Coumarin-Based Noncytotoxicity Fluorescent Dye for Tracking Actin Protein in In-Vivo Imaging.

Drosophila shares maximum homology with the human disease-causing genes and thus has been employed to evaluate the toxicity of numerous compounds. Further, its distinguishable developmental stages, easy rearing, and short lifespan make it a perfect model organism to study toxicological properties of any new compound. The current study evaluates the toxic effect of a coumarin-based organic fluorescent dye, 7-hydroxy-4-methyl-8-((4-(2-oxo-2H-chromen-3-yl)thiazol-2-ylimino)methyl)-2H-chromen-2-one (CTC), using Drosophila melanogaster as a model organism by studying different behavioral, screening, and staining techniques using Oregon-R flies. For toxicity assessment, one control fly group was compared with various flies that had been subjected to fed CTC dye orally of different concentrations (0.5, 1, 2.5, and 5 µg/mL). The 3rd instar larvae were checked for the larvae crawling assay. The crawling assay demonstrates that the speed and path of the treated larvae are almost equal to the control ones, which signifies the non-neurotoxic property of CTC. Trypan blue assay further suggested that the dye does not cause any major damage to the gut. Phalloidin staining revealed that the actin composition remains unaltered even after the CTC treatment, while the DAPI staining experiment indicates that CTC does not cause any nuclear damage to fly gut cells. However, at a concentration of 5 µg/mL, CTC causes developmental delay. The flies hatched after larval treatment of CTC do not show any structural defects, suggesting clearly that CTC is also nongenotoxic to Drosophila. The current studies propose CTC as a noncytotoxic and nongenotoxic dye to track actin protein in the model organism D. melanogaster.

Synthesis of First Coumarin Fluorescent Dye for Actin Visualization.

Selective fluorescence imaging of actin protein hugely depends on the fluorescently labeled actin-binding domain (ABD). Thus, it is always a challenging task to image the actin protein (in vivo or in vitro) directly with an ABD-free system. To overcome the limitations of actin imaging without an ABD, we have designed a facile and cost-effective red fluorescent coumarin dye 7-hydroxy-4-methyl-8-(4-(2-oxo-2H-chromen-3-yl)thiazol-2-ylimino)methyl-2H-chromen-2-one (CTC) for actin binding. The selective binding of the dye was investigated using the gut and eye of the model organism Drosophila melanogaster and C2C12 and SCC-9 cell lines. Our result suggests two major advantages of CTC over the dyes presently used for imaging actin proteins. First, the dye can bind to actin efficiently without any secondary intermediate. Second, it is much more stable at room temperature and exhibits excellent photostability. To the best of our knowledge, this is the first fluorescent dye that can bind to the actin protein without employing any secondary intermediate/actin-binding domain. These findings could pave the way for many biologists and physicists to successfully employ the CTC dye for imaging and tracking actin proteins by fluorescence microscopy in various in vivo and in vitro systems.

Chromo-luminescent selective detection of fluoride ions by a copper(ii) bis(terpyridine) complex solution via a displacement approach.

Herein, for the first time, we have reported a copper(ii) bis(terpyridine) complex solution for instant 'naked eye' chromo-luminescent selective detection of fluoride ions in an acetonitrile medium at micromolar concentration. The copper complex [Cu(ii) (L)2] (NO3)2 [where L = 4'-(4-N,N'-dimethylaminophenyl)-2,2':6',2''-terpyridine] was characterized by mass spectroscopy and the terpyridine ligand by 1H NMR spectroscopy. The complex solution selectively discriminates F- ions from other anions such as AcO-, Br-, Cl-, CN-, H2PO4-, HSO4-, and I- in acetonitrile media via exceptional optical changes. The optical changes were evaluated by UV-visible and fluorescence techniques. Studies on the binding characteristics of the copper complex solution with fluoride ions revealed a displacement of copper ions from the complex solution as CuF2 resulting in the significant optical changes. Furthermore, displacement of Cu(ii) from the complex was established by means of mass spectroscopy in the presence of 20 equivalents of fluoride ions. The limit of detection (LOD) was found to be 5.07 µM which is within the permissible range of fluoride ions in drinking water set by the World Health Organization (WHO).

