Detection of Polymolecular Associations in Hydrophobized Chitosan Derivatives using Fluorescent Probes

The microenvironment formed by lauroyl and stearoyl derivatives of chitosan in solution has been studied using two fluorescent probes, pyrene and nabumetone. Existence or not of microdomains formed by polymolecular associations, the inherent hydrophobicity of them in aqueous solution, and the influence of degree of substitution (DS) of derivatives were investigated by emission properties of pyrene and strengthened by the photophysical behavior of nabumetone. Additionally, the ratio between the fluorescence intensities of first (~372 nm) to the third (~384 nm) bands of the emission spectrum of pyrene was used to determine the critical aggregation concentration (CAC). In a previous work, it was already reported the characterization of chitosan derivatives by three spectroscopic techniques (¹³C-NMR, ¹H-NMR and infrared), as well as data on the solubility and swelling-index of them. In addition of that, the new results show that the investigated lauroyl and stearoyl derivatives of chitosan are expected to be potential models for applications in the medical field.     

Determining the binding affinity and binding site of bensulfuron-methyl to human serum albumin by quenching of the intrinsic tryptophan fluorescence

Bensulfuron-methyl (BM) is a highly active sulfonylurea herbicide for use on paddy rice. Steady state fluorescence, UV/vis absorption, circular dichroism (CD), time-resolved fluorescence and molecular modeling methods have been exploited to determine the binding affinity and binding site of BM to human serum albumin (HSA). From the synchronous fluorescence, UV/vis, CD and three-dimensional fluorescence spectra, it was evident that the interaction between BM and HSA induced a conformational change in the protein. Steady state and time-resolved fluorescence data illustrates that the fluorescence quenching of HSA by BM was the formation of HSA-BM complex at 1:1 molar ratio. Site marker competitive experiments demonstrated that the binding of BM to HSA primarily took place in subdomain IIIA (Sudlow’s site II), this corroborates the hydrophobic probe ANS displacement and molecular modeling results. Thermodynamic analysis displays hydrophobic, electrostatic and hydrogen bonds interactions are the major acting forces in stabilizing the HSA-BM complex.     

芘荧光探针法研究环糊精与有机溶剂的极性微环境性质

基于芘的单体荧光光谱振动结构与其所处环境极性的相关性 ,考察了有机溶剂对环糊精 (α- CD、β-CD)空腔微环境的影响。结果表明 ,在所考察的有机溶剂体系里 ,芘的 I3 / I1 与介电常数呈线性相关 ,可用于探测微环境的极性性质变化规律 ;芘和有机溶剂在疏水驱动力的作用下簇集到 CD周围 ,形成一个有序的有利发光的簇集体系 ,CD与有机溶剂之间存在着协同效应 ,而不是加合效应 ;苯较大地改变了微环境的性质 ,使芘的荧光精细结构消失 ,强度增强。环糊精与有机溶剂间的特殊协同增敏效应和环境的极性有极大的关系。     

Synthesis and Optical Properties of Various Thienyl Derivatives of Pyrene

A series of various thienyl derivatives of pyrene were synthesized by Stille cross-coupling procedure. Their structures were characterized by ¹H NMR, ¹³C NMR and elemental analysis. The spectroscopic characteristics were investigated by UV–vis absorption and fluorescence spectra. Based on quantum chemical calculations, the energy levels of investigated molecules with respect to the pyrene molecule were also discussed.     

From Futile to Fruitful: Diesel Soot as White Light Emitter

The present work describes a solution for the effective use of the hazardous particulate matter (diesel soot) from the internal combustion engines (ICEs) as a potential material emitting white light for white light emitting diodes (WLEDs). The washed soot samples are subjected to Field Emission Scanning Electron Microscopy (FESEM), High- Resolution Transmission Electron Microscopy (HR-TEM), Energy Dispersive Spectroscopy (EDS), UV–Visible, Photoluminescent (PL) Spectroscopy and quantum yield measurements. The CIE plot and Correlated Color Temperature (CCT) reveals the white fluorescence on photoexcitation. The sample on ultraviolet (UV) laser excitation, provides a visual confirmation of white light emission from the sample. The diesel soot collected from public transport buses of different years of manufacture invariably exhibit white fluorescence at an excitation of 350 nm. The sample show a quantum yield of 47.09%. The study is significant in the context of pollution and search for low-cost, rare-earth phosphor free material for white light emission and thereby turning the hazardous, futile material into a fruitful material that can be used for potential applications in photonics and electronics.     

