Free Dye In Solution Research Articles

In Vitro Selection and Characterization of a Light-up DNA Aptamer for Thiazole Orange.

A new DNA aptamer that binds to the target Thiazole Orange-biotin (TO1-biotin) was isolated after nine rounds of in vitro selection. The selection was performed on streptavidin-coated beads with the target bound to the surface and with free dye in solution in higher selection rounds to select for slower off-rate binding. Using next-generation sequencing (NGS), the libraries after the 4th and 9th rounds of selection were sequenced to identify enriched sequences. Several sequence families emerged, showing superior fluorescence enhancement and high affinity for the target compared to the other families obtained by NGS analysis. These sequence families were further studied to understand the binding interactions better. Primary sequence and secondary structure analysis tools were used to identify a hypothetical three-tiered G-quadruplex motif for these families. This indicates that the TO1-biotin DNA aptamer identified here uses a similar ligand-binding topology to the original Mango RNA aptamer.

Immobilization of Solvatochromic Dyes on Transparent Cellulose Films: an Improved Method for the Study of Homeopathic Potencies.

Highly diluted and succussed solutions (homeopathic potencies) have been shown to interact with a wide range of solvatochromic dyes based on changes in their UV-visible spectra. Studies so far have involved free dyes in solution, but there is a pressing need to find ways to investigate the potency-dye interaction using isolated dye molecules to ask more searching physico-chemical questions regarding the fundamental nature of potencies. The aims of the present study have been to look for ways to covalently immobilize solvatochromic dyes onto transparent cellulose films and hence be in a position to investigate dye-potency interactions without the complication of dye-dye interactions, including dye aggregation, which can occur with free dyes in solution. To date, a total of nine different dyes have been immobilized on cellulose films using epoxide activation of hydroxyl groups on the cellulose surface. Using this method, studies have begun looking at the time course of potency action on one of these immobilized dyes, Brooker's merocyanine. Results show that the interaction of Arsenicum 10M with this dye consists of three phases-an initial growth phase, a sustained plateau of interaction, and a final decline phase lasting several days. A method has been developed that successfully immobilizes solvatochromic dyes onto transparent cellulose film. These films can then be used in a spectrophotometer to study at a much more detailed level how potencies interact with dyes compared with using free dyes in solution. Results indicate that the information gained in this way provides new insights regarding the fundamental nature of potencies. Specifically, studies using immobilized Brooker's merocyanine with Arsenicum 10M reveal that the lifetime of the potency is much longer than expected and that its action consists of three distinct phases, suggesting a resonant interaction with the dye. How resonant interaction might help to explain the clinical action of potencies is discussed.

Immobilisation of Solvatochromic Dyes on Transparent Cellulose Films; an Improved Methodology for the Study of Homeopathic Potencies.

Abstract
 Background Highly diluted and succussed solutions (homeopathic potencies) have been shown to interact with a wide range of solvatochromic dyes based on changes in their UV-visible spectra. Studies so far have involved free dyes in solution, but there is a pressing need to find ways to investigate the potency-dye interaction using isolated dye molecules in order to ask more searching physico-chemical questions regarding the fundamental nature of potencies.
 Aims and Methods The aims of the present study have been to look for ways to covalently immobilise solvatochromic dyes onto transparent cellulose films and hence be in a position to investigate dye-potency interactions without the complication of dye-dye interactions, including dye aggregation, which can occur with free dyes in solution.
 Results To date a total of nine different dyes have been immobilised on cellulose films using epoxide activation of hydroxyl groups on the cellulose surface. Using this methodology studies have begun looking at the time course of potency action on one of these immobilised dyes, Brooker’s merocyanine. Results show that the interaction of Arsenicum 10M with this dye consists of three phases – an initial growth phase, a sustained plateau of interaction and a final decline phase lasting several days. 
 Conclusions A methodology has been developed that successfully immobilises solvatochromic dyes onto transparent cellulose film. These films can then be used in a spectrophotometer to study at a much more detailed level how potencies interact with dyes compared with using free dyes in solution. Results indicate that the information gained in this way provides new insights regarding the fundamental nature of potencies. Specifically, studies using immobilised Brooker’s merocyanine with Arsenicum 10M reveal that the lifetime of the potency is much longer than expected and that its action consists of three distinct phases, suggesting a resonant interaction with the dye.

