Characterization of the Binding of Methylene Blue to DNA by Spectroscopic Methods

ABSTRACT

Methylene blue (MB) binds to the double helical DNA with a high affinity, as deduced from the absorption and fluorescence spectral data. Extensive hypochromism and red shifts in the absorption spectra were observed when MB binds to calf thymus DNA(CT DNA), which suggested the intercalation mechanism of MB into DNA bases. Upon binding to DNA, the fluorescence from MB was efficiently quenched by the DNA bases, with no shifts in the emission maximum. The large increases in the polarization upon binding to CT DNA supported the intercalation of MB into the helix. Ferrocyanide quenching studies showed that the magnitude of K_sv of the bound MB was lower than that of the free MB. The results of competitive binding studies showed that ethidium bromide could be displaced by MB from ethidium-DNA complex. The results of all above further studies also proved the intercalation of MB into DNA base stack.     

Nucleation and Growth Mechanism of Electropolymerization of Methylene Blue: The Effect of Preparation Potential on Poly(methylene blue) Structure

Nucleation and growth mechanism of electropolymerization of methylene blue (MB) in a basic medium and the effect of preparation potential on poly(MB) film structure were investigated by using cyclic voltammetry, potentiostatic current‐time transient, scanning tunneling microscopy (STM), atomic force microscopy (AFM), and UV‐vis. absorption spectroscopy techniques. Electropolymerization of MB has been achieved by potentiodynamic (cyclic voltammetry) and potentiostatic (constant potential) techniques. The potentiostatic current‐time transients fitted with a theoretical model and morphological studies indicate that nucleation and growth mechanism of poly(MB) starts with a progressive layer‐by‐layer nucleation and growth besides random adsorption. Nucleation and growth of poly(MB) follows a process between progressive layer‐by‐layer and 3‐D instantaneous mechanism resulting in highly‐oriented poly(MB) nanofibers with increasing poly(MB) film thickness. Cyclic voltammetry and morphological studies exhibit that poly(MB) film structure changes depending on the preparation potential. Poly(MB) films prepared at the potential values of 900 and 950 mV show a well‐ordered, smooth surface but at the potential values higher than 1000 mV, rough polymer surface arises as overoxidation takes place. UV‐vis. absorption spectra of poly(MB) film and MB monomer show three peaks. The peak at 410 nm for poly(MB) shows 100 nm blue shift when compared to the MB monomer and is attributed to poly(MB) formation on the electrode.     

Methylene Blue Interactions with Chromosomal and Plasmid DNA on Screen‐Printed Carbon Electrodes

Methylene blue (MB) is a typical photosensitizing agent and a DNA hybridization indicator, but its modes of interaction with the DNA molecules are not clearly described, particularly in relation to its electrochemical oxidation signals. To probe the DNA‐MB interactions we have used chromosomal salmon testes and supercoiled plasmid sc pUC19 DNA immobilized on home‐made screen‐printed electrodes (SPEs) and a wide range of MB concentrations, from nano‐ to micromolar. The applicability of the home‐made screen‐printed electrodes used for the DNA‐MB studies were tested using standard calf thymus DNA. Two MB oxidation peak signals: MB(I) at ca. ?0.18 V and MB(II) at 0 V vs. Ag/AgCl were detected within ±10–15% standard deviation, signals different from adsorbed MB signal (?0.25 V, pH 4.7). The MB(I) signal, seen when both DNAs were used, showed two plateaus, one at nano‐ and another at micromolar MB concentrations; these were accompanied by the changes in the oxidation signal at 0.98 V, usually attributed to guanine oxidation. In contrast, the MB(II) signal was only seen for salmon testes DNA, indicating various modes of MB interactions with chromosomal and plasmid DNA. In the presence of MB, the guanine related signal (G) at 0.98 V has been amplified significantly (10×), allowing for the identification of the DNAs at low DNA concentrations, the feature particularly useful in the plasmid sc pUC19 detection. The use of another DNA intercalator, riboflavin (RF), aided in the identification of the relation between MB(I), MB(II) and G oxidation signals.     