Combined molecular docking and multi-spectroscopic investigation on the interaction between Eosin B and human serum albumin

The binding of Eosin B to human serum albumin (HSA) was studied using molecular docking, fluorescence, UV–vis, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopy. The mechanism of interaction between Eosin B and HSA in terms of the binding parameters, the thermodynamic functions and the effect of Eosin B on the conformation of HSA were investigated. Protein-ligand docking study indicated that Eosin B bound to residues located in the subdomain IIA of HSA and Eosin B–HSA complex was stabilized by hydrophobic force and hydrogen bonding. In addition, fluorescence data revealed that Eosin B strongly quenched the intrinsic fluorescence of HSA through a static quenching procedure. Furthermore, alteration of the secondary structure of HSA in the presence of the dye was conformed by UV–vis, FT-IR and CD spectroscopy.     

荧光光谱法检测非水体系中蒽和芘

用荧光光谱法检测建立了非水体系中蒽和芘的测试方法,蒽和芘的检测线性范围分别为5.6×10^-9—2.8×10^-6mol/L和4.9×10^-8—1.47×10^-6mol/L,相关系数为0.99959和0.99822,检出限分别为0.356ng/mL,5.230ng/mL,回收率分别为86.61%—98.66%和96.60%—115.80%。考察了常见离子、时间以及温度对荧光强度的影响,结果表明,常见离子对分析检测干扰均不大,可忽略,在2h、5—55℃温度范围内,对荧光强度影响较弱,故此分析检测方法对温度的适应范围较宽。方法可直接用于食品、饮料、酒产品等非水体系中的蒽和芘的测定。     

光谱法研究葛根素与牛血清白蛋白的相互作用

采用紫外、同步荧光和圆二色光谱法研究了葛根素（PUE）与牛血清白蛋白（BSA）的相互作用.紫外光谱表明,BSA在230nm和278nm处的吸收峰,随着葛根素浓度的增加而减小.同步荧光光谱表明,葛根素引起BSA中色氨酸残基所处微环境的疏水性降低.圆二色光谱表明,BSA在208nm和222nm处的负峰随着葛根素浓度的增加而增强,BSA中α-螺旋含量也随之增加.这表明葛根素与BSA的相互作用,可使蛋白质分子的疏水作用增强,导致BSA的肽链结构收缩,蛋白质的构象发生变化.     

Cu(II) recognition materials: Fluorophores grafted on mesoporous silica supports

There were designed and synthesized naphthalene and pyrene derivatives consisting of fluorophore group and of receptor fragment with donor N and O atoms. These fluorosensors were covalently attached by grafting carboxyl group to surfaces of silica xerogel or mesoporous silicas (MCM-41 and MCM-48) functionalized either with 3-aminopropyl or 3-glycidoxypropyl groups. The pyrene derivatives 2 and 3 covalently grafted on MCM-48 silica functionalized with 3-aminopropyl groups are potential recognition elements of a fluorescence chemical sensor.Fluorescence emission of the prepared recognition materials is quenched specifically owing to photoinduced electron transfer (PET) effect after coordination reactions with Cu(II) ions. Moreover, both the materials exhibit selectivity for Cu(II) ions in aqueous solutions in presence of such metal ions as: alkali, alkaline earth and transition. During UV irradiation the studied recognition elements undergo slowly photochemical degradation.     