Sensitive detection of E. coli using bioconjugated fluorescent silica nanoparticles

Fluorescent dye-doped silica nanoparticles have been a major focus of research and development as potential fluorescent probes in biological systems. This is due to their strong fluorescence intensity, excellent photostability, biocompatibility, and ease of surface modification for bioconjugation. In this study, spherical silica nanoparticles doped with fluorescein isothiocyanate (FITC) were synthesized using reverse microemulsion method. The average diameter of the nanoparticles was 78 nm with a narrow size distribution (78 ± 49.50 nm). FITC was incorporated into the silica sphere through 3-aminopropyltriethoxysilane, which minimized the leakage of the dye. Only about 30% of the dye leaked out from these nanoparticles, after they were sonicated in aqueous medium for 60 min and the fluorescent intensity remained comparatively constant over time, showing an increased photostability (∼ 1.5 times higher) compared to the free dye in solution. The fluorescence emission spectrum of nanoparticles showed no apparent shift (maximum emission at 518 nm) when compared with the free dye in solution. The nanoparticle surface was chemically modified with amine and carboxyl functionalities to allow for Escherichia coli antibody conjugation. E. coli bacteria is widely used as a biological indicator to identify fecal contamination in food and water samples. Nanoparticle-based biosensors for the detection of E. coli have a vital importance in water quality, public health, and food safety. The E. coli in water was successfully observed under a fluorescence microscope using the antibody conjugated fluorescent silica nanoparticles synthesized, proving the feasibility of using them in a biosensor for qualitative determination of E. coli. The significance of this study lies in the relatively simple synthetic procedures as compared to those that have been in other studies reported in the literature. The ability to detect live cells under the fluorescence microscope further proves the suitability of these functionalized nanoparticles in live cell imaging.

Lighting Up the Thrombin-Binding Aptamer G-Quadruplex with an Internal Cyanine-Indole-Quinolinium Nucleobase Surrogate. Direct Fluorescent Intensity Readout for Thrombin Binding without Topology Switching.

Fluorescent nucleobases represent an important class of molecular reporters of nucleic acid interactions. In this work, the advantages of utilizing a noncanonical fluorescent nucleobase surrogate for monitoring thrombin binding by the 15-mer thrombin binding aptamer (TBA) is presented. TBA folds into an antiparallel G-quadruplex (GQ) with loop thymidine (T) residues interacting directly with the protein in the thrombin-TBA complex. In the free GQ, T3 is solvent-exposed and does not form canonical base-pairs within the antiparallel GQ motif. Upon thrombin binding, T3 interacts directly with a hydrophobic protein binding pocket. Replacing T3 with a cyanine-indole-quinolinium (4QI) hemicyanine dye tethered to an acyclic 1,2-propanediol linker is shown to have minimal impact on GQ stability and structure with the internal 4QI displaying a 40-fold increase in emission intensity at 586 nm (excitation 508 nm) compared to the free dye in solution. Molecular dynamics (MD) simulations demonstrate that the 4QI label π-stacks with T4 and T13 within the antiparallel GQ fold, which is supported by strong energy transfer (ET) fluorescence from the GQ (donor) to the 4QI label (acceptor). Thrombin binding to 4QI-TBA diminishes π-stacking interactions between 4QI and the GQ structure to cause a turn-off emission intensity response with an apparent dissociation constant (Kd) of 650 nM and a limit of detection (LoD) of 150 nM. These features highlight the utility of internal noncanonical fluorescent surrogates for monitoring protein binding by GQ-folding aptamers in the absence of DNA topology switching.

Functionalized Fluorescent Silica Nanoparticles for Bioimaging of Cancer Cells.