The binding properties of photosensitizer methylene blue to herring sperm DNA: a spectroscopic study

Methylene blue (MB) is a phenothiazinium photosensitizer with promising applications in the photodynamic therapy (PDT) for anticancer treatment. The binding properties of MB to herring sperm DNA have been investigated by the measurements of absorption spectra, quenching experiments and the elucidation of the photobleaching processes. Remarkable hypochromic and bathochromic effects of MB in the presence of increasing amounts of DNA have been observed in the absorption spectra. The quenching of MB by the DNA bases obeys the Stern-Volmer equation and ferrocyanide quenching of MB in the absence and presence of DNA is also measured as extended experiments. Results from the above spectral measurements are all consistent with the intercalative binding mode of MB to DNA with the Kb value of 1.89 x 10(4) M(-1). The photobleaching processes of MB and its DNA complex have also been studies, which indicate that the photobleaching of MB and its DNA complex proceeds with different mechanisms and the reactive oxygen species are responsible for the self-sensitized photooxidation of MB.     

亚甲基蓝在云母表面吸附状态的研究

利用XPS测定吸附前后亚甲基蓝（MB）各原子的电子结合能的变化,以判断原子化学环境的改变,从而确定MB在云母表面的吸附位点是二甲胺基上的氮原子.通过AFM测量得到吸附于云母表面的MB分子的平均高度为0.820 nm,这证实了Hähner吸附模型的正确性,即MB分子的最大横截面以65～70°倾斜在云母的（001）表面上.     

Removal of methylene blue dye from an aqueous media using superabsorbent hydrogel supported on modified polysaccharide

The removal of methylene blue (MB) in water with the superabsorbent hydrogel (SH) formed by modified gum arabic, polyacrylate, and polyacrylamide was investigated. The SH exhibited excellent performance in MB absorption. The maximum absorption capacity was 48 mg of the dye per g of SH, representing 98% of the MB removed. Experimental parameters were used as follows: pH 8, hydrogel mass 50 mg, and initial concentration of MB 50 mg L(-1). In a procedure with an individual solution of orange II, an opposite effect related to the MB was observed: the hydrogel only absorbed water, resulting in an orange II-richer solution. The orange II concentration in solution increased about 50 times (relative to the initial concentration). In another experiment using an aqueous mixture of orange II and MB, the SH absorbed the MB exclusively. Compared to the MB, the orange II is separated from water by SH selectivity-absorption through an inverse process. This effect was attributed to the formation of a ionic complex between the imine groups of MB and the ionized carboxylic groups of SH.     

Binding, aggregation and photochemical properties of methylene blue in mitochondrial suspensions

Methylene Blue (MB) has well-established photochemical properties and has been used in a variety of photochemical applications including photodynamic therapy. Despite the fact that most of MB's cytotoxic effects in cells are attributed to mitochondrial damage, the interactions of this dye with mitochondria and the consequent effects on photochemical properties have not yet been fully determined. We monitored MB binding, aggregation and its ability to release singlet oxygen (1O2) on irradiation when interacting with mitochondrial suspensions. MB actively binds to mitochondria and enters the matrix in a manner stimulated by the mitochondrial proton potential and by the increase in mitochondrial concentrations. The greater accumulation of MB in mitochondria with elevated proton potentials or those treated with high concentrations of MB results in the formation of MB dimers, previously shown to be less effective generators of 1O2. Accumulation of MB within mitochondria with high membrane potentials also results in the reduction of MB to the photochemically inactive leuco-MB. Indeed, irradiation of mitochondria with high proton potentials in the presence of MB results in the generation of approximately half the quantity of 1O2 compared with 1O2 generated in mitochondria with low proton potentials. These differences in photochemical properties should influence the cytotoxic effects of photodynamic treatment in the presence of MB.     

Preparation of metallochelating microbubbles and study on their site-specific interaction with rGFP-HisTag as a model protein

