Water-soluble Fluorescent Dye Research Articles

A Thioflavin T-induced G-Quadruplex Fluorescent Biosensor for Target DNA Detection.

The detection of disease-related DNA is of great significance for early and accurate diagnosis and therapy. In this work, we successfully achieved the sensitive detection of target DNA based on a thioflavin T (ThT)-induced G-quadruplex fluorescent biosensor. ThT, a water-soluble fluorescent dye, can induce G-rich sequences to form G-quadruplexes and obtain an obviously enhanced fluorescence. In this work, it was employed to construct a biosensor for the detection of HIV. When the target HIV existed, the hairpin DNA probes would be opened in succession and release the completely exposed G-rich sequence to combine with ThT. The simple and rapid biosensor performed satisfactory selectivity; it also exhibited sensitivity with a detection limit of 2.4 nM. With good performance in human serum, we believe that this optical biosensor has the potential to be applied to the practical detection of target DNA.

Effect of clay in controlling the non-fluorescence H-dimeric states of a cationic dye Nile Blue Chloride (NBC) in hybrid Langmuir–Blodgett (LB) film

Present communication reports the effect of amphiphilic matrices and nano-clay platelets on the aggregation properties of a water soluble cationic fluorescent dye Nile Blue Chloride (NBC) in Langmuir-Blodgett (LB) films. In-situ Brewster Angle Microscopic (BAM) studies showed distinct domain structures of complex and hybrid Langmuir monolayer at the air-water interface. UV–vis absorption spectra showed non-fluorescent H-dimeric band in concentrated aqueous solution of NBC and in complex LB film of NBC with stearic acid. By changing various parameters, a great control over H-dimeric states has been achieved in clay incorporated hybrid LB films. These films can act as efficient fluorescence probe.

Freeze-dryable lipid vesicles with size tunability and high encapsulation efficiency prepared by the multiple emulsification-solvent evaporation method

We investigated the extent of potential applicability of our recently developed method for preparing lipid vesicles [T. Kuroiwa et al., J. Am. Oil Chem. Soc., 93 (2016) 421], designated as the multiple emulsification-solvent evaporation method, with the intention of controlling the vesicle diameter and achieving high entrapment efficiency for water-soluble compounds. Using this method, the diameter of lipid vesicles could be varied by selecting the methods for preparing the primary water-in-oil emulsion, which contained water droplets as templates for the internal water phases of lipid vesicles. We obtained lipid vesicles with mean diameters of 0.2–4.4μm from water-in-oil-in-water multiple emulsions after solvent evaporation. A high entrapment yield of calcein, a water-soluble fluorescent dye, into the lipid vesicles was obtained for each vesicle sample, depending on their diameter and the type of emulsifier added to the external water phase. The use of polymeric emulsifier was more effective in achieving a high entrapment yield. The obtained lipid vesicles were powderized via freeze-drying. Vesicles could be powderized while maintaining their original diameter, as confirmed by scanning electron microscopy. Furthermore, the powderized vesicles could be rehydrated and resuspended without significant change in their diameter. However, the entrapment yield of calcein decreased after freeze-drying and rehydration. The calcein leakage during the freeze-drying followed by rehydration could be suppressed by adding an appropriate amount of trehalose as a lyoprotectant.

Rhodamine B in dissolved and nano-bound forms: Indicators for light-based advanced oxidation processes

Rhodamine B (RhB) is a water-soluble fluorescent dye that is often used to determine flux and flow direction in biotechnological and environmental applications. In the current research, RhB in soluble (termed free) and virus-bound (termed nano-bound) forms was used as an efficiency indicator for three environmental processes. The degradation of free and nano-bound RhB by (i) direct UV photolysis and (ii) UV/H2O2 advanced oxidation process (AOP) was studied in a collimated beam apparatus equipped with medium-pressure mercury vapor lamp. The degradation by (iii) solar light-induced photocatalysis was studied in a solar simulator with titanium dioxide and bismuth photocatalysts. Results showed negligible RhB degradation by direct UV and solar light, and its nearly linear degradation by UV/H2O2 and photocatalysis/photosensitization in the presence of a solid catalyst. Considerable adsorption of free RhB on bismuth-based catalyst vs. no adsorption of nano-bound RhB on this catalyst or of any form of the dye on titanium dioxide produced two important conclusions. First, the better degradation of free RhB by the bismuth catalyst suggests that close proximity of a catalyst hole and the decomposing molecule significantly influences degradation. Second, the soluble form of the dye might not be the best option for its use as an indicator. Nano-bound RhB showed high potential as an AOP indicator, featuring possible separation from water after the analysis.

