carbocyanines and 5-carboxytetramethylrhodamine-succinimidyl-ester

As VEGF mRNA is an endothelial cell-specific mitogen and a key regulator of angiogenesis in a variety of physiological and pathological processes, high expression levels of VEGF messenger RNA (mRNA) contribute to VEGF-driven angiogenesis in the hypoxic areas of solid tumors and then disrupt the vascular barrier, which may potentiate tumor cell extravasation. Thus, monitoring the changes in VEGF mRNA is necessary to understand the genetic programme under hypoxic conditions and thus facilitate risk assessment and risk reduction in hypoxic environments. Herein, a new fluorescent nanoprobe based on azoreductase-responsive functional metal-organic frameworks (AMOFs) was developed to realize the imaging of VEGF mRNA under hypoxic conditions. Since the azobenzene units in the AMOFs can be reduced to amines by the highly expressed azoreductase in an oxygen-deficient environment, the VEGF mRNA-targeted molecular beacon (MB), which is adsorbed on the surface of AMOFs via electrostatic interactions, can be released due to the structural damage of AMOFs. Moreover, TAMRA (carboxytetramethylrhodamine, donor) and Cy5 (acceptor) were close to each other due to the stem-loop conformation of MB, thus inducing high fluorescence energy resonance transfer (FRET) efficiency. Upon the addition of VEGF mRNA, the hybridization of VEGF mRNA destroyed the stem-loop conformation of MB, and then, the two fluorophores labeled on MB were separated with low FRET efficiency. This constructed fluorescent nanoprobe enables the quantitative analysis and in situ imaging of the VEGF mRNA level in living cells under hypoxic conditions. We expect that it will offer a potentially rich opportunity to understand the physiological processes of genetic programme.

Topics: Carbocyanines; Cell Hypoxia; Cell-Penetrating Peptides; DNA; DNA Probes; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; HeLa Cells; Humans; Metal-Organic Frameworks; Microscopy, Confocal; Microscopy, Fluorescence; NADH, NADPH Oxidoreductases; Nitroreductases; Nucleic Acid Hybridization; Rhodamines; RNA, Messenger; Vascular Endothelial Growth Factor A

MicroRNAs (miRNAs), small noncoding endogenous RNA molecules, are emerging as promising biomarkers for early detection of various diseases and cancers. Practical screening tools and strategies to detect these small molecules are urgently needed to facilitate the translation of miRNA biomarkers into clinical practice. In this study, a label-free biosensing technique based on surface-enhanced Raman scattering (SERS), referred to as plasmonic coupling interference (PCI), was applied for the multiplex detection of miRNA biomarkers. The sensing mechanism of the PCI technique relies on the formation of a nanonetwork consisting of nanoparticles with Raman labels located between adjacent nanoparticles that are interconnected by DNA duplexes. Because of the plasmonic coupling effect of adjacent nanoparticles in the nanonetwork, the Raman labels exhibit intense SERS signals. Such effect can be modulated by the addition of miRNA targets of interest that act as inhibitors to interfere with the formation of this nanonetwork, resulting in a diminished SERS signal. In this study, the PCI technique is theoretically analyzed, and the multiplex capability for detection of multiple miRNA cancer biomarkers is demonstrated, establishing the great potential of PCI nanoprobes as a useful diagnostic tool for medical applications.

Topics: Biomarkers, Tumor; Carbocyanines; DNA Probes; Fluorescent Dyes; Humans; Metal Nanoparticles; MicroRNAs; Neoplasms; Rhodamines; RNA, Neoplasm; Sensitivity and Specificity; Silver; Spectrum Analysis, Raman; Surface Plasmon Resonance

The diverse gut microbial communities are crucial for host health. How the interactions between microbial communities and between host and microbes influence the host, however, is not well understood. To facilitate gut microbiota research, selective imaging of specific groups of microbiotas in the gut is of great utility but remains technically challenging. Here we present a chemical approach that enables selective imaging of Gram-negative and Gram-positive microbiotas in the mouse gut by exploiting their distinctive cell wall components. Cell-selective labeling is achieved by the combined use of metabolic labeling of Gram-negative bacterial lipopolysaccharides with a clickable azidosugar and direct labeling of Gram-positive bacteria with a vancomycin-derivatized fluorescent probe. We demonstrated this strategy by two-color fluorescence imaging of Gram-negative and Gram-positive gut microbiotas in the mouse intestines. This chemical method should be broadly applicable to different gut microbiota research fields and other bacterial communities studied in microbiology.

