Efficient Fluorophores Research Articles

Fluorescence of Size-Expanded DNA Bases: Reporting on DNA Sequence and Structure with an Unnatural Genetic Set

We recently described the synthesis and helix assembly properties of expanded DNA (xDNA), which contains base pairs 2.4 A larger than natural DNA pairs. This designed genetic set is under study with the goals of mimicking the functions of the natural DNA-based genetic system and of developing useful research tools. Here, we study the fluorescence properties of the four expanded bases of xDNA (xA, xC, xG, xT) and evaluate how their emission varies with changes in oligomer length, composition, and hybridization. Experiments were carried out with short oligomers of xDNA nucleosides conjugated to a DNA oligonucleotide, and we investigated the effects of hybridizing these fluorescent oligomers to short complementary DNAs with varied bases opposite the xDNA bases. As monomer nucleosides, the xDNA bases absorb light in two bands: one at approximately 260 nm (similar to DNA) and one at longer wavelength ( approximately 330 nm). All are efficient violet-blue fluorophores with emission maxima at approximately 380-410 nm and quantum yields (Phifl) of 0.30-0.52. Short homo-oligomers of the xDNA bases (length 1-4 monomers) showed moderate self-quenching except xC, which showed enhancement of Phifl with increasing length. Interestingly, multimers of xA emitted at longer wavelengths (520 nm) as an apparent excimer. Hybridization of an oligonucleotide to the DNA adjacent to the xDNA bases (with the xDNA portion overhanging) resulted in no change in fluorescence. However, addition of one, two, or more DNA bases in these duplexes opposite the xDNA portion resulted in a number of significant fluorescence responses, including wavelength shifts, enhancements, or quenching. The strongest responses were the enhancement of (xG)n emission by hybridization of one or more adenines opposite them, and the quenching of (xT)n and (xC)n emission by guanines opposite. The data suggest multiple ways in which the xDNA bases, both alone and in oligomers, may be useful as tools in biophysical analysis and biotechnological applications.

Synthesis and Fluorescence Behavior of Cation Chemosensor Based on Azacrown Ether Fluoroionophore Carrying Styrylanthracene

Recently, the development of chemosensor associated with fluoroionophore has attracted great attention. Fluoroionophore is a compound combining the ionophore and fluorophore, where ionophore binds selectively with a specific ion and fluorophore generates a fluorescence signal upon ion binding. Depending on the nature of fluorophore-ionophore interaction in fluoroionophore, fluorescence properties of fluorophore such as fluorescence intensity or fluorescence wavelength maximum may be influenced by ion capture of ionophore. Sensitivity of fluorophore may be associated with the ionic recognition ability of the ionophore, if the fluorophore may be perturbed by the coordination of cation. The recent increase of interest in the synthesis of crown ether derivatives incorporating different fluoroionophores is recently due to their application as alkali metal ion sensors in biochemical analysis and medical diagnostics. In particular, azacrown ether, a typical ionophore, acts as an electron-rich donor as well as a ligand of the cation. The electron pair on the nitrogen heteroatom in electron-rich azacrown ether could be transferred towards electron deficient fluorophore in a π-conjugated D-A arrangement, through the charge transfer interaction between ionophore, an electron donor, and fluorophore, an electron acceptor. The efficient intramolecular charge transfer takes part in the modification of the first electronic transition state of the fluorophore and may lead to weaken the fluorescence from fluorophore. Upon binding of a cation into ionophore cavity, the electron pair on nitrogen heteroatom of ionophore is stabilized and the intramolecular charge transfer is inhibited. Therefore, the original electronic configuration of the unsubstituted fluorophore is recovered, so that fluorescence may be strengthened. Many fluoroionophores including stilbenyl-, aryl-, or styryl-crown ether derivatives containing a stilbene, styrene or aryl moiety as a fluorophore and a crown ether/azacrown ether moiety as an ionophore have been investigated. However, the main disadvantage of these fluoroionophore is the relatively small changes in fluorescence intensity upon cation binding. Incorporation of more efficient fluorophore such as anthracene into fluoroionophore may lead to more remarkable change of fluorescence signal depending on the cation binding. Recent work has concerned the binding of various fluorophores to the N-phenylaza-15-crown-5 and their spectroscopic and complexation properties. This paper concerns the synthesis of N-phenylaza-15-crown-5 derivative containing anthrylethene chromophore and the effect of the complexation with various alkali metal cations on the absorption and fluorescence behavior. We prepared N-[4-(2-anthracen-9-yl-vinyl)-phenyl]-aza15-crown-5 (1), a azacrown ether derivative containing 1-(9anthryl)-2-phenylethene (9-APE) moiety, by Wittig coupling between N-[4-formyl-phenyl]-aza-15-crown-5 and 9bromomethylanthracene. It is known that t-9-APE, a styrene derivative containing highly fluorescent anthracene, shows fairly strong fluorescence (Φf = 0.45, λf = 476 nm in acetonitrile), coming from the locally excited state which the excitation energy is localized on anthracene ring. For 1-(9-anthryl)-2-(4-methoxyphenyl)ethene (MeO-APE) containing an electron donating substituent methoxy group, fluorescence becomes weak and is red-shifted (Φf = 0.028, λf = 494 nm in acetonitrile) due to the contribution of the intramolecular

