Interactions of Nile Blue with Micelles, Reverse Micelles and a Genomic DNA

In this contribution we report studies on the nature of binding of a small ligand/drug Nile blue (NB) with sodium dodecyl sulfate (SDS) micelles, bis-(2-ethylehexyl) sulfosuccinate (AOT)/isooctane reverse micelles (RM) and a genomic DNA extracted from Salmon sperm. With detailed steady state and picosecond resolved optical spectroscopic techniques, we examined the fluorescence quenching of the ligand upon complexation with the SDS monomers and DNA. Polarization analyzed picosecond-resolved fluorescence measurements reveal geometrical restriction on the probe in SDS micelles, AOT-RM and DNA. Steady state and time resolved studies on the probe in nanocages of AOT RM with various degrees of hydration (w₀) reveal the existence of NB as two distinct species namely, neutral and cationic. This study confirms that the emission of NB in aqueous micelles and DNA solution is due to the cationic form of the drug. Our experiments clearly identified non-specific electrostatic and intercalative modes of interaction of the probe with the DNA at lower and higher DNA concentrations respectively. The nature of binding of NB in presence of the DNA and SDS micelles reveals that the binding affinity of the probe is higher with the micelles than with the DNA. The complex rigidity of NB with DNA and its fluorescence quenching with DNA elucidate a strong recognition mechanism between NB and DNA.     

Combined Quenching Mechanism of Anthracene Fluorescence by Cetylpyridinium Chloride in Sodium Dodecyl Sulfate Micelles

The Stern-Volmer quenching constant (K_SV) for quenching of anthracene fluorescence in sodium dodecyl sulfate (SDS) micelles by pyridinium chloride has been reported previously to be 520 M^?1 based on steady state fluorescence measurements. However, such measurements cannot distinguish static versus dynamic contributions to the overall quenching. In the work reported here, the quenching dynamics of anthracene in SDS micelles by cetylpyridinium chloride (CPC), an analogue of pyridinium chloride, were investigated using both steady state and time resolved fluorescence quenching. Concurrent measurement of the decrease in fluorescence intensity and lifetime of anthracene provide a quantitative evaluation of collision induced (i.e. dynamic) versus complex formation (i.e. static) quenching of the anthracene fluorophore. The results reveal that a combined quenching mechanism is operative with approximately equal constants of 249?±?6 M^?1 and 225?±?12 M^?1 for dynamic and static quenching, respectively.     

Fluorescence study of the core/shell interface in polyelectrolyte micelles. Binding of fluorescent surfactants in the interfacial region

Properties of the interfacial region between the nonpolar core and the polar shell in polystyreneblock-poly (methacrylic acid) micelles were studied by fluorescence techniques using 5-(N-octadecanoyl) aminofluorescein (OAF) as a probe for microfluidity and local pH. The block copolymer used was tagged between blocks by one 9, 10-diphenylanthracene (DPA) group, which allowed us to study binding of OAF at the interface by means of nonradiative energy transfer between DPA and OAF. A shift in the pK _a of OAF and appreciable changes in anisotropy and quenching efficiency due to immobilization of the fluorophore head-group in hydrophobic poly(methacrylic acid) domains were observed after binding of the probe at the interface.     

Synthesis,Characterization, DNA Binding Properties,Fluorescence Studies and Antioxidant Activity of Transition Metal Complexes with Hesperetin-2-hydroxy Benzoyl Hydrazone

A novel Schiff-base ligand (H₅L), hesperetin-2-hydroxy benzoyl hydrazone, and its copper (II), zinc (II) and nickel (II) complexes (M·H₃L) [M(II) = Cu, Zn, Ni], have been synthesized and characterized. The ligand and Zn (II) complex exhibit green and blue fluorescence under UV light and the fluorescent properties of the ligand and Zn (II) complex in solid state and different solutions were investigated. In addition, DNA binding properties of the ligand and its metal complexes have been investigated by electronic absorption spectroscopy, fluorescence spectra, ethidium bromide displacement experiments, iodide quenching experiments, salt effect and viscosity measurements. Results suggest that all the compounds bind to DNA via an intercalation binding mode. Furthermore, the antioxidant activity of the ligand and its metal complexes was determined by superoxide and hydroxyl radical scavenging methods in vitro. The metal complexes were found to possess potent antioxidant activity and be better than the free ligand alone and some standard antioxidants like vitamin C and mannitol.     