Topics: Animals; Azides; Carbocyanines; Cell Wall; Click Chemistry; Diagnostic Techniques, Digestive System; Dysbiosis; Fluorescent Dyes; Gastrointestinal Microbiome; Gastrointestinal Tract; Gram-Negative Bacteria; Gram-Positive Bacteria; Lipopolysaccharides; Mice, Inbred C57BL; Microbial Viability; Optical Imaging; Pilot Projects; Porphobilinogen; Rhodamines; Specific Pathogen-Free Organisms; Sugar Acids; Vancomycin

DNA origami nanostructures are a versatile tool to arrange metal nanostructures and other chemical entities with nanometer precision. In this way gold nanoparticle dimers with defined distance can be constructed, which can be exploited as novel substrates for surface enhanced Raman scattering (SERS). We have optimized the size, composition and arrangement of Au/Ag nanoparticles to create intense SERS hot spots, with Raman enhancement up to 10(10), which is sufficient to detect single molecules by Raman scattering. This is demonstrated using single dye molecules (TAMRA and Cy3) placed into the center of the nanoparticle dimers. In conjunction with the DNA origami nanostructures novel SERS substrates are created, which can in the future be applied to the SERS analysis of more complex biomolecular targets, whose position and conformation within the SERS hot spot can be precisely controlled.

Topics: Carbocyanines; Dimerization; DNA; DNA, Single-Stranded; Gold; Metal Nanoparticles; Microscopy, Atomic Force; Microscopy, Electron, Scanning; Nanotechnology; Nucleic Acid Hybridization; Rhodamines; Scattering, Radiation; Silicon; Silver; Spectrum Analysis, Raman

We demonstrate the successful construction of fluorescently labeled magnetic antibody-like nanoparticles (ANPs) via a facile one-step surface-initiated in situ molecular imprinting approach over silica coated magnetite (Fe3O4@SiO2) core-shell nanocomposites. The as-prepared ANPs had a highly compact structure with an overall size of 83 ± 5 nm in diameter and showed excellent aqueous dispersion stability. With the predetermined high specificity to the target protein and high biocompatibility, the ANPs enabled rapid, efficient, selective and optically trackable sequestration of target proteins within living cells. This work represents the first example of fully artificially engineered multifunctional ANPs for the intracellular protein-sequestration without disruption of the cells. The established approach may be further extended to generate ANPs for various proteins of interest and provide useful tools for related biological research and biomedical applications.

Topics: Antibodies; Carbocyanines; Cell Survival; Deoxyribonuclease I; Ferrosoferric Oxide; HeLa Cells; Humans; Magnetic Fields; Magnetite Nanoparticles; Microscopy, Fluorescence; Molecular Imprinting; Particle Size; Rhodamines; Silicon Dioxide; Spectrometry, Fluorescence; Surface Properties

Our objective was to create a novel fluorogenic substrate for efficient in vitro kinetic assays on caspase-3. We designed a TAMRA (5'-tetramethylrhodamine-5(6)-carboxamide)- and Cy5 (cyanine 5)-labeled probe that allowed us to evaluate the caspase-3 activity via the changes in fluorescence intensity and wavelength. The prepared probe was found to be an efficient and selective substrate of caspase-3, with V(max) of 41.4±3.3 nM/min and K(M) of 1.60±0.23 μM. The strategy used in the design of this fluorogenic substrate can be applied in future endeavors to development of substrates for caspase-3 inhibitor screening assays or for real-time detection of apoptosis in living cells.

