Oxazine Dyes Research Articles

Oxazine Dye-Conjugated DNA Oligonucleotides: Förster Resonance Energy Transfer in View of Molecular Dye–DNA Interactions

In this work, the photophysical properties of two oxazine dyes (ATTO 610 and ATTO 680) covalently attached via a C6-amino linker to the 5'-end of short single-stranded as well as double-stranded DNA (ssDNA and dsDNA, respectively) of different lengths were investigated. The two oxazine dyes were chosen because of the excellent spectral overlap, the high extinction coefficients, and the high fluorescence quantum yield of ATTO 610, making them an attractive Förster resonance energy transfer (FRET) pair for bioanalytical applications in the far-red spectral range. To identify possible molecular dye-DNA interactions that cause photophysical alterations, we performed a detailed spectroscopic study, including time-resolved fluorescence anisotropy and fluorescence correlation spectroscopy measurements. As an effect of the DNA conjugation, the absorption and fluorescence maxima of both dyes were bathochromically shifted and the fluorescence decay times were increased. Moreover, the absorption of conjugated ATTO 610 was spectrally broadened, and a dual fluorescence emission was observed. Steric interactions with ssDNA as well as dsDNA were found for both dyes. The dye-DNA interactions were strengthened from ssDNA to dsDNA conjugates, pointing toward interactions with specific dsDNA domains (such as the top of the double helix). Although these interactions partially blocked the dye-linker rotation, a free (unhindered) rotational mobility of at least one dye facilitated the appropriate alignment of the transition dipole moments in doubly labeled ATTO 610/ATTO 680-dsDNA conjugates for the performance of successful FRET. Considering the high linker flexibility for the determination of the donor-acceptor distances, good accordance between theoretical and experimental FRET parameters was obtained. The considerably large Förster distance of ~7 nm recommends the application of this FRET pair not only for the detection of binding reactions between nucleic acids in living cells but also for monitoring interactions of larger biomolecules such as proteins.

Synthesis of Near-IR Fluorescent Oxazine Dyes with Esterase-Labile Sulfonate Esters

Near-IR oxazine dyes are reported that contain sulfonate esters which are rapidly cleaved by esterase activity to unmask highly polar anionic sulfonates. Strategies for the synthesis of these dyes included the development of milder dye condensation conditions with improved functional compatibility and the use of an alkyl halide that allows for the introduction of esterase-labile sulfonates without the need for sulfonation of the target molecule.

Molecular interaction of oxazine dyes in aqueous solution: Temperature dependent molecular disposition of the aggregates

Temperature dependent molecular interaction of oxazine dyes, viz., brilliant cresyl blue (C.I. Basic dye), cresyl violet (C.I. Basic violet 3) and nile blue (C.I. Basic blue 12) are studied in aqueous media within a concentration range of 5.0 × 10 − 6 M to 8.0 × 10 − 4 M by UV–visible absorption spectroscopy. The effect of temperature on the geometrical structure of the dimer in solution along with the dimerization equilibria is explained in terms of electrostatic and hydrophobic interactions. Modified non-covalent interaction between two monomer molecules in a dimer as a function of temperature affects the extinction coefficient as well as the geometrical disposition of the dimers and this is well manifested in the exciton splitting of the dimer spectra. The angle θ between main oscillators of the two monomer molecules in a dimer increases by 1.94° for brilliant cresyl blue, whereas an increase of 4.32° and 1.73° were observed for cresyl violet and nile blue respectively due to the increase of temperature from 20 °C to 60 °C.

A comparative study on the aggregate formation of two oxazine dyes in aqueous and aqueous urea solutions

The visible absorption spectra of two oxazine dyes, brilliant cresyl blue and nile blue, in aqueous and aqueous urea solutions (0.01–1 M) were studied as a function of the dye concentration. Due to structural similarity of the investigated dyes, their spectral features were compared. The dimerization behavior of the dyes in water with or without urea was analyzed in terms of monomer–dimer equilibrium. The nature and structure of the interacting pairs for these dyes were discussed using the exciton theory. The dimerization constant values of the dyes in aqueous and aqueous urea solutions were calculated. For both the dyes in aqueous urea solutions, a reduction in dimer formation was observed. The excitonic parameters were compared with those of oxazine-1, which is structurally similar to the investigated dyes.