Aggregation and microenvironmental properties of gemini and conventional mixed surfactants systems: A fluorometric study

Aggregation behavior of cationic gemini (hexanediyl-1,5-bis(dimethylcettylammonium bromide) (16-5-16)) surfactant with conventional single chain surfactants cetyltrimethylammonium bromide (CTAB) and tetradecyltrimethylammonium bromide (TTAB) were studied with the help of fluorescence measurements. Fluorescence probe is a proficient technique for examining the surfactant-surfactant interaction and aggregation. The micelle aggregation number (N _agg) was measured using steady-state fluorescence quenching method. The micelle aggregation numbers of binary combinations fall between those of constituent surfactants. The micropolarity (I ₁/I ₃), binding constant (K _sv) and dielectric constant (D _exp) of mixed systems were determined from the ratio of peaks intensity in the pyrene fluorescence spectrum. The I ₁/I ₃ values were found to be more than >1, showing more polar environment around pyrene in the mixed micelle as compared to the pure micelles.     

Elucidation of the Binding Properties of A Photosensitizer to Salmon Sperm DNA and Its Photobleaching Processes by Spectroscopic Methods

Methylene blue (MB) is a tricyclic heteroaromatic photosensitizer with a promising application in the photodynamic therapy (PDT) for anticancer treatment. The binding properties of MB to salmon sperm DNA have been investigated by the measurements of absorption spectra, quenching experiments and 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 K _b value of 5.6?×?10³?M^?1. The photobleaching processes of MB and its DNA complex have also been studied, which indicate that the photobleaching of MB and its DNA complex proceed with different mechanisms and the reactive oxygen species are responsible for the self-sensitized photooxidation of MB.     

Modulated photophysics of 2-anthracene sulphonate in micellar assembly

The association of a non-ionic surfactant of polyoxyethylene-p-(1,1,3,3-tetramethylbutyl)phenyl ether (Triton X) series with 2-AS in aqueous solution has been studied by means of steady-state, time-resolved fluorescence and fluorescence anisotropy techniques. The effect of the hydrophobic chain length on the structural dynamism of the fluorophore has been reported. Experimental results demonstrate that the equilibrium of this dynamism is sensitive to the environment. The association constant of the probe molecule with the non-ionic micelles of Triton X (TX), location of the probe in the micellar environment, have been determined from the change in emission characteristics of the probe as a function of surfactant concentration. The rate constant of quenching and mode of quenching of probe in micellar media have been ascertained. Quantitative estimates of the micropolarity at the binding sites of the probe molecule have been determined. Some of the environment-dependent relevant fluorescence parameters, fluorescence anisotropy (r), have been monitored for exploring the imposed motional restriction of the microenvironment around the probe. An attempt has been made to correlate the steady-state results with time resolved study.     

Study of Protein–Probe Interaction and Protective Action of Surfactant Sodium Dodecyl Sulphate in Urea-Denatured HSA using Charge Transfer Fluorescence Probe Methyl Ester of N,N-Dimethylamino Naphthyl Acrylic Acid

We have demonstrated that the intramolecular charge transfer (ICT) probe Methyl ester of N,N-dimethylamino naphthyl acrylic acid (MDMANA) serves as an efficient reporter of the proteinous microenvironment of Human Serum Albumin (HSA). This work reports the binding phenomenon of MDMANA with HSA and spectral modulation thereupon. The extent of binding and free energy change for complexation reaction along with efficient fluorescence resonance energy transfer from Trp-214 of HSA to MDMANA indicates strong binding between probe and protein. Fluorescence anisotropy, red edge excitation shift, acrylamide quenching and time resolved measurements corroborate the binding nature of the probe with protein and predicts that the probe molecule is located at the hydrophobic site of the protein HSA. Due to the strong binding ability of MDMANA with HSA, it is successfully utilized for the study of stabilizing action of anionic surfactant Sodium Dodecyl Sulphate to the unfolding and folding of protein with denaturant urea in concentration range 1M to 9M.     

Modulation of Intramolecular Charge Transfer Emission Inside Micelles: A Fluorescence Probe for Studying Microenvironment of Micellar Assemblies

Modulation of intramolecular charge transfer reaction of ethyl ester of N,N-Dimethylaminonaphthyl-(acrylic)-acid (EDMANA) in anionic sodium dodecyl sulfate (SDS), cationic cetyltrimethylammonium bromide (CTAB) and non-ionic p-tert-octylphenoxy polyoxyethanol (Triton-X 100, TX-100) micelles has been addressed using steady state and time resolved spectroscopy. The interaction of the CT probe EDMANA with micelles and its location inside the micelles have been investigated by the study of fluorescence spectral band position of EDMANA in micelle, the effective polarity of micelle-water interface and cetyl pyridinium chloride induced fluorescence quenching measurement. The effects of urea on the properties of the micelles such as Critical Micelle Concentration and the interaction between EDMANA and micelles have been explored using EDMANA as emission probe.     