Matrix dependent changes in metachromasy of crystal violet in Langmuir-Blodgett films

This communication reports the effect of building matrices and nano clay platelets on the aggregation pattern of a water soluble cationic fluorescent dye Crystal Violet (CV) in Langmuir-Blodgett (LB) Films. When stearic acid (SA) was used as a building matrix, pressure induced changes in metachromasy was observed in the SA-CV complex LB films with a characteristic J-band formed at the longer wavelength side. On the other hand, clay incorporated hybrid LB films showed high degree of control over H-dimeric band. Isotherm characteristics, Brewster Angle Microscopic (BAM) images, UV–vis absorption and fluorescence characteristic studies were employed to investigate this result.

Clarithromycin Dose-Dependently Stabilizes Rat Peritoneal Mast Cells

Background: Macrolides, such as clarithromycin, have antiallergic properties. Since exocytosis in mast cells is detected electrophysiologically via changes in membrane capacitance (Cm), the absence of such changes due to the drug indicates its mast cell-stabilizing effect. Methods: Employing the whole-cell patch clamp technique in rat peritoneal mast cells, we examined the effects of clarithromycin on Cm during exocytosis. Using a water-soluble fluorescent dye, we also examined its effect on deformation of the plasma membrane. Results: Clarithromycin (10 and 100 μ<smlcap>M</smlcap>) significantly inhibited degranulation from mast cells and almost totally suppressed the GTP-γ-S-induced increase in Cm. It washed out the trapping of the dye on the surface of mast cells. Conclusions: This study provides for the first time electrophysiological evidence that clarithromycin dose-dependently inhibits the process of exocytosis. The mast cell-stabilizing action of clarithromycin may be attributable to its counteractive effect on plasma membrane deformation induced by exocytosis.

Synthesis and Cell markers of Novel Water-soluble Asymmetric Fluorescent Dyes with Nonionic Hydrophilic Group

Synthesis and Cell markers of Novel Water-soluble Asymmetric Fluorescent Dyes with Nonionic Hydrophilic Group

Adsorption of a cationic water-soluble dye onto cationic Langmuir–Blodgett films via nano clay platelets: An efficient approach to control the H-dimer

ABSTRACTThis communication reports the successful adsorption of a water-soluble cationic fluorescent dye Acridine Orange (AO) onto Langmuir–Blodgett (LB) films of a cationic amphiphile octadecylamine (ODA) in the presence of nano-clay platelets hectorite. Acridine orange (AO) has been widely used as a stainer for the characterization of biopolymers. But AO has a tendency to form non-florescent H-dimer even in the aqueous solution. Anionic nano-clay platelets hectorite played an important role in controlling the H-dimer formation of AO in the hybrid film. Effects of various parameters in the adsorption process were investigated in detail.

Synthesis, Aggregation-Induced Emission Enhancement and Cells Imaging of Water-Soluble Pyridine Naphthalimide-Polyamidoamine Dendrimer

将具有聚集诱导荧光的4-（2-吡啶乙烯基）-1,8-萘酰亚胺单元通过酰胺化反应连接到聚酰胺-胺（PAMAM）树形分子上,制备了一种水溶性吡啶萘酰亚胺-聚酰胺-胺荧光树形分子PN-PAMAM.结构经核磁共振氢谱、核磁共振碳谱、红外及高分辨质谱表征.吡啶萘酰亚胺-聚酰胺-胺树形分子PN-PAMAM具有明显的聚集诱导荧光增强（AIEE）特性.在固体态时的最大荧光发射波长为532 nm,紫外灯下发出黄绿色荧光.在纯水中的最大荧光发射波长为494 nm,荧光量子产率为4.42%.在水含量为60%的水/四氢呋喃混合溶液中,其荧光强度达到最大,荧光发射波长为485 nm,荧光量子产率增大到17.54%.制备了一种负载聚集诱导荧光染料PN-PAMAM的二氧化硅纳米粒子PN-PAMAM/Si O_2,测得该纳米粒子的荧光发射波长为473 nm,粒径约为40 nm.测定了水溶液中树形分子PN-PAMAM与乳腺癌细胞MCF-7共同孵化后的共聚焦荧光成像,得到清晰的蓝场荧光照片.研究表明,吡啶萘酰亚胺-聚酰胺-胺树形分子PN-PAMAM是一种水溶性荧光分子,具有明显的聚集诱导荧光增强特性,可广泛应用于肿瘤定位、生物追踪及纳米材料等重要领域.