Predicting the UV–vis spectra of oxazine dyes

In the current work we have investigated the ability of time-dependent density functional theory (TD-DFT) to predict the absorption spectra of a series of oxazine dyes and the effect of solvent on the accuracy of these predictions. Based on the results of this study, it is clear that for the series of oxazine dyes an accurate prediction of the excitation energy requires the inclusion of solvent. Implicit solvent included via a polarizable continuum approach was found to be sufficient in reproducing the excitation energies accurately in the majority of cases. Moreover, we found that the SMD solvent model, which is dependent on the full electron density of the solute without partitioning into partial charges, gave more reliable results for our systems relative to the conductor-like polarizable continuum model (CPCM), as implemented in Gaussian 09. In all cases the inclusion of solvent reduces the error in the predicted excitation energy to <0.3 eV and in the majority of cases to <0.1 eV.

Fluorescence Correlation Spectroscopy of Fast Chain Dynamics within Denatured Protein L

Intramolecular dynamics in the denatured state of a protein are of importance for protein folding. Native-like contact formation within the ensemble of denatured conformations of a protein may guide its transformation towards the native conformation. The efficiency of folding is thus dependent on the diffusion of chain fragments, which facilitates contact formation. Herein we investigate intramolecular diffusion of denatured molecules of the small two-state-folding protein L with fluorescence correlation spectroscopy (FCS). We utilize the specific quenching of the fluorescence of the oxazine dye Atto655 labeling a cysteine at position 64 (the C-terminus of the protein) by the side chain of a tryptophan at position 47. FCS measurements allow us to probe processes ranging in timescales from tens of nanoseconds to seconds. Two fast photophysical processes can be distinguished in the fluorescence correlation curves. The slower of the two is found to be due to triplet dynamics, while the faster process is attributed to the quenching of the Atto655 by the tryptophan upon transient ground-state complex formation. We study the dependence of the intrachain dynamics of the denatured protein on the concentration of the denaturant guanidinium chloride (GdmCl), and extract complex association and dissociation rates. While the dissociation rate does not depend on the denaturant, the association rate decreases as denaturant concentration is increased from 3 to 7 M GdmCl. This decrease in contact formation rate tracks the expansion of denatured protein L, measured in our previous work. Thus, the intramolecular diffusion coefficient calculated from the results is found to be essentially independent of the denaturant concentration over this range, even as the protein expands by more than 20%.

In situ synthesis of fluorescent poly(norbornene)/oxazine-1 dye loaded fluoromica hybrids: supramolecular control over dye arrangement

We report on the in situ synthesis of fluorescent poly(norbornene)/oxazine-1 (Ox1) dye loaded fluoromica mineral Somasif (SME) hybrid materials. SME acts as host for supramolecular arrangement of Ox1 molecules in their 2D interlayer region. The dye arrangement is easily tuned by modulating the dye loading as well as by competitive intercalation with an ammonium surfactant. The dye molecules bend at most by 30° with respect to the SME layer surface increasing the dye loading up to 69% of the clay cation exchange capacity (CEC). This Ox1 state provides accessible interlayer voids which in turn facilitate the entry of polymerizable monomers (i.e., norbornene and ethylene) and the in situgrowth of the polymer. In this way a highly processable polymer hybrid, in which the dye molecules are highly oriented in the inorganic framework, is formed. The optical spectroscopy shows unique J-band characteristics along with the Ox1 monomer features. The formation of the J-aggregates is likely induced by the interlayer segregated poorly polar norbornene phase that forces the Ox1 molecules to increase the head-to-tail interaction between them. These features strongly encourage the technical application of these materials for fabricating functional devices with unique photoluminescence properties.