Spectroscopic Characterization of the Interaction of Phenosafranin and Safranin O with Double Stranded, Heat Denatured and Single Stranded Calf Thymus DNA

Interaction of phenosafranin and safranin O with double stranded, heat denatured and single stranded calf thymus DNA has been studied by fluorescence, absorbance and circular dichroic techniques. Binding to the double stranded and heat denatured DNA conformations induced strong quenching in the fluorescence spectra of both dyes. Linear Scatchard plots indicated the binding to be of one type and the affinity evaluated to be of the order of 10(5) M(-1) with double stranded and heat denatured DNAs. Fluorescence quenching was much weaker with the single stranded DNA and the binding affinity was one order lower. Ferrocyanide quenching studies revealed that the fluorescence emission of the dye molecules bound to the double stranded and heat denatured DNAs was quenched much less compared to that bound to the single stranded DNA. Further, there was significant emission polarization for the bound dyes and strong energy transfer from the DNA base pairs to the dye molecules indicating intercalative binding. Salt dependence of the binding phenomenon revealed that electrostatic forces have significant role in the binding process. The intercalation of these molecules to double stranded and heat denatured DNA and simple stacking to single strands was proved by these fluorescence techniques. Support to the fluorescence results have been derived from absorption and circular dichroic results. Phenosafranin was revealed to be a stronger binding species compared to safranin O.     

Enhancement of the Fluorescence Quenching Efficiency of DPPH<Superscript>•</Superscript> on Colloidal Nanocrystalline Quantum Dots in Aqueous Micelles

Luminescent CdS quantum dots capped with thioglycolic acid (CdS-TGA QDs) were demonstrated to serve as a fluorescence probe for a model organic radical, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH^•), employing the quenching of the CdS-TGA QDs emission signal by the radical. Under the optimum conditions, the quenching efficiency of DPPH^• on CdS-TGA QDs was proportional to the concentration of DPPH^•, following Stern-Volmer relationship. Different types of surfactants (cationic, anionic and neutral surfactants) were introduced to CdS-TGA QDs in order to increase the detection sensitivity. The fluorescence intensity of CdS-TGA QDs was greatly enhanced by cationic and neutral surfactants. Moreover, the quenching efficiency of DPPH^• on the QDs in the presence of micelles was remarkably ca. 13 times higher than that in the system without micelles. Effects of pH and concentration of surfactants on the fluorescence quenching of CdS-TGA QDs were investigated. Electron spin resonance (ESR) spectroscopy was also used to monitor the DPPH radical species in CdS-TGA QDs mixtures with and without micelles. Fluorescence quenching mechanisms of CdS-TGA QDs by DPPH^• in the presence and in the absence of CTAB were proposed.     

Interactions between imazethapyr and bovine serum albumin: Spectrofluorimetric study

The interaction between imazethapyr (IMA) and bovine serum albumin (BSA) was investigated by fluorescence spectroscopy. The Stern–Volmer quenching constant (K_SV) at three temperatures was evaluated in order to determine the quenching mechanism. The dependence of fluorescence quenching on viscosity was also evaluated for this purpose. The results showed that IMA quenches the fluorescence intensity of BSA through a static quenching process. The values of the binding constant for the formed BSA–IMA complex and the number of binding sites were found to be 1.51×10⁵ M^?1 and 0.77, respectively, at room temperature. Based on the calculated thermodynamic parameters, the forces that dominate the binding process are hydrogen bonds and van der Waals forces, and the binding process is spontaneous and exothermic. The quenching of protein fluorescence by iodide ion was used to probe the accessibility of tryptophan residues in BSA and the change in accessibility induced by the presence of IMA. According to the obtained results, the BSA–IMA complex is formed in the site where the Trp-134 is located, causing it to become less exposed to the solvent.     

Characterization of the Binding of Adriamycin to Dna by Spectroscopic Methods

Adriamycin(ADM) binds to the double helical DNA with a high affinity, as deduced from the absorption and fluorescence spectral data. Extensive hypochromism, red shifts, and an isosbestic point in the absorption spectra were observed when ADM binds to calf thymus DNA(CT DNA), which suggested the intercalation mechanism of ADM into DNA bases. Upon binding to DNA, the fluorescence from ADM 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 ADM into the helix. Iodide quenching studies showed that the magnitude of K_sv of the bound ADM was lower than that of the free ADM. the results of competitive binding studies showed that ethidium bromide could be displaced by Adm. Thermal denaturation experiments exhibited that the quenching of the fluorescence from ADM by single strand(ssDNA) was smaller than that by double strand(dsDNA). the results of all further studies also proved the intercalation of ADM into DNA base stack.     