Functionalized fluorescent silica nanoparticles were designed and synthesized to selectively target cancer cells for bioimaging analysis. The synthesis method and characterization of functionalized fluorescent silica nanoparticles (50–60 nm), as well as internalization and subcellular localization in HeLa cells is reported here. The dye, rhodamine 101 (R101) was physically embedded during the sol–gel synthesis. The dye loading was optimized by varying the synthesis conditions (temperature and dye concentration added to the gel) and by the use of different organotriethoxysilanes as a second silica precursor. Additionally, R101, was also covalently bound to the functionalized external surface of the silica nanoparticles. The quantum yields of the dye-doped silica nanoparticles range from 0.25 to 0.50 and demonstrated an enhanced brightness of 230–260 fold respect to the free dye in solution. The shell of the nanoparticles was further decorated with PEG of 2000 Da and folic acid (FA) to ensure good stability in water and to enhance selectivity to cancer cells, respectively. In vitro assays with HeLa cells showed that fluorescent nanoparticles were internalized by cells accumulating exclusively into lysosomes. Quantitative analysis showed a significantly higher accumulation of FA functionalized fluorescent silica nanoparticles compared to nanoparticles without FA, proving that the former may represent good candidates for targeting cancer cells.

Adsorption of New Azo Dyes Derived From 4-Aminoantipyrine from Aqueous solution by A New Type of Activated Carbon: Equilibrium and Kinetic Studies

This study is aimed to the synthesis of new azo dyes starting with 4-Aminoantipyrine. The thermodynamic behavior of the dyes adsorption on a new kind of activated carbon prepared from (Asphalt : Polymer) mixtures are studied. The research involved the selection of the optimum conditions for the adsorption process by studying the factors affecting it such as the effect of adsorbent dose, contact time, initial concentration and temperature, the work also included the fitting of two models of isotherm equations (Freundlich, Langmuir isotherms) on experimental data of adsorption models under consideration. This study was carried at different temperature and at range of concentrations. The constants of these isotherms were used to describe the nature of the studied systems and the type of interaction between dye and carbon surface. Depending on the results obtained from adsorption study at different temperature, the values of thermodynamic functions of adsorption were calculated at different concentration, using the equilibrium constant which obtained from the ratio between the concentration of the adsorbed and the free dye in solution. The above results showed that, the adsorption of these dyes are of physical nature and the experiential data were better obeyed Langmuir isotherm. The study also involved employing four kinetic models on the experimental data; pseudo first order, pseudo second order, Elovich and intraparticle diffusion equations. The results of this study showed that, the adsorption system under investigation obeyed the Pseudo second order model and the intraparticle diffusion is not the only mechanism controlling the adsorption systems under study.

A Mechanistic Investigation of Methylene Blue and Heparin Interactions and Their Photoacoustic Enhancement.

We recently reported a real-time method to measure heparin in human whole blood based on the photoacoustic change of methylene blue (MB). Intriguingly, the MB behaved unlike other "turn on" photoacoustic probes-the absorbance decreased as the photoacoustic signal increased. The underlying mechanism was not clear and motivated this study. We studied the binding mechanism of MB and heparin in water and phosphate buffer saline (PBS) with both experimental and computational methods. We found that the photoacoustic enhancement of the MB-heparin mixture was a result of MB-heparin aggregation due to charge neutralization and resulting sequestration of MB in these aggregates. The sequestration of MB in the MB-heparin aggregates led to decreased absorbance-there was simply less free dye in solution to absorb light. The highest photoacoustic signal and aggregation occurred when the number of negatively charged sulfate groups on heparin was approximately equal to the number of positively charged MB molecule. The MB-heparin aggregates dissociated when there were more sulfated groups from heparin than MB molecules because of the electrostatic repulsion between negatively charged sulfate groups. PBS facilitated MB dimer formation regardless of heparin concentration and reprecipitated free MB in aggregates due to ionic strength and ionic shielding. Further molecular dynamics experiments found that binding of heparin occurred at the sulfates and glucosamines in heparin. Phosphate ions could interact with the heparin via sodium ions to impair the MB-heparin binding. Finally, our model found 3.7-fold more MB dimerization upon addition of heparin in MB solution confirming that heparin facilitates MB aggregation. We conclude that theaddition of heparin in MB decreases the absorbance of the sample because of MB-heparin aggregation leading to fewer MB molecules in solution; however, the aggregation also increases the PA intensity because the MB molecules in the MB-heparin aggregate have reduced degrees of freedom and poor heat transfer to solvent.