The histidine-metallochelating lipid complex is one of the smallest high affinity binding units used as tools for rapid noncovalent binding of histidine tagged molecules, especially recombinant proteins. The advantage of metallochelating complex over protein-ligand complexes (e.g., streptavidine-biotin, glutathiontransferase-glutathion) consists in its very low immunogenicity, if any. This concept for the construction of surface-modified metallochelating microbubbles was proved with recombinant green fluorescent protein (rGFP) containing 6His-tag. This protein is easy to be detected by various fluorescence techniques as flow cytometry and confocal microscopy. Microbubbles (MB) composed of DPPC with various contents of metallochelating lipid DOGS-NTA-Ni were prepared by intensive shaking of the liposome suspension under the atmosphere of sulfur hexafluoride. For this purpose, the instrument 3M ESPE CapMix was used. Various techniques (static light scattering, flow cytometry, and optical microscopy) were compared and used for the measurements of the size distribution of MB. All three methods demonstrated that the prepared MB were homogeneous in their size, and the mean diameter of the MB in various batches was within the range of 2.1-2.8 μm (the size range of 1-10 μm). The presence of large MB (8-10 μm) was marginal. Counting of MB revealed that the average amount of MB prepared of 10 mg of phospholipid equaled approximately 10(9) MB/mL. Lyophilized MB were prepared with saccharose as a cryoprotectant. These MB were shown to be stable both in vitro (the estimated half-live of the MB in bovine serum at 37 °C was 3-7 min) and in vivo (mouse). The stability of the MB was affected by molar content of DOGS-NTA-Ni. DPPC-based metallochelating MB provided a clear and very contrast image of the ventricular cavity soon after the injection. Site selective and stable binding of rGFP-HisTag (as a model of His-tagged protein) onto the surface of metallochelating MB was demonstrated by confocal microscopy.     

An Electrochemical Sensing Strategy for Amantadine Detection Based on Competitive Host‐guest Interaction of Methylene Blue/β‐cyclodextrin/Poly(N‐acetylaniline) Modified Electrode

A novel electrochemical strategy for the detection of amantadine (AMD) has been developed based on the competitive host‐guest interaction of AMD and methylene blue (MB) with β‐cyclodextrin (β‐CD). Due to the host‐guest interaction, MB molecules can enter into the hydrophobic inner cavity of β‐CD, and the MB/β‐CD/poly(N‐acetylaniline)/glassy carbon electrode displays a remarkable reduction peak due to MB. In the presence of AMD, competitive association to β‐CD occurs and the MB molecules are displaced by AMD, resulting in a decrease of reduction peak current of MB. The difference value of the cathodic peak current showed a linear relationship with the AMD concentration.     

Increased cytotoxicity and phototoxicity in the methylene blue series via chromophore methylation

The cytotoxic and photodynamic activities of the commercially-available biological stains methylene blue (MB), 1,9-dimethyl MB (Taylor's Blue) and a newly synthesised compound, 1-methyl MB, were measured against the murine mammary tumour cell line, EMT-6 Both 1-methyl MB and 1,9-dimethyl MB exhibited increased dark toxicity with concomitant higher phototoxicity compared to MB at a light dose of 7.2 J cm⁻². While increasing the light dose as a function of the fluence rate increased the photocytotoxicity of MB, this had little effect on the methylated derivatives. In vitro chemical testing proved that successive methylation rendered the phenothiazinium chromophore both more resistant to reduction to its inactive leuco form, and also led to increased levels of singlet-oxygen production, thus providing a possible explanation for the increased toxicities of the methylated derivatives. Comparisons are made with the benzo[a]phenothiazinium photosensitizer, EtNBS.     

Surface‐Confined Electropolymerization of Methylene Blue on Gold Electrodes

We report on the electrochemical behaviour and electropolymerization of self‐assembled monolayers (SAMs) of methylene blue (MB) on gold electrodes. The SAMs of MB on gold electrodes were prepared by immersing the substrates into a solution of 1.0 mM MB in absolute ethanol for different times at room temperature. Cyclic voltammetry experiments exhibited that reductive desorption of MB monolayer takes place at three different potentials on polycrystalline gold electrodes, while reductive desorption of MB monolayer consists of only one peak on single crystal Au(111) substrates. Calculated charge densities for different immersion times indicated that optimal immersion time for self‐assembly of MB is 96 h. Electropolymerization of SAMs of MB on gold electrode was achieved by applying 0.95 V for 1 s in 0.1 M borate buffer solution (pH: 9.0). It was observed that poly(MB) monolayers are highly stable in acidic media. ATR‐FTIR and UV‐vis spectra exhibited differences between monomer and polymer monolayers, which are attributed to surface‐confined electropolymerization. STM image of poly(MB) monolayer on Au(111) substrate revealed a surface that is covered by well‐ordered, collateral nanowires with an average size of 3 nm.     

