yields, and nucleophiles other than thiols do not react. After the addition of thiol groups in dichloromethane, the emission maximum of TAP-VK1 shifts to a shorter wavelength and the fluorescence intensity is substantially increased. The utility of TAP-VK1 as a compact fluorescent labeling reagent is clearly demonstrated by the labeling of Captopril, which is a small molecular drug for hypertension. The successful imaging of Captopril, one of the most compact drugs, in this study demonstrates the usefulness of compact fluorophores for mechanistic studies.

Sulfolobus acidocaldarius Microvesicles Exhibit Unusually Tight Packing Properties as Revealed by Optical Spectroscopy

In this study, we used optical spectroscopy to characterize the physical properties of microvesicles released from the thermoacidophilic archaeon Sulfolobus acidocaldarius (Sa-MVs). The most abundant proteins in Sa-MVs are the S-layer proteins, which self-assemble on the vesicle surface forming an array of crystalline structures. Lipids in Sa-MVs are exclusively bipolar tetraethers. We found that when excited at 275 nm, intrinsic protein fluorescence of Sa-MVs at 23 °C has an emission maximum at 303 nm (or 296 nm measured at 75 °C), which is unusually low for protein samples containing multiple tryptophans and tyrosines. In the presence of 10–11 mM of the surfactant n-tetradecyl-β-d-maltoside (TDM), Sa-MVs were disintegrated, the emission maximum of intrinsic protein fluorescence was shifted to 312 nm, and the excitation maximum was changed from 288 nm to 280.5 nm, in conjunction with a significant decrease (>2 times) in excitation band sharpness. These data suggest that most of the fluorescent amino acid residues in native Sa-MVs are in a tightly packed protein matrix and that the S-layer proteins may form J-aggregates. The membranes in Sa-MVs, as well as those of unilamellar vesicles (LUVs) made of the polar lipid fraction E (PLFE) tetraether lipids isolated from S. acidocaldarius (LUVPLFE), LUVs reconstituted from the tetraether lipids extracted from Sa-MVs (LUVMV) and LUVs made of the diester lipids, were investigated using the probe 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan). The generalized polarization (GP) values of Laurdan in tightly packed Sa-MVs, LUVMV, and LUVPLFE were found to be much lower than those obtained from less tightly packed DPPC gel state, which echoes the previous finding that the GP values from tetraether lipid membranes cannot be directly compared with the GP values from diester lipid membranes, due to differences in probe disposition. Laurdan’s GP and red-edge excitation shift (REES) values in Sa-MVs and LUVMV decrease with increasing temperature monotonically with no sign for lipid phase transition. Laurdan’s REES values are high (9.3–18.9 nm) in the tetraether lipid membrane systems (i.e., Sa-MVs, LUVMV and LUVPLFE) and low (0.4–5.0 nm) in diester liposomes. The high REES and low GP values suggest that Laurdan in tetraether lipid membranes, especially in the membrane of Sa-MVs, is in a very motionally restricted environment, bound water molecules and the polar moieties in the tetraether lipid headgroups strongly interact with Laurdan’s excited state dipole moment, and “solvent” reorientation around Laurdan’s chromophore in tetraether lipid membranes occurs very slowly compared to Laurdan’s lifetime.

Tuning the Bi3+-photoemission color over the entire visible region by manipulating secondary cations modulation in the ScVxP1−xO4:Bi3+ (0 ≤ x ≤ 1) solid solution

Manipulating the secondary cation ratio in the ScVxP1−xO4:Bi3+ solid solution allows tuning of the excitation tail from 340 to 425 nm and the corresponding emission maximum from 455 to 641 nm, without significant reabsorption.

Novel Phosphonium Dye TDV1 as a Potential Fluorescent Probe to Monitor DNA Interactions with Lysozyme Amyloid Fibrils

The applicability of the novel cationic phosphonium dye TDV1 to monitor the complexation between DNA and pathologically aggregated proteins, amyloid fibrils, was tested using the optical spectroscopy and molecular docking techniques. TDV1 has been found to be highly emissive in buffer solution and is characterized by one well-defined fluorescence peak attributed to the dye monomers. The association of the dye with the double stranded DNA was followed by the enhancement of monomer fluorescence coupled with a bathochromic shift of the emission maximum. The addition of fibrillar lysozyme (LzF) to TDV1-DNA mixture led to the further enhancement of fluorescence intensity of the monomeric dye form coupled with a hypsochromic shift of the emission maximum and an appearance of a second long-wavelength peak. An assumption has been made that the fluorescence enhancement augmenting with increasing the protein concentration in the TDV1/DNA system is produced by the interaction of the free TDV1 monomers with lysozyme fibrils as well as by the LzF-induced conformational alterations of DNA. The long-wavelength peak emerging in the presence of LzF is presumably a consequence of the J-aggregate formation upon the TDV1 association with lysozyme fibrils. The molecular docking studies showed that TDV1 monomers are incorporated into the fibril grooves associating with 7 β-strands in such a way that the dye long axis is parallel to the fibril axis. The most energetically favorable position of TDV1 is the S60-W62/G54-L56 groove in the lysozyme fibril core. In contrast, the TDV1 dimers seem to associate with the more hydrophilic side of the model β-sheet. Cumulatively, the results from the absorption and fluorescence measurements, together with the molecular docking analysis are consistent with the minor groove mode of the TDV1 binding to dsDNA. The electrostatic interactions seem to play a predominant role in the TDV1 complexation with the double stranded DNA, while the hydrophobic interactions and steric hindrances are supposed to be influential in the association of TDV1 with fibrillar lysozyme.

Monomerization of far-red fluorescent proteins

Anthozoa-class red fluorescent proteins (RFPs) are frequently used as biological markers, with far-red (λem ∼ 600–700 nm) emitting variants sought for whole-animal imaging because biological tissues are more permeable to light in this range. A barrier to the use of naturally occurring RFP variants as molecular markers is that all are tetrameric, which is not ideal for cell biological applications. Efforts to engineer monomeric RFPs have typically produced dimmer and blue-shifted variants because the chromophore is sensitive to small structural perturbations. In fact, despite much effort, only four native RFPs have been successfully monomerized, leaving the majority of RFP biodiversity untapped in biomarker development. Here we report the generation of monomeric variants of HcRed and mCardinal, both far-red dimers, and describe a comprehensive methodology for the monomerization of red-shifted oligomeric RFPs. Among the resultant variants is mKelly1 (emission maximum, λem = 656 nm), which, along with the recently reported mGarnet2 [Matela G, et al. (2017) Chem Commun (Camb) 53:979–982], forms a class of bright, monomeric, far-red FPs.