Adsorbed Dyes onto Nanoparticles: Large Wavelength Dependence in Second Harmonic Scattering

Spectrally and polarization resolved second harmonic scattering (SHS) has been used to probe the adsorption of an hemicyanine dye onto polystyrene nanoparticles. The polarization resolved SHS measurements in both the vertically and horizontally output polarization configuration demonstrate the ability of this technique to monitor simultaneously the amount of free dye in solution and the amount of dye species adsorbed onto nanoparticles. Indeed, those two contributions can be discriminated when changing the laser incident polarization angle. Moreover, a large modification appears for the horizontally polarization resolved pattern when the laser excitation wavelength is tuned from 790 to 810 nm. This result is discussed within the framework of nonlinear Mie theory.

Determination of the chemical warfare agents Sarin, Soman and Tabun in natural waters employing fluorescent hybrid silica materials

A novel mesoporous silica material containing boron–dipyrromethene (BODIPY) moieties (I) is employed for the detection of nerve agent simulants (NASs) and the organophosphate nerve or chemical warfare agents (CWAs) Sarin (GB), Soman (GD), and Tabun (GA) in aqueous environments. The reactive BODIPY dye with an optimum positioned hydroxyl group undergoes acylation reactions with phosph(on)ate substrates, yielding a bicyclic ring. Due to aggregation of the dyes in water, the sensitivity of the free dye in solution is very low. Only after immobilization of the BODIPY moieties into the silica substrates is aggregation inhibited and a sensitive determination of the NASs diethyl cyanophosphonate (DCNP), diethyl chlorophosphate (DCP) and diisopropyl fluorophosphate (DFP) possible. The signaling mode is a strong quenching of the fluorescence, reaching LODs in the pM range. The best performing hybrid material was singled out from a library of hybrid silicas varying in morphology and surface functionalization. The response to actual CWAs such as GB, GD, and GA has also been tested, offering similar behavior as for the simulants. The proposed reaction mechanism has been verified by investigation of other model materials, containing for instance BODIPY moieties without an optimum hydroxyl group (III) or a BODIPY dye with an all-aliphatic counterpart (IV). The latter can only form a monocyclic reaction product, showing much less reactivity as I. Assays with other possible competitors have been additionally carried out, showing favorably low cross-reactivities. Finally, the determination of NASs in several natural waters has been demonstrated.

Chiral, J-Aggregate-Forming Dyes for Alternative Signal Modulation Mechanisms in Self-Immolative Enzyme-Activatable Optical Probes.

Enzyme-activatable optical probes are important for future advances in cancer imaging, but may easily suffer from low signal-to-background ratios unless not optimized. To address this shortcoming, numerous mechanisms to modulate the fluorescence signal have been explored. We report herein newly synthesized probes based on self-immolative linkers containing chiral J-aggregate-forming dyes. Signal modulation by formation of chiral J-aggregates is yet unexplored in optical enzyme probe design. The comprehensive characterization of the probes by absorption, CD, fluorescence, and time-resolved fluorescence spectroscopy revealed dye-dye interactions not observed for the free dyes in solution as well as dye-protein interactions with the enzyme. This suggested that J-aggregate formation is challenging to achieve with current probe design and that interactions of the dyes with the enzyme may interfere with achieving high signal-to-background ratios. The detailed understanding of the interactions provided herein provides valuable guidelines for the future design of similar probes.

Anionic chromogenic chemosensors highly selective for cyanide based on the interaction of phenyl boronic acid and solvatochromic dyes

Solutions of two solvatochromic dyes, Reichardt's pyridinium N-phenolate betaine (1) and Brooker's merocyanine (2), are colored in acetonitrile but become colorless when phenyl boronic acid (PBA) is added due to the formation of covalently linked dye-PBA species. Of the various anions added, only F− and CN− caused the reappearance of the original color related to the free dye in solution. These anions act as nucleophiles in acetonitrile and displace the dye leaving group. On the addition of very small amounts of water the system becomes highly selective toward CN− because of the ability of water to strongly solvate F−, inhibiting its nucleophilic action, while CN− is sufficiently naked to react with dye-PBA species. The titrations of the dye-PBA complexes with CN− revealed that the shape of the curves is dependent on the experimental conditions. With the use of large amounts of PBA, the dye, after being displaced, interacts with the free PBA forming hydrogen-bonded complexes, which are disrupted with the addition of an excess of the anion. This was not observed in aqueous acetonitrile since water can form hydrogen bonds with the boronic acid and with the dye, preventing the formation of dye-PBA hydrogen-bonded complexes.