Topics: Amino Acid Sequence; Carbocyanines; Caspase 3; Enzyme Assays; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Kinetics; Rhodamines

The fluorescence correlation spectroscopy (FCS)-based competitive binding assay to screen for protein-protein interaction inhibitors is a highly sensitive method as compared with the fluorescent polarization assay used conventionally. However, the FCS assay identifies many false-positive compounds, which requires specifically designed orthogonal screenings. A two-colored application of the FCS-based screening was newly developed, and inhibitors of a protein-protein interaction, involving selective autophagy, were selected. We focused on the interaction of LC3 with the adaptor protein p62, because the interaction is crucial to degrade the specific target proteins recruited by p62. First, about 10,000 compounds were subjected to the FCS-based competitive assay using a TAMRA-labeled p62-derived probe, and 29 hit compounds were selected. Next, the obtained hits were evaluated by the second FCS assay, using an Alexa647-labeled p62-derived probe to remove the false-positive compounds, and six hit compounds inhibited the interaction. Finally, we tested all 29 compounds by surface plasmon resonance-based competitive binding assay to evaluate their inhibition of the LC3-p62 interaction and selected two inhibitors with IC50 values less than 2 µM. The two-colored FCS-based screening was shown to be effective to screen for protein-protein interaction inhibitors.

Topics: Adaptor Proteins, Signal Transducing; Amino Acid Sequence; Binding, Competitive; Carbocyanines; Dose-Response Relationship, Drug; Escherichia coli; Fluorescent Dyes; Glutathione Transferase; High-Throughput Screening Assays; Humans; Kinetics; Microtubule-Associated Proteins; Molecular Sequence Data; Peptides; Protein Binding; Recombinant Fusion Proteins; Rhodamines; Sequestosome-1 Protein; Small Molecule Libraries; Spectrometry, Fluorescence

We have developed a new technique using fluorescent silica nanotubes for simple and sensitive DNA detection. The quantum-dot-embedded silica nanotubes (QD-SNTs) were fabricated by a sol-gel reaction using anodic aluminum silica oxide (AAO) as a template. The fluorescent QD-SNTs of different colors were then immobilized with single-stranded DNA and used as nanoprobes for DNA detection. The optical and structural properties of QD-SNT nanoprobes were examined using photoluminescence spectroscopy, confocal microscopy and transmission electron microscopy (TEM). The QD-SNT nanoprobes were applied to detect dye-labeled target DNA in a solution phase. The obvious color change of the QD-SNT nanoprobes was observed visually under a simple microscope after the successful detection with target DNA. The quantitative analyses indicated that ∼ 100 attomole of target DNA in one nanoprobe can generate a distinguishable and observable color change. The detection results also demonstrated that our assay exhibited high specificity, high selectivity and very low nonspecific adsorption. Our simple DNA assay based on QD-SNT nanoprobes is expected to be quite useful for the needs of fast DNA screening and detection applications.

Topics: Adsorption; Carbocyanines; DNA; DNA Probes; DNA, Complementary; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Microscopy, Electron, Transmission; Microscopy, Fluorescence; Nanotubes; Nucleic Acid Hybridization; Particle Size; Quantum Dots; Rhodamines; Silicon Dioxide; Spectrometry, Fluorescence

Fluorescent imaging of cytoskeletal structures permits studies of both organization within the cell and dynamic reorganization of the cytoskeleton itself. Traditional fluorescent labels of microtubules, part of the cytoskeleton, have been used to study microtubule localization, structure, and dynamics, both in vivo and in vitro. However, shortcomings of existing labels make imaging of microtubules with high precision light microscopy difficult. In this paper, we report a new fluorescent labeling technique for microtubules, which involves a GTP analog modified with a bright, organic fluorophore (TAMRA, Cy3, or Cy5). This fluorescent GTP binds to a specific site, the exchangeable site, on tubulin in solution with a dissociation constant of 1.0±0.4 µM. Furthermore, the label becomes permanently incorporated into the microtubule lattice once tubulin polymerizes. We show that this label is usable as a single molecule fluorescence probe with nanometer precision and expect it to be useful for modern subdiffraction optical microscopy of microtubules and the cytoskeleton.