Clean Photothermal Heating and Controlled Release from Near-Infrared Dye Doped Nanoparticles without Oxygen Photosensitization.

The photothermal heating and release properties of biocompatible organic nanoparticles, doped with a near-infrared croconaine (Croc) dye, were compared with analogous nanoparticles doped with the common near-infrared dyes ICG and IR780. Separate formulations of lipid-polymer hybrid nanoparticles and liposomes, each containing Croc dye, absorbed strongly at 808 nm and generated clean laser-induced heating (no production of (1)O2 and no photobleaching of the dye). In contrast, laser-induced heating of nanoparticles containing ICG or IR780 produced reactive (1)O2, leading to bleaching of the dye and also decomposition of coencapsulated payload such as the drug doxorubicin. Croc dye was especially useful as a photothermal agent for laser-controlled release of chemically sensitive payload from nanoparticles. Solution state experiments demonstrated repetitive fractional release of water-soluble fluorescent dye from the interior of thermosensitive liposomes. Additional experiments used a focused laser beam to control leakage from immobilized liposomes with very high spatial and temporal precision. The results indicate that fractional photothermal leakage from nanoparticles doped with Croc dye is a promising method for a range of controlled release applications.

Synthesis of a water soluble red fluorescent dye and its application to living cells imaging

A water soluble red fluorescent dye (TD-mPEG750) has been prepared by treatment of 2-(3,5,5-trimethylcyclohex-2-en-1-ylidene)malononitrile with 4-(diethylamino)-2-mPEG benzaldehyde (mPEG-OH, average MW = 750). TD-mPEG750 exhibits red emission at λem = 664 nm in water, a small fluorescence quantum yield (ϕf = 0.01) and a large Stoke's shift (Δλ = 145 nm) are obtained. Using HeLa cells as prototype, the application of TD-mPEG750 to living cells imaging has been investigated. It is found that TD-mPEG750 can be clearly expressed in mitochondria with high contrast in HeLa cells imaging.

Visualizing monolayers with a water-soluble fluorophore to quantify adsorption, desorption, and the double layer

Contrast in confocal microscopy of phase-separated monolayers at the air-water interface can be generated by the selective adsorption of water-soluble fluorescent dyes to disordered monolayer phases. Optical sectioning minimizes the fluorescence signal from the subphase, whereas convolution of the measured point spread function with a simple box model of the interface provides quantitative assessment of the excess dye concentration associated with the monolayer. Coexisting liquid-expanded, liquid-condensed, and gas phases could be visualized due to differential dye adsorption in the liquid-expanded and gas phases. Dye preferentially adsorbed to the liquid-disordered phase during immiscible liquid-liquid phase coexistence, and the contrast persisted through the critical point as shown by characteristic circle-to-stripe shape transitions. The measured dye concentration in the disordered phase depended on the phase composition and surface pressure, and the dye was expelled from the film at the end of coexistence. The excess concentration of a cationic dye within the double layer adjacent to an anionic phospholipid monolayer was quantified as a function of subphase ionic strength, and the changes in measured excess agreed with those predicted by the mean-field Gouy-Chapman equations. This provided a rapid and noninvasive optical method of measuring the fractional dissociation of lipid headgroups and the monolayer surface potential.

Adsorption of a water soluble cationic dye into a cationic Langmuir monolayer

This communication reports the successful adsorption of a water soluble cationic fluorescent dye Rhodamine B (RhB) into a cationic Langmuir monolayer of Octadecylamine (ODA). Anionic nano clay platelets Hectorite played an important role in the process of adsorption. Surface pressure vs. area per molecule (π–A) isotherms were studied to monitor the adsorption process. In-situ fluorescence Imaging Microscopic (FIM) technique was employed to visualize the domain structures formed at the air–water interface. Atomic Force Microscopic (AFM) image of the monolayer Langmuir–Blodgett (LB) films were taken to study the morphology and ultrastructure of the film. Detailed spectroscopic investigations were carried out on the mono- and multilayer Langmuir–Blodgett (LB) films.

