New Class Of Fluorescent Probes Research Articles

Carrier-free, water dispersible and highly luminescent dye nanoparticles for targeted cell imaging

We develop a new strategy of using surface functionalized small molecule organic dye nanoparticles (NPs) for targeted cell imaging. Organic dye (2-tert-butyl-9,10-di(naphthalen-2-yl)anthracene, TBADN) was fabricated into NPs and this was followed by surface modification with an amphipathic surfactant poly(maleic anhydride-alt-1-octadecene)-polyethylene glycol (C18PMH-PEG) through hydrophobic interactions to achieve good water dispersibility and bio-environmental stability. It should be noted that no additional inert materials were added as carriers, thus the dye-loading capacity of the resulting TBADN NPs is obviously higher than those of previously reported carrier-based structures. This would lead to much larger absorption and then much higher brightness. The resulting TBADN NPs possess comparable, if not higher, brightness than CdSe/ZnS quantum dots under the same conditions, with favorable biocompatibility. Significantly, TBADN NPs are readily conjugated with folic acid, and successfully applied in targeted cell imaging. These results show that water dispersible and highly stable organic NPs would be a promising new class of fluorescent probe for bioapplications in cellular imaging and labeling. This strategy may be straightforwardly extended to other organic dyes to achieve water dispersible NPs for cell imaging and drug delivery.

Fluorescent silver nanoclusters for user-friendly detection of Cu2+ on a paper platform

The development of a user-friendly sensing platform for the detection of Cu(2+) in water is necessary as there are wide concerns due to the substantial impact of Cu(2+) on human health, environmental monitoring, and so on. Motivated by this, we report a fluorescent silver nanoclusters (AgNCs)-based sensor for the detection of Cu(2+). These water-soluble AgNCs, as a new class of fluorescent probes, were synthesized by using azobenzene modified poly(acrylic acid) (MPAA) as templates. Their fluorescence can be quenched in the presence of Cu(2+), which enables the label-free detection of Cu(2+) in real water samples. Furthermore, such AgNCs can be integrated onto cellulose filter paper and used as fluorescent indicators for Cu(2+). The fluorescence quenching can be observed by the naked eye under UV light. It should be noted that this AgNCs-based paper assay performs successfully in barrelled drinking water and river water samples. Therefore, it opens up new avenues to the development of robust clusters-based sensing platforms.

Expression of Caveolin-1 in tongue squamous cell carcinoma by quantum dots

Quantum dots (QDs) are a new class of fluorescent probes to detect biomarker expression. The role of caveolin-1 (Cav-1) in tongue squamous cell carcinoma (TSCC) is still unknown. This study aimed to investigate the expression profile of Cav-1 in carcinogenesis and development of TSCC by QDs immunofluorescence histochemistry (QDs-IHC) and discuss the relationship between the Cav-1 expression and the clinicopathological outcomes. QDs-IHC was used to detect Cav-1 expression in tissue microarrays including normal tongue mucosa (NTM; n=10), hyperplastic tongue mucosa (HTM; n=10), tongue pre-cancer lesions (TPL; n=15) and primary tongue squamous cell carcinoma (PTSCC; n=61). Correlations between the Cav-1 expression and clinicopathologic variables were evaluated statistically. Cells positive for Cav-1 were clearly detected and bright images were obtained in a fine, granular pattern at the cell membrane and cytoplasm using QDs-IHC. The rate of Cav-1 immunoreactivity increased progressively from NTM (0%), HTM (0%), TPL (36%) to PTSCC (74%). When compared with each other, there was statistical significance among PTSCC, TPL and NTM as well as among PTSCC, TPL and HTM. Moreover, Cav-1 expression level in PTSCC was correlated positively with clinical stage and histologic grade. QDs-IHC could accurately detect protein location in tongue mucosa. An increased expression of Cav-1 in the stepwise carcinogenesis from NTM, HTM, TPL to PTSCC suggested that Cav-1 might be an oncogene in the development of tongue squamous cell carcinoma.