Topics: Animals; Binding Sites; Carbocyanines; Cattle; Fluorescent Dyes; Guanosine Triphosphate; Microtubules; Rhodamines; Staining and Labeling; Tubulin

Detection of single, fluorescently labeled biomolecules is providing a powerful approach to measuring molecular transport, biomolecular interactions, and localization in biological systems. Because the biological molecules of interest rarely exhibit sufficient intrinsic fluorescence to allow observation of individual molecules, they are usually labeled with fluorescent dye molecules, fluorescent proteins, semiconductor nanocrystals or quantum dots, or fluorescently doped silica or polymer nanospheres to allow their detection. Differences in the photophysical and spectral properties of different labels allow one to identify individual molecules by distinguishing their corresponding labels. A simple approach to measuring fluorescence spectra of individual fluorescent labels can be implemented in a standard wide-field fluorescence microscope, where a grating or prism is incorporated into the path from the microscope to an imaging detector to disperse the emission spectrum. In this work, principal components and cluster analysis are applied to the identification of fluorescence spectra from single fluorescent labels, with statistical tests of the classification results. Spectra are determined from diffracted images of fluorescent nanospheres labels, where emission maxima are separated by less than 20 nm, and of single dye-molecule labels with 30 nm separation. Clusters of points in an eigenvector representation of the spectra correctly classify known labels (both nanospheres and single molecules) and unambiguously identify unknown labels in mixtures.

Topics: Benzenesulfonates; Carbocyanines; Cluster Analysis; Equipment Design; Fluorescent Dyes; Image Processing, Computer-Assisted; Microscopy, Fluorescence; Nanospheres; Polystyrenes; Principal Component Analysis; Rhodamines; Spectrometry, Fluorescence

Topics: Amino Acid Sequence; Carbocyanines; Checkpoint Kinase 1; Fluorescence Resonance Energy Transfer; Metals; Peptides; Phosphates; Protein Kinases; Rhodamines; Substrate Specificity

We have developed a multi-color fluorescence in situ hybridization (FISH) method which detects, by a single reaction, all seven species of Bifidobacterium (B. adolescentis, B. angulatum, B. bifidum, B. breve, B. catenulatum, B. dentium, and B. longum), the dominant bacteria in human feces. First, eight new types of oligonucleotide probe were designed, complementary with the 16S rRNA sequence specific to genus Bifidobacterium and each bifidobacterial species described above. Using whole cell hybridization, the fluorescent intensity was measured against the bacterial species targeted by each probe, to show that each probe is specific to the targeted bacteria and that the relative fluorescent intensity (RFI) as an indicator of probe accessibility is high at 61-117%. Then, bacterial species-specific probes were labeled with fluorochromes (FITC, TAMRA and Cy5) in seven different ways, singly or in combination. Using these probes, seven species of Bifidobacterium were differentially stained in mixed samples of cultured bacteria and feces from adult volunteers, proving the efficacy of this technique.

Topics: Bifidobacterium; Carbocyanines; DNA, Bacterial; Feces; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Humans; In Situ Hybridization, Fluorescence; Microscopy, Fluorescence; Rhodamines; RNA, Ribosomal, 16S; Sensitivity and Specificity

Patterning of the ventral head has been attributed to various cell populations, including endoderm, mesoderm, and neural crest. Here, we provide evidence that head and heart development may be influenced by a ventral midline endodermal cell population. We show that the ventral midline endoderm of the foregut is generated directly from the extreme rostral portion of Hensen's node, the avian equivalent of the Spemann organizer. The endodermal cells extend caudally in the ventral midline from the prechordal plate during development of the foregut pocket. Thus, the prechordal plate appears as a mesendodermal pivot between the notochord and the ventral foregut midline. The elongating ventral midline endoderm delimits the right and left sides of the ventral foregut endoderm. Cells derived from the midline endoderm are incorporated into the endocardium and myocardium during closure of the foregut pocket and fusion of the bilateral heart primordia. Bilateral ablation of the endoderm flanking the midline at the level of the anterior intestinal portal leads to randomization of heart looping, suggesting that this endoderm is partitioned into right and left domains by the midline endoderm, thus performing a function similar to that of the notochord in maintaining left-right asymmetry. Because of its derivation from the dorsal organizer, its extent from the forebrain through the midline of the developing face and pharynx, and its participation in formation of a single midline heart tube, we propose that the ventral midline endoderm is ideally situated to function as a ventral organizer of the head and heart.

Topics: Animals; Body Patterning; Carbocyanines; Chick Embryo; Chimera; Coturnix; Digestive System; Endoderm; Gene Expression Regulation, Developmental; Genes, Homeobox; Head; Heart; Homeodomain Proteins; Models, Biological; Organizers, Embryonic; Rhodamines