Translocation of Cationic Amphipathic Peptides Across Phospholipid Bilayers

We have developed a method to measure translocation of amphipathic peptides across lipid membranes that uses giant unilamellar vesicles (GUVs) containing inner vesicles. When the GUVs are added to a peptide solution containing a water-soluble fluorescent dye, permeabilization of the membrane of the outer vesicle by the peptide results in the appearance of fluorescence in its lumen. The inner vesicles remain dark if the peptide does not translocate. However, the appearance of dye in the inner vesicles indicates peptide translocation across the outer membrane of the GUVs. This is because to cause dye flux into the inner vesicles, the peptide must have crossed the membrane of the outer vesicle. The method does not require that the peptide itself be fluorescently labeled; but if it is, this approach can be used to measure the kinetics of translocation. Initially we tested the method with three peptides derived from each class of antimicrobial, cytolytic, and cell-penetrating peptides. Those three peptides also had very different Gibbs energies of insertion into the bilayer, approximated by the difference between the Gibbs energy of binding to the membrane and the Gibbs energy of transfer from water to octanol. The initial measurements appeared to indicate that the probability of translocation was inversely correlated with the Gibbs energy of insertion. Now we extended this study to several variants of the original peptides. Two hypotheses were tested: (1) The probability of translocation simply decreases as the Gibbs energy of peptide insertion increases; (2) The probability of translocation increases if the distribution of positively charged residues along the peptide sequence is such that the inserted peptide has similar probabilities of returning the outside and crossing the membrane, to the inside of the vesicle. The results of these new tests are discussed.

Olopatadine Inhibits Exocytosis in Rat Peritoneal Mast Cells by Counteracting Membrane Surface Deformation

Backgroud/Aims: Besides its anti-allergic properties as a histamine receptor antagonist, olopatadine stabilizes mast cells by inhibiting the release of chemokines. Since olopatadine bears amphiphilic features and is preferentially partitioned into the lipid bilayers of the plasma membrane, it would induce some morphological changes in mast cells and thus affect the process of exocytosis. Methods: Employing the standard patch-clamp whole-cell recording technique, we examined the effects of olopatadine and other anti-allergic drugs on the membrane capacitance (Cm) in rat peritoneal mast cells during exocytosis. Using confocal imaging of a water-soluble fluorescent dye, lucifer yellow, we also examined their effects on the deformation of the plasma membrane. Results: Low concentrations of olopatadine (1 or 10 µM) did not significantly affect the GTP-γ-S-induced increase in the Cm. However, 100 µM and 1 mM olopatadine almost totally suppressed the increase in the Cm. Additionally, these doses completely washed out the trapping of the dye on the cell surface, indicating that olopatadine counteracted the membrane surface deformation induced by exocytosis. As shown by electron microscopy, olopatadine generated inward membrane bending in mast cells. Conclusion: This study provides electrophysiological evidence for the first time that olopatadine dose-dependently inhibits the process of exocytosis in rat peritoneal mast cells. Such mast cell stabilizing properties of olopatadine may be attributed to its counteracting effects on the plasma membrane deformation in degranulating mast cells.

Indocyanine green plasma disappearance rate as dynamic liver function test in critically ill patients.

Indocyanine green (ICG) is a water-soluble fluorescent dye that is bound to plasma protein when administered intravenously. Removal of ICG from the blood depends on hepatic blood flow, function of the parenchymal cells and biliary excretion. ICG elimination is described as a useful dynamic liver function test. In this review, we looked at the most recent literature to clarify why ICG is useful in critically ill patients, the validity of the ICG plasma disappearance rate (ICG-PDR) measured transcutaneously and whether ICG-PDR has any prognostic value. In conclusion, measuring ICG-PDR is a valuable method for dynamic assessment of liver function, and is found to be a valuable prognostic tool in predicting survival for septic patients, patients presenting with acute liver failure and critically ill patients.

A highly stable and water-soluble fluorescent dye for fluorescence imaging of living cells.