A quenching fluoroimmunoassay for analysis of the pesticide propazine in an apolar organic solvent, reverse micelles of AOT inn-octane: Effect of the micellar matrix and labeled antigen structure

A simple way of directly observing antigen-antibody binding in a reverse micellar system,n-octane containing reverse micelles of aerosol OT (AOT), using the hydrophobic pesticide propazine as antigen, is described. We observed two processes during fluorescein-labeled propazine (FP)-antibody (Ab) interaction in reverse micelles: (1) quenching of the fluorescence of FP after mixing of Ab and FP (due immune complex formation) and (2) restoration of FP fluorescence after addition of excess propazine to the immune complex formed. We found that the quenching efficiency depends on both the properties of the reverse micellar system (surfactant concentration, hydration degreeW ₀ = [water]/[surfactant]) and the structure of the labeled antigen. A quenching fluoroimmunoassay of propazine both in apolar organic solvents and in water is developed. The method is homogeneous. The quenching time is 10–30 min, and the detection limit of propazine is 100 nM (20 Μg/L) in organic solvent and 10nM (2 Μg/L) in water. Propazine can be added to the reverse micellar system when dissolved in AOT/octane, or in an octane/chloroform mixture, or in chloroform. This makes possible the use of the analysis directly for pesticide extracts in nonpolar organic solvents.     

Quenching of Long Lifetime Emitting Fluorophores with Paramagnetic Molecules

In this work, we have studied quenching of the fluorescence of two well-known oxygen probes, 1-pyrene butyric acid (PBA) and tris(2,2’-bipyridine)ruthenium ([Ru(bpy)₃]²⁺) by reactive oxygen species (superoxide anion, nitric oxide derivative, hydrogen peroxide) and by the O₂ molecule. Both, time-resolved and steady state fluorescence measurements were performed in solution (ethanol, dimethyl sufoxide, water) and in micelles of Sodium Dodecyl Sulfate that serve as a model for membrane-containing biological structures. We have found that only the free radicals and O₂ can actively quench for the two probes, but not the diamagnetic H₂O₂. Our data correspond to the classical Stern–Volmer equation. H₂O₂ has an effect only at high molar concentrations (>0.1 M). In contrast, effective concentrations of free radicals and O₂ that lead to quenching are in millimolar range. In conclusion, our methods allows for detecting global ROS that are small free radicals without interference from the reactive hydroxyl radical. Our data suggest that the method can be used for the quantification of ROS in individual living cells based on the measurement of fluorescence lifetime of those probes.     

Determination of the Constants of Binding of Acridine Dyes with Micelles of Sodium Dodecyl Sulfate Using the Quenching of Delayed Fluorescence by Heavy Atoms

The constants of binding dye molecules with the micelles of sodium dodecyl sulfate are determined using quenching of delayed fluorescence of acridine dyes by sodium iodide in aqueous–micellar solutions. Kinetic equations have been composed that describe the processes of deactivation of the excited states of dyes. By solving these equations at the concentration of the quencher sodium iodide corresponding to the minimum lifetime of triplet states and at the concentration of micelles corresponding to the least value of the delayed fluorescence quenching rate constants, we obtained the constants of binding dyes with micelles equal to 1.3·10⁷, 2.9·10⁷, and 3.1·10⁷ M^–1 for trypaflavine, acridine orange, and acridine yellow, respectively. We calculated the rate constants of quenching of the triplet states of the molecules of dyes by iodide ions (I ^–) that decreased in transition from trypaflavine to acridine orange and acridine yellow.     

Study of the Interaction Between Apis mellifera Venom and Micro-Heterogeneous Systems