Long distance energy transfer in a polymer matrix doped with a perylene dye

Exciton migration over long distances is a key issue for various applications in organic electronics. We investigate a disordered material system which has the potential for long exciton diffusion lengths in combination with a high versatility. The perylene bisimide dye Perylene Red is incorporated in a polymer matrix with a high concentration. The dye molecules represent active sites with a narrow energy distribution for the electronically excited states. Excitons can be efficiently exchanged between them by Förster resonance energy transfer (FRET). The narrow energy distribution reduces drastically the trapping probability of the excitons compared to polymers and allows for long transfer distances. To characterize the mobility of the excitons and their diffusion length the dye Oxazine 1 is added as an acceptor in low concentration and the transfer probability to the acceptor is determined by measuring the reduction of Perylene Red fluorescence. The quenched quantum yield is measured for dye concentrations varying from 0.05M to 0.15 M for Perylene Red and from 0.3 mM to 3 mM for Oxazine 1. The experimental results are compared to a model which assumes that excitons can diffuse through the material by FRET between Perylene Red sites and are trapped at an acceptor with a final hetero FRET step. We find a quite good match between theory and experiment though the observed diffusion constant is about two times smaller than the calculated one. The exciton diffusion length extracted from the data is 30 nm for a Perylene Red concentration of 0.1 M and demonstrates that long distance energy transfer is possible in this disordered material system.

Controlling the Photoluminescence from a Laser Dye through the Oxidation Level of Polypyrrole

AbstractIn situ spectroelectrochemistry was used to study the emission of the laser dye oxazine 720 (oxa720) embedded into a polypyrrole (ppy) matrix. The photoluminescence (PL) of the dye was found to be controlled by the oxidation level of ppy. It is suggested that a Förster resonance energy transfer (FRET) mechanism is involved in the potential‐controlled PL. FRET calculations were used to determine the dependence of the apparent distance between the dye molecules and the polymer chain with the oxidation level of ppy. In the reduced state, at −0.9 V, an efficient FRET process quenches the PL from the dye due to the short distance between the dye molecules and the polymer chains. In the oxidized state, at +0.9 V, the FRET efficiency is only 24% leading to higher PL intensity, due to the larger distance between the donor (oxa720) and the acceptor (ppy).magnified image

Synthesis and Characterization of Oxazine-doped Silica Nanoparticles for Their Potential Use as Stable Fluorescent Reagents

The synthesis process to obtain silica nanoparticles (NPs) doped with two oxazine dyes, nile blue and cresyl violet, has been investigated using a modification of the reverse micelle microemulsion method and a procedure based on the Stöber method. A micellar medium provided by the non-ionic surfactant Triton X-100 in a hexanol:water mixture and an ethanol:water mixture, have been used to provide the synthesis medium in each case. Tetraethoxysilane has been used as the initiator of the polymerization and condensation reactions after its hydrolysis in basic medium using ammonium hydroxide. Dye-silane precursor NPs have been also synthesized in order to compare their potential advantages against the NPs obtained by the direct encapsulation of the oxazine dyes. Size distribution and fluorescence of the synthesized NPs, which were monitored using Transmision Electron Microscopy (TEM) and a microplate reader, respectively, depend on the molar ratio and total concentration of the reagents involved in the synthesis. NPs obtained using the developed synthesis procedures had sizes below 400 nm in most instances and the best luminescent properties were observed for NPs with sizes ranging from 100 to 300 nm. Lower sizes result in a decrease in the fluorescence intensities of these nanomaterials. Parameters related with the luminescence features of these NPs were calculated in order to compare the feasibility of both synthesis approaches. The repeatability of the reverse-micelle microemulsion procedure performed in different days gave a relative standard deviation of 10% for the fluorescence intensity values.

Nonlinear optical properties of two oxazine dyes in aqueous solution and polyacrylamide hydrogel using single beam Z-scan

The nonlinear optical properties of the two oxazine dyes, i.e. oxazine 720 (OX720) and oxazine 750 (OX750), in aqueous solution and in polyacrylamide hydrogel (PAA) matrix were studied by Z-scan technique using CW He–Ne laser at 632.8 nm. All the polymeric and aqueous samples showed a negative nonlinearity and large nonlinear refractive index of the order of 10 −8 cm 2/W. The concentration-dependent nonlinear refractive index was also investigated. In the case of PAA–dye systems, two types of transit (fast) and steady state (slow) nonlinear effects were observed. It is believed that these effects are due to the thermo-optical induced refractive index gradient and a concentration gradient induced by a temperature gradient present in a medium.

Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy

Fluorescent molecular switches have widespread potential for use as sensors, material applications in electro-optical data storages and displays, and superresolution fluorescence microscopy. We demonstrate that adjustment of fluorophore properties and environmental conditions allows the use of ordinary fluorescent dyes as efficient single-molecule switches that report sensitively on their local redox condition. Adding or removing reductant or oxidant, switches the fluorescence of oxazine dyes between stable fluorescent and nonfluorescent states. At low oxygen concentrations, the off-state that we ascribe to a radical anion is thermally stable with a lifetime in the minutes range. The molecular switches show a remarkable reliability with intriguing fatigue resistance at the single-molecule level: Depending on the switching rate, between 400 and 3,000 switching cycles are observed before irreversible photodestruction occurs. A detailed picture of the underlying photoinduced and redox reactions is elaborated. In the presence of both reductant and oxidant, continuous switching is manifested by "blinking" with independently controllable on- and off-state lifetimes in both deoxygenated and oxygenated environments. This "continuous switching mode" is advantageously used for imaging actin filament and actin filament bundles in fixed cells with subdiffraction-limited resolution.

Optical properties of molecular aggregates of oxazine dyes in dispersions of clay minerals

The molecular aggregation of oxazine 1 (Ox1) and oxazine 4 (Ox4) in reduced charge montmorillonite (RCM) colloids was investigated by absorption and fluorescence spectroscopies. The aggregation was significantly influenced by the structure of dye cations. Presence of four hydrophobic ethyl groups attached to the ammonium substituents in Ox1 cation prevented formation of closely packed sandwich-type assemblies (H-aggregates). Significant effect of the layer charge was observed for Ox4/RCMs dispersions. Large amounts of the Ox4 H-aggregates were formed in the systems with RCMs of the highest layer charge and reflected in quenched fluorescence. The presence of J-aggregates was proven by absorption spectra for the systems with Ox4 and low-charge RCMs. The flocculation of the lowest charge RCM colloids led to an extensive reduction of the luminescence. The trends and effects of the dye molecular structure and RCM properties are compared with the results previously published for other types of dyes.

Hybrid FMT–CT imaging of amyloid-β plaques in a murine Alzheimer's disease model

The need to study molecular and functional parameters of Alzheimer's disease progression in animal models has led to the development of disease-specific fluorescent markers. However, curved optical interfaces and a highly heterogeneous internal structure make quantitative fluorescence imaging of the murine brain a particularly challenging tomographic problem. We investigated the integration of X-ray computed tomography (CT) information into a state-of-the-art fluorescence molecular tomography (FMT) scheme and establish that the dual-modality approach is essential for high fidelity reconstructions of distributed fluorescence within the murine brain, as compared to conventional fluorescence tomography. We employ this method in vivo using a fluorescent oxazine dye to quantify amyloid-β plaque burden in transgenic APP23 mice modeling Alzheimer's disease. Multi-modal imaging allows for accurate signal localization and correlation of in vivo findings to ex vivo studies. The results point to FMT–CT as an essential tool for in vivo study of neurodegenerative disease in animal models and potentially humans.

The effect of chromophore-chromophore interaction on the spectral luminescent properties of oxazine dye dimers

Spectral and luminescent properties were studied for chemical dimers and molecular aggregates of Nile Red oxazine dye in solutions. Chemical bonding of two oxazine dye molecules leads to the formation of a dimer whose spectral behavior is in close agreement with the exciton model constructed for “physical” molecular aggregates. Chemical dimers luminesce, whereas dimers formed because of van der Waals interactions do not.

Vibrational Spectroscopy of Interfaces Based on Fourth- and Third-Order Time-Domain Raman Processes

Vibrational spectroscopy of interfaces was developed by taking advantage of fourth- and third-order Raman processes. An example making use of the fourth-order Raman process is interface-specific vibrational spectroscopy using only visible light. An oxazine dye at an air-solution interface was irradiated with visible 16-fs pump and probe pulses. The fourth-order signal as a function of the pump-probe delay represents the Raman-active nuclear motion. The Fourier transform of the motion provided the vibrational spectrum from 0 to 1000 cm-1 for the dye at the interface. An example making use of the third-order Raman process is vibrational spectroscopy of molecular films of submonolayer coverage. For a cresyl-violet dye of submonolayer coverage (4 × 1013 molecules cm-2) on a fused silica, the observation of time-resolved reflectance due to the third-order Raman process provided the sensitivity six times higher than in time-resolved transmittance. The vibrational spectrum of the dye at the interface was obtained from 0 to 1000 cm-1 by the Fourier transform of the reflection signal.