A highly stable and water-soluble bis-benzoxazole-based fluorescent dye (BBT-mPEG) is prepared by the treatment of 2,5-bis(benzoxazol-2-yl)thiophene-3,4-diol (BBT) with methoxypolyethylene glycol (mPEG-OH, average Mw = 350). BBT-mPEG exhibits a blue emission at λem = 445 nm in water upon excitation with 365 nm light, and a moderate fluorescence quantum yield (ϕf = 0.33, in H2O) is obtained using quinine sulfate (ϕf = 0.557, in H2SO4) as the reference. It is found that BBT-mPEG shows good photo-stability and large Stokes shift (Δλ = 80 nm) in aqueous solution in entire pH range (pH = 1-14). Moreover, it is also found that BBT-mPEG exhibits low cytotoxicity and enhanced fluorescence in cells; using HeLa cells as a prototype, a clear cellular imaging is obtained.

4,4′-Diarylsulfanyl-2,2′,5,5′-tetraoxybiaryl Derivatives as a Water-Soluble Fluorescent Dye

4,4'-Disulfanyl-2,2',5,5'-tetrahydrobiaryl (5,5'-disulfanyl hydroquinone dimer) derivatives were readily synthesized from benzoquinone and thiols via an oxidative coupling reaction. The hydroquinone dimers showed strong fluorescence upon excitation at 330 nm, and it was observed that the presence of the sulfanyl groups at the C4 and C4' positions is important for achieving strong photoluminescence. The tetrapotassium salts of the hydroquinone dimers also showed good water solubility, but the fluorescence disappeared rapidly on dissolution in water. 2,2'- and 5,5'-protected biaryls were synthesized. The dipotassium salt of the 5,5'-dimethoxy-2,2'-dihydroxy derivative was observed to show good and stable fluorescence in water, while the dipotassium salt of the 2,2'-dimethoxy-5,5'-dihydroxy derivative showed less water solubility. Introduction of propargyl groups was demonstrated to provide a convenient method for installing amino acids derivatives. This derivatization afforded potentially useful compounds for attaching the biologically active fragment to the fluorescent unit.

Entry of Cell-Penetrating Peptide Transportan 10 into a Single Vesicle by Translocating Across Lipid Membrane and Its Induced Pores

The cell-penetrating peptide, transportan 10 (TP10), can translocate across the plasma membrane of living cells and thus can be used for the intracellular delivery of biological cargo such as proteins. However, the mechanisms underlying its translocation and the delivery of large cargo remain unclear. In this report we investigated the entry of TP10 into a single giant unilamellar vesicle (GUV) and the TP10-induced leakage of fluorescent probes using the single GUV method. GUVs of 20% dioleoylphosphatidylglycerol (DOPG)/80% dioleoylphosphatidylcholine (DOPC) were prepared, and they contained a water-soluble fluorescent dye, Alexa Fluor 647 hydrazide (AF647), and smaller vesicles composed of 20% DOPG/80% DOPC. The interaction of carboxyfluorescein (CF)-labeled TP10 (CF-TP10) with these loaded GUVs was investigated using confocal microscopy. The fluorescence intensity of the GUV membrane increased with time to a saturated value, then the fluorescence intensity due to the membranes of the smaller vesicles inside the GUV increased prior to leakage of AF647. This result indicates that CF-TP10 entered the GUV from the outside by translocating across the lipid membrane before CF-TP10-induced pore formation. The rate constant of TP10-induced pore formation in lipid membranes increased with an increase in TP10 concentration. Large molecules such as Texas Red Dextran 40,000, and vesicles with a diameter of 1-2 μm, permeated through the TP10-induced pores or local rupture in the lipid membrane. These results provide the first direct experimental evidence that TP10 can deliver large cargo through lipid membranes, without the need for special transport mechanisms such as those found in cells.

Stable label-free fluorescent sensing of biothiols based on ThT direct inducing conformation-specific G-quadruplex

In this work, a new, label-free, turn-on fluorescent sensor for biothiols detection based on ThT direct inducing conformation-specific G-quadruplex is developed. The sensing approach is based on a conformational switch of oligonucleotide controlled by Hg2+ and a commercially available water-soluble fluorescent dye, Thioflavin T (ThT). A noticeable fluorescence light-up in ThT on binding to the G-quadruplex grants the sensor excellent sensitivity. The specific quadruplex conformation induced directly by ThT and pronounced structural selectivity of ThT for G-quadruplexes could generate more stable luminescence and make sure high specificity in complex biological samples. The present assay allows for the selective determination of cysteine and glutathione in the range of 2.0×10−8–2.5×10−6M and 3.0×10−8–2.0×10−6M with a detection limit of 8.4nM and 13.9nM respectively. The diagnostic capability and potential in practical applications of this method have been demonstrated by detecting biothiols in human blood serum.