Methylene blue mediated photodynamic therapy in experimental colorectal tumors in mice

Methylene blue (MB+) is a well-known dye in medicine and has been discussed as an easily applicable drug for the topical treatment during photodynamic therapy (PDT). The therapeutic response of MB+ was investigated in vivo by local injection of MB+ in a xenotransplanted subcutanous tumor (adeno-carcinoma, G-3) in female nude mice. MB+ in a concentration of 1% was applied both undiluted and diluted to 0.1 and 0.01% with isotonic sodium chloride. Treatment with 1% MB+ and subsequent irradiation at 662 nm with 100 J/cm2 led to complete tumor destruction in 79% of the treated animals. A decrease of the fluence rate from 100 to 50 mW/cm2 increased the phototoxic response as well as fractionated light application. Small sensitizer concentrations reduced the PDT effect significantly. It seems that the light induced reaction of MB+ could be correlated with the rapid production of reactive oxygen species. Below a threshold dose of MB+ oxidative damage of the tissue is prevented. However, above this dose, as a point of no return, MB+ acts as an extremely potent oxidant.     

Ignition and combustion characteristics of compound of magnesium and boron

Compound of magnesium and boron (MB) is promising to be the ideal substitute of amorphous boron which is usually used as the raw material of boron-based fuel-rich propellants. In this study, the physical characteristics of MB and amorphous boron were studied by the scanning electron microscope, X-ray diffraction and X-ray photoelectron spectroscopy. The thermal reaction characteristics and the ignition and combustion characteristics were investigated through TG/DSC experiments and laser ignition experiments. The experimental results show that the MB particle is much more regular than amorphous boron, which favors for the preparation of boron-based fuel-rich propellants. Magnesium exists in the form of elementary substance, and boron oxide is produced during the preparation of MB which results in the longer ignition delay time of MB. The content of magnesium and the pressure have effects on the MB combustion performance. Although the combustion of magnesium can provide much heat for the combustion of boron, MB with moderate content of magnesium shows the best combustion performance. On the contrast, the ignition delay time of MB is independent on the content of magnesium and the pressure.     

Effects of Experimental Conditions and Kinetics of Photodegradation of Methylene Blue over TiO2

The authors investigated the catalytic activity of TiO2 for methylene blue（MB） degradation under solar light.The reaction parameters such as reaction time,TiO2 content,temperature,pH,MB concentration and light irradiation were in attention.Then,the experimental data was analyzed to investigate the adsorption order and adsorption model.The results indicate that the optimum conditions for the removal of MB are a TiO2 content of 0.5 g/L,0.50 mg/L MB solution,a temperature of 30 ℃ and reaction time of 60 min.It was found that the amount of MB removal was decreased when the pH and temperature increased.This suggests that the removal process is exothermic.However,the solar light irradiation plays a vital role in enhancing the removal amount of MB.In the dark reaction,the ability of TiO2 to remove MB was increased when the pH increased.The kinetics studies confirm that the adsorption of MB is the Pseudo-second-order.And the adsorption model was fitted with the Freundlich isotherm.     

Anticancer Photodynamic Therapy Properties of Sulfur‐Doped Graphene Quantum Dot and Methylene Blue Preparations in MCF‐7 Breast Cancer Cell Culture

Photodynamic therapy (PDT) is a field with many applications including chemotherapy. Graphene quantum dots (GQDs) exhibit a variety of unique properties and can be used in PDT to generate singlet oxygen that destroys pathogenic bacteria and cancer cells. The PDT agent, methylene blue (MB), like GQDs, has been successfully exploited to destroy bacteria and cancer cells by increasing reactive oxygen species generation. Recently, combinations of GQDs and MB have been shown to destroy pathogenic bacteria via increased singlet oxygen generation. Here, we performed a spectrophotometric assay to detect and measure the uptake of GQDs, MB and several GQD‐MB combinations in MCF‐7 breast cancer cells. Then, we used a cell counting method to evaluate the cytotoxicity of GQDs, MB and a 1:1 GQD:MB preparation. Singlet oxygen generation in cells was then detected and measured using singlet oxygen sensor green. The dye, H₂DCFDA, was used to measure reactive oxygen species production. We found that GQD and MB uptake into MCF‐7 cells occurred, but that MB, followed by 1:1 GQD:MB, caused superior cytotoxicity and singlet oxygen and reactive oxygen species generation. Our results suggest that methylene blue's effect against MCF‐7 cells is not potentiated by GQDs, either in light or dark conditions.     