The photophysical properties of Nile red and Nile blue in ordered anisotropic media

The absorption and fluorescence spectra of a highly fluorescent laser dye, Nile red, were investigated in nematic solvents and isotropic liquids as a function of the solvent polarity and type. The interaction of the dye with the anisotropic surrounding and with that of the isotropic solvents was investigated and compared using optical spectroscopy. The spectral behavior of the cationic oxazine dye, Nile blue, was also investigated in anisotropic and isotropic environments for comparison. The spectral shifts were correlated by the solvent polarity and nature. The electro-optical effect of the guest–host systems was also investigated in an electro-optical system using polarized spectroscopic method and the dichroic ratios R and degree of anisotropies S of these dyes in the liquid crystalline hosts were investigated.

Electron Storage in Single Wall Carbon Nanotubes. Fermi Level Equilibration in Semiconductor–SWCNT Suspensions

The use of single wall carbon nanotubes (SWCNTs) as conduits for transporting electrons in a photoelectrochemical solar cell and electronic devices requires better understanding of their electron-accepting properties. When in contact with photoirradiated TiO(2) nanoparticles, SWCNTs accept and store electrons. The Fermi level equilibration with photoirradiated TiO(2) particles indicates storage of up to 1 electron per 32 carbon atoms in the SWCNT. The stored electrons are readily discharged on demand upon addition of electron acceptors such as thiazine and oxazine dyes (reduction potential less negative than that of the SWCNT conduction band) to the TiO(2)-SWCNT suspension. The stepwise electron transfer from photoirradiated TiO(2) nanoparticles --> SWCNT --> redox couple has enabled us to probe the electron equilibration process and determine the apparent Fermi level of the TiO(2)-SWCNT system. A positive shift in apparent Fermi level (20-30 mV) indicates the ability of SWCNTs to undergo charge equilibration with photoirradiated TiO(2) particles. The dependence of discharge capacity on the reduction potential of the dye redox couple is compared for TiO(2) and TiO(2)-SWCNT systems under equilibration conditions.

Simultaneous Measurement of Orientational and Spectral Dynamics of Single Molecules in Nanostructured Host−Guest Materials

Nanostructured host-guest materials are important for various applications in nanoscience, and therefore, a thorough understanding of the dynamics of the guest molecules within the host matrix is needed. To this aim we used single-molecule fluorescence techniques to simultaneously examine the spectral and the orientational behavior of single molecules in nanostructured porous host materials. Two types of host-guest systems have been investigated. First, oxazine-1 dye molecules were fixed rigidly in the channels of microporous AlPO4-5 crystals. Second, it was shown that terrylenediimide (TDI) dye molecules move in the mesoporous network of an uncalcined M41S thin film. In the first sample both spectral fluctuations ( approximately 5 nm) and rare spectral jumps (>10 nm) of the emission maximum were observed. However, the orientation of the emission dipole of the dye molecules remained constant. In contrast, the second system showed orientational dynamics as well as substantially more spectral dynamics. In this system the molecules were found to move between different regions in the host. The typical motion of the TDI molecules in the pores of M41S was not continuous but characterized by jumps between specific sites. Moreover, the spectral and orientational dynamics were correlated and arose directly from the different environments that were being explored by the mobile molecule.

Formation of oxazine dye by photochemical reaction of N-acyl oxazine derivatives

The synthesis and photochemical reaction mechanism of N-benzoyl oxazine derivatives have been investigated. The synthesized materials were found to be photosensitive and capable of generating oxazine dye upon light irradiation. The initial reactions were found to include homolytic bond cleavage between the oxazine nitrogen and benzoyl carbon atoms proceeding through the excited singlet state and the formation of oxazine and benzoyl radicals. Subsequent reaction steps include direct electron transfer between two oxazine radicals or disproportionation of the oxazine dimer species formed through the recombination of two oxazine radicals. These reactions lead to the formation of the oxazine dye cation and anion pair.