PH-Sensitive Fluorescent Lipids as Novel Probes to Monitor Vesicle Recycling

During synaptic transmission, neurotransmitters stored in presynaptic vesicles are released by excocytosis through fusion of vesicles with the plasma membrane. In a subsequent step, membranes and proteins at the synapse are reinternalized by a reverse process, endocytosis. In the analysis of this synaptic vesicle cycle genetically encoded pH-sensitive fluorescent proteins like the GFP-derivative pHluorin have become indispensable tools. These probes are capable of detecting changes in pH that accompany excocytosis and subsequent reacidification of endocytosed vesicles. Here we describe a new class of fluorescent probes, based on pH-sensitive organic dyes coupled to phospholipids, as promising alternative to genetically encoded fluorescent proteins like pHluorin. Moreover the pH-dependent fluorescence properties of these dyes are opposite to those of pHluorin. In hippocampal neurons, cell membranes can be stained in a pH dependent manner, and upon quenching of the fluorescence at the plasma membrane by a slightly basic pH, vesicle recycling can be monitored yielding fluorescence transients with kinetics mirroring those of the well characterized pHluorin signal. Furthermore, this approach can be used to study vesicle recycling in acute preparations like bipolar cells of the retina, where application of genetically encoded probes was not possible so far. This experimental approach using pH-dependent fluorescent lipids has not only the potential of being used in a variety of cellular and slice preparations, but in addition will shed light on an important presynaptic mechanism neglected so far, namely lipid recycling. Comparison of vesicle incorporation of different dye-labeled lipid moieties will bring new insights into lipid organisation and trafficking at the synapse.

A novel carbazole-based fluorescent probe: 3,6-Bis-[( N-ethylcarbazole-3-yl)-propene-1-keto]- N-ethylcarbazole

A novel carbazole-based compound 5, 3,6-bis[( N-ethylcarbazole-3-yl)-propene-1-keto]- N-ethylcarbazole has been designed, synthesized and characterized. The absorption and fluorescence spectra in solvents of different polarities prove that the compound has a distinct intramolecular charge transfer character. Compound 5 can be used as a new class of fluorescent probe or biosensor due to its sensitivity to the local microenvironment such as solvent polarity.

Immunolabeling and NIR-Excited Fluorescent Imaging of HeLa Cells by Using NaYF4:Yb,Er Upconversion Nanoparticles

Upconversion fluorescent nanoparticles can convert a longer wavelength radiation (e.g., near-infrared light) into a shorter wavelength fluorescence (e.g., visible light) and thus have emerged as a new class of fluorescent probes for biomedical imaging. Rare-earth doped beta-NaYF(4):Yb,Er upconversion nanoparticles (UCNPs) with strong UC fluorescence were synthesized in this work by using a solvothermal approach. The UCNPs were coated with a thin layer of SiO(2) to form core-shell nanoparticles via a typical Stober method, which were further modified with amino groups. After surface functionalization, the rabbit anti-CEA8 antibodies were covalently linked to the UCNPs to form the antibody-UCNP conjugates. The antibody-UCNP conjugates were used as fluorescent biolabels for the detection of carcinoembryonic antigen (CEA), a cancer biomarker expressed on the surface of HeLa cells. The successful conjugation of antibody to the UCNPs was found to lead to the specific attachment of the UCNPs onto the surface of the HeLa cells, which further resulted in the bright green UC fluorescence from the UCNP-labeled cells under 980 nm near-infrared (NIR) excitation and enabled the fluorescent imaging and detection of the HeLa cells. These results indicate that the amino-functionalized UCNPs can be used as fluorescent probes in cell immunolabeling and imaging. Because the UCNPs can be excited with a NIR light to exhibit strong visible fluorescence and the NIR light is safe to the body and can penetrate tissue as deep as several inches, our work suggests that, with proper cell-targeting or tumor-homing peptides or proteins conjugated, the NaYF(4):Yb,Er UCNPs can find potential applications in the in vivo imaging, detection, and diagnosis of cancers.

The environmental influence on the photoluminescence behavior of thiol-capped CdTe quantum dots in living cells

Water-soluble colloidal semiconductor quantum dots (QDs) as a new class of fluorescent probes with excellent optical properties have attracted considerable attention in bioapplications. Because of the large surface-to-volume ratio of QDs, especially in the case of QDs passivated with organic ligands, the photoluminescence (PL) of QDs is sensitive to the environmental conditions. Generally, the PL behavior of intracellular QDs is more complicated than that in solutions due to the complex cellular environment. In this review, the environmental influence on the PL behavior of intracellular thiol-capped CdTe QDs, such as the time-dependent spectral blueshift, photobleaching and lifetime shortening, is introduced and its mechanisms are discussed.