Anomalous Diffusion in Thermoresponsive Polymer–Clay Composite Hydrogels Probed by Wide-Field Fluorescence Microscopy

Thermoresponsive materials exhibit an enormous potential for tissue engineering, separation systems, and drug delivery. We investigated the diffusion of laponite clay nanoparticles, which serve as physical cross-linkers to achieve improved material properties in poly(N-isopropylacrylamide) (PNIPAM)-clay composite hydrogels close to the gel point. The networks are formed through physical interactions between PNIPAM chains and clay nanoparticles after these two components are mixed. In contrast to previous studies, a covalent labeling strategy was chosen to minimize the amount of free dyes in solution. Single-particle tracking of the labeled clay nanoparticles showed that their diffusion is anomalous at all temperatures used in this study, reflecting the viscoelastic behavior as a cross-linker. Stepwise heating from 24 to 38 °C resulted in a slight increase of the diffusion coefficient and the anomality parameter α up to the volume phase transition temperature of ca. 31 °C, which was followed by a significant drop of both parameters, reflecting strongly hindered motion of the collapsed nanoparticle aggregates.

Transient state imaging of live cells using single plane illumination and arbitrary duty cycle excitation pulse trains

We demonstrate the applicability of Single Plane Illumination Microscopy to Transient State Imaging (TRAST), offering sensitive microenvironmental information together with optical sectioning and reduced overall excitation light exposure of the specimen. The concept is verified by showing that transition rates can be determined accurately for free dye in solution and that fluorophore transition rates can be resolved pixel-wise in live cells. Furthermore, we derive a new theoretical framework for analyzing TRAST data acquired with arbitrary duty cycle pulse trains. By this analysis it is possible to reduce the overall measurement time and thereby enhance the frame rates in TRAST imaging.

Preparation and Characterization of Uniform Near IR Polystyrene Nanoparticles

Biomaterials for in vivo fluorescence imaging are required to be biocompatible, nontoxic, photostable and highly fluorescent. Fluorescence must be in the near infrared (NIR) region of the electromagnetic spectrum to avoid absorption and autofluorescence of endogenous tissues. NIR fluorescent polystyrene nanoparticles may be considered ideal biomaterials for in vivo imaging applications. These NIR nanoparticles were prepared by a swelling process of polystyrene template nanoparticles with a hydrophobic NIR dye dissolved in a water-miscible swelling solvent, a method developed for preparation of nonbiodegradable nanoparticles, for NIR fluorescent bioimaging applications. This method overcomes common problems that occur with dye entrapment during nanoparticle formation such as loss of fluorescence and size polydispersity. Fluorescence intensity of the nanoparticles was found to be size dependent, and was optimized for differently sized nanoparticles. The resulting NIR nanoparticles were also found to be more fluorescent and highly photostable compared to the free dye in solution, showing their potential as biomaterials for in vivo fluorescence imaging.

Aggregation and Electrolyte Composition Effects on the Efficiency of Dye-Sensitized Solar Cells. A Case of a Near-Infrared Absorbing Dye for Tandem Cells

Time-resolved laser spectroscopy studies of complete solar cells sensitized with a near-infrared absorbing dye (HY103) and filled with different electrolytes are applied to explain their macroscopic parameters (efficiency and short-circuit current). Particular attention is paid to the effect of coadsorbent, size of cations in electrolyte (lithium vs guanidine ones), and addition of tert-butylpyridine. A complete deactivation scheme in the cell is revealed, and the rates of electron injection and all other processes are explored. For the most efficient electrolyte, the electron injection rate constants are 0.21 ps–1 from monomers and 0.07 ps–1 from H-aggregates. Moreover, two important and novel findings are revealed: energy transfer from the excited state of monomers to H-aggregates (with rate constants from 0.04 to 0.25 ps–1) and the decrease of internal conversion rate in HY103 attached to the nanoparticles (0.01 ps–1) with respect to that of free dye in solution (0.06 ps–1). Thus, our study gives more ...