N-Dansyl-carbazoloquinone; a chemical and electrochemical fluorescent switch

A new ‘push–pull’ molecule having an efficient fluorophore (dansyl) in electronic communication with an active redox quencher (phenyl-carbazoloquinone) through an NH-bridge was designed and synthesized. This all-organic molecule is suggested as a highly reversible ‘on/off’ molecular switching system. Chemical and electrochemical inter-conversion between the quinone acceptor and the dansyl donor were demonstrated via UV–vis, cyclic voltammetry and fluorescence measurements.

Reactions and luminescence in passivated Si nanocrystallites induced by vacuum ultraviolet and soft-x-ray photons

Alkyl-modified silicon nanocrystallites are efficient fluorophores which are of interest for fundamental spectroscopic studies and as luminescent probes in biology because of their stability in aqueous media. In this work we have investigated these particles using scanning tunneling microscopy, synchrotron-radiation excited photoemission, and x-ray excited optical luminescence (XEOL). During the course of illumination with 145-eV photons we have monitored the evolution of the Si2p core level and, in samples which have suffered prolonged atmospheric exposure, observed in real time the growth of an extra Si2p component attributed to in situ photoinduced oxidation of the Si nanocrystallites. XEOL reveals that two emission bands are active upon soft-x-ray photon excitation and that photoluminescence intensity decreases with photon exposure, which is attributed to charge trapping within the film.

Novel cyanine-AMP conjugates for efficient 5' RNA fluorescent labeling by one-step transcription and replacement of [ -32P]ATP in RNA structural investigation

Two novel fluorescent cyanine-AMP conjugates, F550/570 and F650/670, have been synthesized to serve as transcription initiators under the T7 φ2.5 promoter. Efficient fluorophore labeling of 5′ RNA is achieved in a single transcription step by including F550/570 and F650/670 in the transcription solution. The current work makes fluorescently labeled RNA readily available for broad applications in biochemistry, molecular biology, structural biology and biomedicine. In particular, site-specifically fluorophore-labeled large RNAs prepared by the current method may be used to investigate RNA structure, folding and mechanism by various fluorescence techniques. In addition, F550/570 and F650/670 may replace [γ-32P]ATP to prepare 5′ labeled RNA for RNA structural and functional investigation, thereby eliminating the need for the unstable and radio-hazardous [γ-32P]ATP.

Chemiluminescence from the oxidation of urea and ammonia with hypobromite and N-bromosuccinimide

The spectral distribution for the chemiluminescent oxidation of ammonia with hypobromite is significantly different to that for the oxidation of ammonia with N-bromosuccinimide. Therefore, in contrast to the assumptions of several authors, the action of N-bromosuccinimide is not solely derived from the in situ formation of hypobromite. Neither the oxidation of urea with hypobromite nor the oxidation of urea with N-bromosuccinimide involves an initial hydrolysis of urea to ammonia in the alkaline solution. However, these two reactions lead to a common emitter. The addition of xanthene dyes, such as dichlorofluorescein, enhance the chemiluminescence intensity by energy transfer to the efficient fluorophore, but reaction between the sensitiser and hypobromite can result in a significant increase in the background signal. A list of potential interferences has been compiled; particular attention was paid to guanidino compounds, as the chemiluminescence accompanying the oxidation of this functional group has not been previously discussed.

Chemiluminescence from the oxidation of urea and ammonia with hypobromite and N-bromosuccinimide

The spectral distribution for the chemiluminescent oxidation of ammonia with hypobromite is significantly different to that for the oxidation of ammonia with N-bromosuccinimide. Therefore, in contrast to the assumptions of several authors, the action of N-bromosuccinimide is not solely derived from the in situ formation of hypobromite. Neither the oxidation of urea with hypobromite nor the oxidation of urea with N-bromosuccinimide involves an initial hydrolysis of urea to ammonia in the alkaline solution. However, these two reactions lead to a common emitter. The addition of xanthene dyes, such as dichlorofluorescein, enhance the chemiluminescence intensity by energy transfer to the efficient fluorophore, but reaction between the sensitiser and hypobromite can result in a significant increase in the background signal. A list of potential interferences has been compiled; particular attention was paid to guanidino compounds, as the chemiluminescence accompanying the oxidation of this functional group has not been previously discussed.