Emission under Hypoxia: One‐Electron Reduction and Fluorescence Characteristics of an Indolequinone–Coumarin Conjugate

A characteristic feature of the reactivity of indolequinone derivatives, substituents of which can be removed by one-electron reduction under hypoxic conditions, was applied to the development of a new class of fluorescent probes for disease-relevant hypoxia. A reducing indolequinone parent molecule conjugated with fluorescent coumarin chromophores could suppress efficiently the fluorescence emission of the coumarin moieties by an intramolecular electron-transfer quenching mechanism and a conventional internal-filter effect. Under hypoxic conditions, however, the conjugate, denoted IQ-Cou, underwent a one-electron reduction triggered by X irradiation or the action of a reduction enzyme to release a fluorescent coumarin chromophore, whereupon an intense fluorescence emission with a maximum intensity at 420 nm was observed. The one-electron reduction of IQ-Cou was suppressed by molecular oxygen under aerobic conditions. IQ-Cou also showed intense fluorescence in a hypoxia-selective manner upon incubation with a cell lysate of the human fibrosarcoma cell line HT-1080. The IQ-Cou conjugate has several unique properties that are favorable for a fluorescent probe of hypoxia-specific imaging.

Fluorescence Analysis with Quantum Dot Probes for Hepatoma Under One- and Two-Photon Excitation

A new class of fluorescent probe produced by conjugating semiconductor quantum dots (QDs) with protein molecule is proposed as an alternative to conventional organic labels. However the fluorescence characteristics of the QD bioconjugates are not clear while they are excitied with one- or two-photon laser pulse. We synthesized specific immunofluorescent probes by linking QDs to alpha fetoprotein (AFP) antibody for specific binding alpha-fetoprotein -an important marker for hepatocellular carcinoma cell lines, and archived specific fluorescence detection with the QDs-Anti-AFP in nude mice. Then, we have analyzed the fluorescence characteristics of QDs-Anti-AFP and original QDs both under one- and two-photon excitations. The results demonstrated that QDs-Anti-AFP's fluorescent spectral and lifetime haven't varied much from that of original QDs. Moreover, QDs-Anti-AFP have exhibited higher fluorescence efficiency than QDs under two-photon examination.

Boronate-Based Fluorescent Probes for Imaging Cellular Hydrogen Peroxide

The syntheses, properties, and biological applications of the Peroxysensor family, a new class of fluorescent probes for hydrogen peroxide, are presented. These reagents utilize a boronate deprotection mechanism to provide high selectivity and optical dynamic range for detecting H2O2 in aqueous solution over similar reactive oxygen species (ROS) including superoxide, nitric oxide, tert-butyl hydroperoxide, hypochlorite, singlet oxygen, ozone, and hydroxyl radical. Peroxyresorufin-1 (PR1), Peroxyfluor-1 (PF1), and Peroxyxanthone-1 (PX1) are first-generation probes that respond to H2O2 by an increase in red, green, and blue fluorescence, respectively. The boronate dyes are cell-permeable and can detect micromolar changes in H2O2 concentrations in living cells, including hippocampal neurons, using confocal microscopy and two-photon microscopy. The unique combination of ROS selectivity, membrane permeability, and a range of available excitation/emission colors establishes the potential value of PR1, PF1, PX1, and related probes for interrogating the physiology and pathology of cellular H2O2.

In vivo molecular and cellular imaging with quantum dots

Quantum dots (QDs), tiny light-emitting particles on the nanometer scale, are emerging as a new class of fluorescent probe for in vivo biomolecular and cellular imaging. In comparison with organic dyes and fluorescent proteins, QDs have unique optical and electronic properties: size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescence colors. Recent advances have led to the development of multifunctional nanoparticle probes that are very bright and stable under complex in vivo conditions. A new structural design involves encapsulating luminescent QDs with amphiphilic block copolymers and linking the polymer coating to tumor-targeting ligands and drug delivery functionalities. Polymer-encapsulated QDs are essentially nontoxic to cells and animals, but their long-term in vivo toxicity and degradation need more careful study. Bioconjugated QDs have raised new possibilities for ultrasensitive and multiplexed imaging of molecular targets in living cells, animal models and possibly in humans.

Molecular beacons: fluorogenic probes for living cell study.

Molecular beacons are a new class of fluorescent probes that can report the presence of specific nucleic acids with high sensitivity and excellent specificity. In addition to their current wide applications in monitoring the progress of polymerase chain reactions, their unique properties make them promising probes for the detection and visualization of target biomolecules in living cells. This article is focused on our recent research in exploring the potential of using molecular beacon for living-cell studies in three important areas: the monitoring of mRNA in living cells, the development of ultrasmall DNA/RNA biosensors, and the novel approach of combining molecular beacon's signal transduction mechanism with aptamer's specificity for real-time protein detection. These applications demonstrate molecular beacon's unique properties in bioanalysis and bioassay development.