Signal-on electrochemical Y or junction probe detection of nucleic acid

A methylene-blue (MB)-labeled molecular beacon junction probe allows for a signal-on electrochemical detection of nucleic acids via target recycling using endonucleases. Electron transfer is reduced when the MB is intercalated in the stem of the molecular beacon, but then electron transfer from MB to a gold electrode is enhanced upon cleavage of the junction probe due to increased probability of MB approaching the electrode when attached to the more flexible ssDNA.     

Spectroscopic studies of the interaction between methylene blue-naphthol orange complex and anionic and cationic surfactants

Initially, the absorbance of MB at 665 nm decreased rapidly with increasing sodium dodecyl sulfate (SDS) concentration, but increased gradually above about 2.3x10(-3) M. However, the absorbance of MB was almost independent of the cetyltrimethylammonium bromide (CTAB) concentration. On the other hand, the absorbance of NpO was affected by addition of CTAB in a similar manner to the change in MB-SDS system, but not by addition of SDS. The absorption band of MB showed a small red-shift and a decrease in intensity upon addition of NpO, while that of NpO showed only the decrease in the intensity upon the addition of MB. These spectral changes were attributed to the formation of complexes between these dyes. The effects of anionic and cationic surfactants on the absorption spectra of mixtures of MB and NpO were also examined. Contrary to the effects on the absorption spectrum of MB and NpO, respectively, the absorbances of MB and NpO in the mixtures were changed with the additions of both anionic and cationic surfactants. The spectral changes were explained by changes in the forms of MB and NpO in the solutions with increasing surfactant concentrations.     

亚甲蓝在分子有序组合体中的聚集行为

研究了生物染色剂亚甲蓝(MB)在分子有序组合体(胶束、O/W微乳液和层状液晶)介质中的聚集行为。根据MB特征峰处的吸收光谱值,计算了MB的二聚平衡常数和相应单体、二聚体的摩尔消光系数。并用稳态荧光法和无外加探针的循环伏安法进一步论证了计算结果。研究发现,当MB总浓度一定时,在胶束体系中MB单体量较少,在层状液晶体系中单体量较多,在O/W微乳液体系中则介于胶束和层状液晶之间。表明层状液晶体系是获取更多MB单体的理想介质。     

Voltammetric Study of Methylene Blue at Thiol SAMs‐Modified Gold Electrodes

The voltammetric behavior of methylene blue (MB) at thiol self‐assembled monolayers modified gold electrodes (SAMs/Au) has been investigated. MB exhibited a redox peak at about ?0.35 V (vs.SCE) in alkaline solution at bare gold electrodes. When the gold electrodes were modified with thiol SAMs, the peak grew due to the accumulation of MB at SAMs. With the solution pH rising, more MB was accumulated, hence the peak height increased, which differed from that at bare gold electrodes. The electrode process at SAMs/Au featured the characteristics of adsorption and/or electrode reaction controlled. The enhancing action of glutathione monolayer (GSH SAM), 3‐mercaptopropionic acid monolayer (3MPA SAM) and other thiol SAMs was compared. Among these, GSH SAM made the MB peak increase more. At GSH SAM/Au, the peak height varied linearly with MB concentration over the range of 2 μM to 400 μM. So this can be developed for the determination of MB and studies concerned. The accumulation behavior caused by GSH SAM and native fish sperm dsDNA was compared. The interaction between DNA and MB was also discussed under this condition.     

A Novel and Selective Methylene Blue Imprinted Polymer Modified Carbon Paste Electrode

In this study, methylene blue (MB) imprinted microbeads were synthesized and characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM).Differential pulse voltammetric responses of carbon paste electrodes modified with MB imprinted polymer were used to evaluate the adsorption and selectivity features of the polymer. For selectivity studies two basic dyestuffs (thionine blue and toluidine blue) which have similar structure to MB were chosen. Comparison of the voltammetric responses obtained with pure carbon paste and carbon paste modified with either imprinted or nonimprinted polymer electrodes revealed that MB imprinted polymer presented a higher selectivity to the template molecule MB in contrast to structurally similar molecules, thionine blue and toluidine blue.