Synthesis, photophysical properties and application of dye doped water soluble silica-based nanoparticles to label bacteria E. coli O157:H7

Organically modified silicate (ORMOSIL) nanoparticles (NPs) doped with rhodamine 6G and rhodamine B (RB) dyes were synthesized by Stöber method from methyltriethoxysilane CH3Si(OCH3)3 precursor (MTEOS). The NPs are surface functionalized by cationic amino groups. The optical characterization of dye-doped ORMOSIL NPs was studied in comparison with that of free dye in solution. The synthesized NPs were used for labeling bacteria E. coli O157:H7. The number of bacteria have been counted using the fluorescent spectra and microscope images of labeled bacteria. The results show the ability of NPs to work as biomarkers.

Synthesis, characterisation and functionalisation of luminescent silica nanoparticles

The synthesis of highly monodispersed, homogeneous and stable luminescent silica nanoparticles, synthesized using a process based on the Stober method is reported here. These particles have been functionalised with the ruthenium and europium complexes: bis (2,2′-bipyridine)-(5-aminophenanthroline) Ru bis (hexafluorophosphate), abbreviated to (Ru(bpy)2(phen-5-NH2)(PF6)), and tris (dibenzoylmethane)-mono (5-aminophenanthroline) europium(III), abbreviated to (Eu:TDMAP). Both dyes have a free amino group available, facilitating the covalent conjugation of the dyes inside the silica matrix. Due to the covalent bond between the dyes and the silica, no dye leaching or nanoparticle diameter modification was observed. The generic and versatile nature of the synthesis process was demonstrated via the synthesis of both europium and ruthenium-functionalised nanoparticles. Following this, the main emphasis of the study was the characterisation of the luminescence of the ruthenium-functionalised silica nanoparticles, in particular, as a function of surface carboxyl or amino group functionalisation. It was demonstrated that the luminescence of the ruthenium dye is highly affected by the ionic environment at the surface of the nanoparticle, and that these effects can be counteracted by encapsulating the ruthenium-functionalised nanoparticles in a plain 15 nm silica layer. Moreover, the ruthenium-functionalised silica nanoparticles showed high relative brightness compared to the free dye in solution and efficient functionalisation with amino or carboxyl groups. Due to their ease of fabrication and attractive characteristics, the ruthenium-functionalised silica nanoparticles described here have the potential to be highly desirable fluorescent labels, particularly, for biological applications.

Synthesis and Optical Characterization of Dye Doped in Ormosil Nanospheres for Bioapplications

Dye-doped ORMOSIL (organically modified silicate) nanoparticles (NPs) have significant advantages over single-dye labeling in signal amplification, photostability and surface modification for various biological applications. The dyes: Rhodamine 6G (R6G) and Rhodamine B (RB) were successfully incorporated into ORMOSIL nanoparticles fabricated by micellar nanochemistry from Trimethoxysilane CH$_3$Si(OCH$_3$)$_3$ precursor. The optical characterization of dye-doped ORMOSIL NPs was studied in comparison with it’s of free dye in solution. The results shown that the photostability of ORMOSIL dye doped nanospheres is much improved in comparison with it’s of dye in solution. Other studies of the photophysical properties such as anisotropy, fluorescence lifetime and energy transfer were also done.

Influence of Ionic Liquids on the Sorption of Acid Dyes by Nylon Fibers

In the present study, the influence of four 1-alkyl-3-methylimidazolium bromine ionic liquids (ILs), CnMImBr (n=4, 8, 12, 16) on the sorption of acid dyes by nylon fibers was investigated. The quantity of dye sorption at pH 5 and pH 7 was found to be greatly dependent on the concentrations of ILs and the length of alkyl groups in ILs. The quantity of dyes adsorbed by nylon increased in the presence of C8MImBr at all the concentrations employed, and in the presence of C12MImBr and C16MImBr at appropriate concentrations. The mechanism of the increase in dye sorption in the presence of ILs at appropriate concentrations is due to the existence of the new positively charged dyeing sites in nylon induced by the sorption of ILs on nylon. The further increasing concentrations of C12MImBr and C16MImBr decreased the extent of dye sorption, which can be explained by the greatly reduced quantity of free dyes in solution induced by the interaction between anionic dyes and cationic ILs in solution as well as the increasing aggregation of the complex of dyes and ILs.