Paper alert: Biotechnology

Quantum dots (QDs), tiny light-emitting particles on the nanometer scale, are emerging as a new class of fluorescent probe for in vivo biomolecular and cellular imaging. In comparison with organic dyes and fluorescent proteins, QDs have unique optical and electronic properties: size-tunable light emission, improved signal brightness, resistance against photobleaching, and simultaneous excitation of multiple fluorescence colors. Recent advances have led to the development of multifunctional nanoparticle probes that are very bright and stable under complex in vivo conditions. A new structural design involves encapsulating luminescent QDs with amphiphilic block copolymers and linking the polymer coating to tumor-targeting ligands and drug delivery functionalities. Polymer-encapsulated QDs are essentially nontoxic to cells and animals, but their long-term in vivo toxicity and degradation need more careful study. Bioconjugated QDs have raised new possibilities for ultrasensitive and multiplexed imaging of molecular targets in living cells, animal models and possibly in humans.

Spectroscopic Measurements of Temperature with Uranyl Compounds

A new class of fluorescent probes for measuring temperature is described. The intensities of the strong fluorescence bands of uranyl acetate, uranyl nitrate hexahydrate, and uranyl orthophosphate were recorded when the uranyl compound was illuminated with an argon-ion laser operating at 488 nm. The intensity ratio of two fluorescence bands of uranyl acetate at room temperature were determined for five freshly prepared samples and shown to be independent of the intensity of the laser excitation and optical alignment. The intensities of the fluorescence bands lying between 500 and 570 nm were measured at various temperatures ranging from 77 to 295 K. The ratio of the intensities of two fluorescence bands was calculated and shown to vary linearly with temperature. A linear fit provided the slope. The largest positive slope of all the intensity ratios associated with uranyl acetate was 6.02 × 10−3 K−1, that associated with uranyl orthophosphate was 4.68 × 10−3 K−1, and that associated with uranyl nitrate hexahydrate was 1.81 × 10−3 K−1. The potential of uranyl compounds as a new group of temperature probes is discussed.

Characterization of a Series of Far Red Absorbing Perylene Diones: A New Class of Fluorescent Probes for Biological Applications

The far red region of the spectrum is increasingly being utilised in many applications in the biosciences. However, apart from the cyanine group of dyes, there are relatively few far red fluorescent probes available which are of practical use. We have synthesised and characterised a new class of far red fluorescent probes based on the perylene dione chromophore. The 2,10 di-substituted perylene diones possess broad absorption spectra (>90 nm bandwidths), large Stokes shifts (>60 nm) and quantum yields of up to 0.5 with a maximum absorption at 610–640 nm in organic solvents or in solutions of non-ionic detergents. A number of derivatives have been synthesised that can be used as membrane probes, as chromogenic substrates for alkaline phosphatase, and for the labelling of macromolecules such as proteins and DNA. These novel far red fluorophores have potential applications in diagnostic and research applications.

Dibutyltin‐3‐hydroxyflavone bromide: A fluorescent inhibitor of F1F0‐ATPase

AbstractDibutyltin‐3‐hydroxyflavone bromide [Bu2SnBr‐(of)] is a fluorescent inhibitor (excitation max, 395 nm; emission max., 450 nm) of mitochondrial F1F0–ATPase which does not inhibit F1–ATPase. Bu2SnBr(of) binding to mitochondria and submitochondrial particles results in a 10‐fold fluorescence enhancement which correlates with the amount of F1F0–ATPase in the inner membrane. Enhancement is not affected by respiratory‐chain substrates, ATP, uncoupling agents, ionophores or respiratory‐chain inhibitors. It is reversed by tributyltin chloride (Bu3SnCl), indicating competition for a common triorganotin‐binding site on the F0 segment of F1F0–ATPase. Enhancement is not reversed by dialkyltins, monoalkyltins, tributyl‐lead acetate, efrapeptin or oligomycin. Bu2SnBr(of) is thus a new class of fluorescent probe of the F0 segment of F1F0–ATPase which titrates F0.

Organotin-flavone complexes: A new class of fluorescent probes for F 1F 0ATPase

Fluorescent 5-coordinate organotin-flavone complexes of 3-hydroxyflavone (Hof) and 3,5,7,2′,4′,-pentahydroxyflavone (morin) are good inhibitors of mitochondrial F 1F 0ATPase but do not inhibit F 1-ATPase and they have been examined as possible fluorescent probes of F 1F 0ATPase. R 2SnX (morin) complexes exhibit low fluorescence enhancement on binding to mitochondrial membranes with no displacement by equimolar tributyltin. In contrast R 2SnX (of) complexes exhibit high fluorescence enhancement whose extent is variable and is displacable by equimolar tributyltin. Fluorescence enhancement by R 2SnX (of) complexes correlates with the ATPase I 50 values. Dialkyltin-3-hydroxy flavone, R 2SnX(of), complexes act as a new class of fluorescent probes which titrate the F 0 segment of F 1F 0ATPase.