Butterfly pea (Clitoria ternatea) extract as a green analytical tool for selective colorimetric detection of bisulphate (HSO4-) ion in aqueous medium.

The blue color of butterfly pea (Clitoria ternatea) was extracted by Milli-Q water and evaluated for selective detection of bisulphate (HSO4-) ions. The stability of the Clitoria ternatea extract was established by UV-visible and fluorescence techniques. The blue water extract from Clitoria ternatea selectively recognizes HSO4- ions over various anions via a distinct visual color change from blue to purple with a significant hypsochromic shift of 68 nm in the UV-visible absorption spectra. Thus Clitoria ternatea extract provides a selective real time colorimetric monitoring of HSO4- ions which would pave the way for the development of low cost green analytical tool. This type of detection technique enhances the environmental and economic benefits and can emerge as an alternative form of synthetic chelating sensors.

Through bond energy transfer (TBET)-operated fluoride ion sensing via spirolactam ring opening of a coumarin-fluorescein bichromophoric dyad.

The detection of fluoride ions in a competitive environment often poses several challenges. In this work, we have designed and synthesized a coumarin functionalized fluorescein dyad (R3) which represents an ideal through bond energy transfer (TBET) fluorophore with the coumarin unit as donor and fluorescein unit as acceptor. The bichromophoric dyad demonstrates the detection of fluoride ions in the parts per billion (ppb) concentration level (22.8 ppb) with high selectivity via a TBET emission signal at 548 nm with a diagnostic bright yellow colour fluorescence output. Based on UV-visible, fluorescence, 1H NMR and DFT studies, it is shown that the fluoride ion induces the opening of the spirolactam ring of the fluorescein moiety and provides a π-conjugation link between the donor and acceptor units enabling a TBET phenomenon with a larger pseudo-Stokes shift of 172 nm. To the best of our knowledge, this is the first report where the fluoride ion is detected via a TBET signal between the coumarin and fluorescein units in a bichromophoric dyad.

Sequential displacement strategy for selective and highly sensitive detection of Zn2+, Hg2+ and S2- ions: An approach toward a molecular keypad lock.

A thiocarbonohydrazone locked salicylidene based macrocycle ligand L has been synthesized and its ion sensing properties were examined by UV-visible and fluorescence spectroscopy. The macrocycle serves as a highly selective colorimetric sensor for Hg2+ ions while it acts as an excellent fluorescent sensor for Zn2+ ions by exhibiting a green fluorescence at 498 nm even in the presence of interfering ions. A detailed analysis of binding characteristics such as complex stoichiometry, association constant and detection limits of L toward Hg2+ and Zn2+ ions were evaluated by UV-visible and fluorescence experiments which revealed a stronger binding affinity and higher detection limit of L toward the mercury ions. Further, the sequential displacement strategy for the chromofluorogenic detection of Zn2+, Hg2+ and S2- ions by ligand L, has been studied comprehensively. Finally, the ion-responsive fluorescence output signal of L were employed to design a molecular keypad lock which could be accessible by two users having two different set of chemical passwords (inputs) through distinguishable optical trajectories.

Fluorescence-based ion sensing in lipid membranes: a simple method of sensing in aqueous medium with enhanced efficiency.

Detection of ions in chemical, biological and environmental samples has gathered tremendous momentum considering the beneficial as well as adverse effects of the ions. Generally, most of the ions are beneficial up to an optimum concentration, beyond which they are toxic to human health. However, most of the fluorescence-based ion sensors are only active in non-aqueous solution because of the low solubility of the sensor molecules in aqueous buffer medium. In the present work, we have demonstrated that encapsulation of an aqueous insoluble thiocarbonohydrazone-locked salicylidene-based macrocyclic ligand in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes allows the selective detection of Zn2+ in aqueous medium with approximately 3-fold enhanced efficiency compared to its efficiency in DMSO medium. We have further modulated the charge of the membrane surface by adding various concentrations of a negatively charged lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), and showed that negative surface charge further enhances the Zn2+ sensing efficiency up to approximately 6-fold. This strategy opens up a new avenue of utilizing organic sensors to detect vital ions in aqueous medium.