The bee venom, used in treatment of inflammatory and articular diseases, is a complex mixture of peptides and enzymes and the presence of tryptophan allows the investigation by fluorescence techniques. Steady state and time-resolved fluorescence spectroscopy were used to study the interaction between bee venom extracted from Apis mellifera and three micro heterogeneous systems: sodium dodecylsulphate (SDS) micelles, sodium dodecylsulphate-poly(ethylene oxide) (SDS-PEO) aggregates, and the polymeric micelles LUTROL^® F127, formed by poly(ethylene oxide)-poly(propylene oxide)- poly(ethylene oxide). Fluorescence parameters in buffer solution were typical of peptides containing tryptophan exposed to the aqueous medium, and they gradually changed upon the addition of surfactant and polymeric micelles, demonstrating the interaction of the peptides with the micro heterogeneous systems. Quenching experiments were carried out using the N-alkylpyridinium ions (ethyl, hexyl, and dodecyl) as quenchers. In buffer solution the quenching has low efficiency and is independent of the alkyl chain length of the quencher. In the presence of the micro heterogeneous systems the extent of static and dynamic quenching enhanced, showing that both fluorophore and quenchers reside in the microvolume of the aggregates. The more hydrophobic quencher (dodecyl pyridinium ion) provides higher values for K _SV and dynamic quenching constants, and SDS-PEO aggregates are most efficient to promote interaction between peptides and alkyl pyridinium ions. The results proved that bee venon interacts with drug delivery micelles of the copolymer LUTROL^® F127.     

Detection of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> Carrying the Gene for Toxic Shock Syndrome Toxin 1 by Quantum-Dot-Probe Complexes

In this study, a high-sensitive and high-specific method to detect the toxic shock syndrome toxin-1 (TSST-1)-producing Staphylococcus aureus was developed based on quantum dot (QD) and oligonucleotide probe complexes. S. aureus carrying tst gene which is responsible for the production of TSST-1 were detected based on fluorescence resonance energy transfer (FRET) occurring between CdSe/ZnS QD donors and black hole quencher (BHQ) acceptors. QD-DNA probe was prepared by conjugating the carboxyl-modified QD and the amino-modified DNA with the EDC. Photoluminescence (PL) quenching was achieved through FRET after the addition of BHQ-DNA which was attached to tst gene probe by match sequence hybridization. The PL recovery was detected in the presence of target DNA by BHQ-DNA detached from QD-DNA probe because of the different affinities. In contrast, mismatch oligonucleotides and DNAs of other bacteria did not contribute to fluorescence intensity recovery, which exhibits the higher selectivity of the biosensor. The experimental results showed clearly that the intensity of recovered QD PL is linear to the concentration of target DNA within the range of 0.2–1.2 μM and the detection limit was 0.2 μM.     

Interaction Between Methylene Blue and CalfThymus Deoxyribonucleic Acid by Spectroscopic Technologies

Characterization of the interaction between methylene blue (MB) and calf thymus deoxyribonucleic acid (ctDNA) was investigated by UV absorption spectra, fluorescence spectra, fluorescence polarization and fluorescence quenching experiments by ferrocyanide. The above results indicated that the binding modes of MB to ctDNA were relative to the molar ratio γ (γ=[DNA]/[MB]). At low γ ratios (γ < 4), remarkable hypochromic effect with no shift of λ_max in the absorption spectra of MB was observed in the presence of increasing amounts of ctDNA, the fluorescence of MB was efficiently quenched by the ctDNA bases and the fluorescence polarization of MB was slightly increased, which indicated that MB cations bound to phosphate groups of ctDNA by electrostatic interaction and then stacked on the surface of ctDNA helix. While at high γ ratios (γ > 6), besides the fluorescence of MB was quenched efficiently by the ctDNA bases, a red shift (about 3 nm) in the absorption spectra of MB was observed and the fluorescence polarization of MB was obviously increased, which indicated the intercalation binding that MB molecules were intercalated into the space of two neighbouring DNA base pairs was the preferred mode. Effects of K₄Fe(CN)₆ on the fluorescence quenching of the MB-ctDNA system at low and high γ ratios were also performed. The results showed that at γ = 1.7, the quenching effect by ferrocyanide was higher than that of pure MB, while at γ = 13.6 a decreased quenching of the fluorescence intensity was observed as compared with that of pure MB, which further proved the above conclusion. In addition, the mechanisms of the hypochromic effect and the fluorescence quenching were also discussed in detail.     

Spectrophotometric studies on the binding of Vitamin C to lysozyme and bovine liver catalase

The patterns of Vitamin C (ascorbic acid) binding to lysozyme (LYSO) and bovine liver catalase (BLC) were investigated at 298, 308 and 316 K at pH 7.40 using spectrophotometric techniques. The quenching mechanism, binding constant and the number of binding sites were determined by fluorescence experiments. Moreover, the Stern-Volmer fluorescence quenching constant (K_SV) of LYSO by Vitamin C was more sensitive to the temperature changes than that of BLC by Vitamin C. The thermodynamic data suggest that hydrogen bonds were the predominant intermolecular forces in the binding reaction. The effect of Vitamin C on the conformation of LYSO or BLC was analyzed using synchronous fluorescence, UV-vis absorption and circular dichroism (CD) spectra.