Synthesis and characterization of novel dipyridylbenzothiadiazole and bisbenzothiadiazole derivatives.

Novel dipyridyl compounds containing a mono- and bisbenzothiadiazole unit were synthesized using the Stille coupling reaction. Their pyridinium salts, viologen analogues, were also prepared by the N-alkylation. The X-ray crystallographic analysis of the compounds containing a benzothiadiazole ring revealed nonplanar molecular structures and unique crystal structures depending on the nitrogen positions. The dipyridyl compounds are efficient fluorophores with high electron affinity. The derivative afforded complexes with chloranilic acid and cyanuric acid composed of hydrogen bonding networks. The methyl viologen analogues showed two-stage one-electron reduction waves.

Toward a new genetic system with expanded dimensions: size-expanded analogues of deoxyadenosine and thymidine.

We describe the design, preparation, and properties of two key building blocks of a size-expanded genetic system. Nucleoside analogues of the natural nucleosides dA and dT are reported in which the fusion of a benzo ring increases their size by ca. 2.4 A. The expanded dA analogue (dxA), having a tricyclic base, was first reported by Leonard nearly three decades ago. We describe a shortened and more efficient approach to this compound. The expanded dT analogue (dxT), a methylquinazolinedione C-glycoside, was previously unknown; we describe its preparation in eight steps from 5-methylanthranilic acid. The key glycoside bond formation employed Pd-mediated coupling of an aryl iodide precursor with a dihydrofuran derivative of deoxyribose. Both nucleosides are shown to be efficient fluorophores, emitting light in the blue-violet range. The base-protected phosphoramidite derivatives were prepared, and short oligonucleotides containing them were characterized. The two size-expanded nucleosides are key components of a new four-base genetic system designed to form helical paired structures having a diameter greater than that of natural DNA. Elements of the design of this expanded genetic molecule, termed xDNA, are discussed, including the possibility of up to eight base pairs of information storage capability.

Real-time detection of PAH mixtures in the vapor phase at high temperatures

Laser-induced fluorescence (LIF) spectroscopy can serve as a rapid analytical method for the quantitative analysis of polycyclic aromatic hydrocarbons (PAHs). The high quantum yields of this class of compounds, along with large absorption cross-sections, make fluorescence a very attractive technique for trace analysis determination of PAHs. Furthermore, fluorescence can provide in situ and online information regarding the composition and concentration of PAHs formed during combustion or pyrolysis processes. In this paper, we utilize a fiber-optic probe coupled to a spectrometer to collect fluorescence spectra of several PAH vapors at elevated temperatures using excitation at 337 nm. We have collected the fluorescence of standards as well as complex mixtures of PAHs at 300 °C. The expected band broadening induced by temperature effects was observed, but did not compromise the spectrum signature for each PAH studied. We have measured fluorescence spectra of benzo[a]pyrene, pyrene, anthracene, and phenanthrene vapors. Determination of trace amounts of the toxicologically important benzo[a]pyrene has been shown to be feasible. Experimental results show that at 337 nm, benzo[a]pyrene is the most quantum efficient fluorophore among the PAHs studied. Furthermore, fluorescence lifetime measurements were found to be helpful in the characterization of PAH mixtures.

ChemInform Abstract: Proquinoid Acceptors as Building Blocks for the Design of Efficient π-Conjugated Fluorophores with High Electron Affinity.

ChemInform Abstract: Proquinoid Acceptors as Building Blocks for the Design of Efficient π-Conjugated Fluorophores with High Electron Affinity.

Proquinoid acceptors as building blocks for the design of efficient π-conjugated fluorophores with high electron affinity

The association of aromatic electron donor groups with proquinoid acceptors leads to π-conjugated fluorophores combining tunable emission wavelength at constant geometry, high fluorescence efficiency and high electron affinity.

Three-dimensional optical circuitry using two-photon-assisted polymerization

An approach using an as-formed solid bulk sample containing a blend of photocurable and thermally curable epoxies together with a highly efficient two-photon excitable fluorophore is demonstrated for in situ fabrication of three-dimensional optical circuitry with a precise control of dimension and spatial locations. Confocal volume images of a 1×2 splitter and a grating coupler are shown. End-fire coupling of a He–Ne laser beam into the 1×2 splitter has been achieved.

Three Dimensional Optical Data Storage Using Two-Photon Induced Photobleaching in a Polymer Matrix

The intensity of two-photon induced fluorescent shows quadratic dependency to the excitation intensity, as a result, one can achieve optical sectioning in fluorescent microscopy without the use of a confocal aperture. When the excitation power is properly adjusted, the fluorescent and photobleached volume is restricted to the vicinity of the focal points; therefore, it is possible to use this mechanism to achieve 3D data storage and microstructure fabrication (stereolithography).The recording medium used in this experiment was poly-HEMA block doped with a high efficient two-photon fluorophore, APSS, (4-[N-(2-hydroxyethyl)-N-methyl) amino phenyl]-4’-(6-hydroxyhexyl sulfonyl) stilbene). The block was made by dissolving APSS in HEMA with an initiator, 2,2’-azobisisobutyronitril (AiBN) (1% mole ratio) and polymerized at 60° C for 48 hours. The polymer was then trimmed into a small rectangular block (3×5×1 mm3) and surfaced by using a ultramicrotome with a glass knife. A modified Biorad MC500 LSM equipped with a home-made computer controlled stage scanner was used for both writing (stage scanning) and read-back (beam scanning).

Three-dimensional laser scanning two-photon fluorescence confocal microscopy of polymer materials using a new, efficient upconverting fluorophore.

Three-dimensional confocal imaging of polymer samples was achieved by the use of two-photon excited fluorescence in both positive and negative contrast modes. The fluorophore was a new and highly efficient two-photon induced upconverter, resulting in improved signal strength at low pumping power. Because of the relatively long wavelength of the excitation source (798 nm from a mode-locked Ti:Sapphire laser), this technique shows a larger penetration depth into the samples than provided by conventional single-photon fluorescence confocal microscopy. Single-photon and two-photon images of the same area of each sample show significant differences. The results suggest the possibility of using two-photon confocal microscopy, in conjunction with highly efficient fluorophores, as a tool to study the surface, interface, and fracture in material science applications.

Properties of singlet excited N-(pyrimidin-2-one-4-yl)pyridinium chloride and structurally related organic cations

The UV absorption and fluorescence spectra, fluorescence quantum yields and lifetimes of four new fluorescent N-(pyrimidin-2-one-4-yl)pyridinium and N-(pyrimidin-2-one-4-yl)methylimidazolium cations were measured in several solvents at room temperature and in ethanol glasses at 77 K. The spectroscopic studies were supplemented by theoretical calculations of the electronic transition energies, oscillator strengths and dipole moment changes by the INDO/S CI method. It is concluded that the lowest excited singlet state of the compounds studied has charge transfer character. The significantly different charge distribution in the S 1 state and the rotational relaxation of the excited molecules are responsible for the large Stokes shift observed in the spectra of pyridinium cations. N-(Pyrimidin-2-one-4-yl)methylimidazolium cations are more efficient fluorophores than pyridinium cations and this is due to the increased rate of non-radiative decay in the latter compounds.

A condensed thiadiazolo-pyrimidine as a new efficient fluorophore. Theoretical and experimental investigation of the electronic spectra and photophysics

A recently synthesized condensed thiadiazolo-pyrimidine (MTP) has been found to exhibit a very intense fluorescence emission in aprotic solvents. The origins of such an unusual property for a compound containing many different heteroatoms (four nitrogens, two sulphurs and one oxygen) have been investigated by a combined theoretical and experimental approach. The nature and peculiarity of the MTP chromophore have been analysed using both computed electron densities and crystallographic data from the literature. The singlet and triplet spectra have been measured and calculated, and a good agreement has been found between the two sets of results. On this basis, we have attempted to interpret the rather peculiar photophysical behaviour of MTP.

Erythrosin as energy acceptor in a biphasic chemiluminescent system for glucose oxidase detection

Chemiluminescence from the reaction of bis(2,4,6-trichlorophenyl) oxalate (TCPO) with hydrogen peroxide in the presence of erythrosin as fluorescer was investigated as a suitable means of determining glucose oxidase, with the aim of enzyme immunoassay applications. The chemiluminescence intensity is measured in the organic phase of a liquid biphasic system where TCPO is dissolved in ethyl acetate, whereas hydrogen peroxide and erythrosin are contained in an underlying aqueous buffer solution. Erythrosin has the advantage over various other efficient fluorophores of being stable when stored in aqueous solution. Moreover, it was found that cetyltrimethylammonium bromide strongly enhances the emission, thus improving the sensitivity of hydrogen peroxide detection about 200-fold.

Fluorescence digital imaging microscopy in cell biology.

Developments in microscope, sensor, and image-processing technologies have led to integrated systems for the quantification of low-light-level emission signals from biological samples. Specificity is provided in the form of monoclonal antibodies and other ligands or enzyme substrates conjugated with efficient fluorophores. Fluorescent probes are also available for cellular macromolecular constituents and for free ions of biological interest such as H+ and Ca2+. The entire spectrum of photophysical phenomena can be exploited. Representative data are presented from studies of DNA conformation and architecture in polytene chromosomes and from studies of receptor-mediated endocytosis, calcium distribution, and the organization of the contractile apparatus in muscle cells.