Ilaprazole metabolites,ilaprazole sulfone and ilaprazole sulfide decreased the affinity of ilaprazole to bovine serum albumin

The interaction of ilaprazole (IPZ), ilaprazole sulfone (IPZO) and ilaprazole sulfide (IPZI) with bovine serum albumin (BSA), and the effect of IPZO and IPZI on the interaction of IPZ with BSA have been investigated by fluorescence, synchronous fluorescence, ultraviolet–visible (UV–vis), Fourier transform infrared spectroscopy (FT-IR) and circular dichroism (CD). The results indicated that IPZ, IPZO and IPZI had a strong ability to quench the intrinsic fluorescence of BSA, and the binding affinities were significantly affected by structures in the order IPZ>IPZO>IPZI, while the van der Waals force and hydrogen bond played major roles in their binding with BSA. The analysis of synchronous fluorescence, FT-IR and CD spectra showed the change in secondary structure of BSA upon interaction with IPZ, IPZO or IPZI. Site marker competitive experiments indicated that their binding to BSA primarily took place in subdomain IIA. The presence of IPZO and IPZI decreased the quenching constants of IPZ with BSA by about 68.4% and 95.1%, respectively, which possibly resulted from the existence of competitive binding between IPZ and its metabolites with BSA. However, IPZO and IPZI did not change the quenching mechanism of IPZ with BSA, while all the fluorescence quenching was initiated by static quenching procedure combined with non-radiative energy transfer. Our results may have relevant insight into IPZ's bioavailability and efficacy affected by its metabolites.     

Investigation of proton pump inhibitors binding with bovine serum albumin and their relationship to molecular structure

The interactions of three proton pump inhibitors (PPIs), omeprazole, pantoprazole and ilaprazole with bovine serum albumin (BSA) have been investigated by fluorescence, synchronous fluorescence, ultraviolet–visible (UV–vis) and circular dichroism (CD). Various binding parameters have been calculated at various temperatures. The results indicated that omeprazole, pantoprazole and ilaprazole had a strong ability to quench the intrinsic fluorescence of BSA with static quenching mechanism, and the binding affinities were significantly affected by different substituents and polarities as the order ilaprazole>pantoprazole>omeprazole. The site marker competitive experiments indicated that the binding of omeprazole, pantoprazole and ilaprazole to BSA primarily took place in subdomain IIA. The results of thermodynamic parameters ΔG, ΔH and ΔS indicated that electrostatic interaction played a major role for PPIs–BSA association. The distance r between PPIs and BSA was evaluated according to the theory of Förster's energy transfer. The quantitative analysis of synchronous fluorescence and CD spectra showed the change in secondary structure of the BSA upon interaction with PPIs by a reduction of α-helix. All the above results many have relevant insight into the PPIs' availability and distribution.     

Green Fluorescent Protein (GFP) as a Marker for Cell Viability After UV Irradiation

Generation of Chinese Hamster Ovary (CHO) cell lines stably expressing green fluorescent protein (GFP) was achieved using a plasmid vector that encoded the red-shifted pCX-xGFP under the control of a strong hybrid promoter composed of a CMV enhancer and a -actin/-globin gene promoter. Cotransfection of the promoter-less pSV2-Neo helper plasmid transmitting neomycin resistance was followed by selection with the antibiotic G418. Constitutive GFP expression could be visualized in living and fixed cells using fluorescence spectroscopy, fluorescence microscopy, and flow cytometry. DNA repair-proficient (AA8) and deficient (UV5) CHO strains were used for survival tests after UVC irradiation. Cells carrying the GFP construct (AA8-pGFP, UV5-pGFP) show the same response to UV irradiation (colony forming ability) as their nontransformed parental cell lines (AA8, UV5). Using GFP as a marker for cell viability, cells were harvested after certain postirradiation growth periods and the numbers of GFP expressing cells and fluorescence intensities were determined by FACS analysis. Generally, GFP fluorescence in irradiated cells is not seen when cell membranes are damaged (leak-out of the soluble GFP). Irradiated cells without membrane damage express GFP continuously (leading to a dose-dependent increase in GFP contents).     

A fluorescence based assay for DNA damage induced by radiation,chemical mutagens and enzymes

A simple and rapid assay to detect DNA damage is reported. This novel assay is based on changes in melting/annealing behavior and facilitated using certain dyes that increase their fluorescence upon association with double stranded (ds)DNA. Damage caused by ultraviolet (UV) radiation, chemical mutagens or restriction enzymes produced an assay response. UV radiation at 254 nm (approximating UV-C) and 360 nm (approximating UV-A) were used to induce the damage in dsDNA. Chemical damage was induced using several compounds with known effects on nucleic acids. Restriction enzymes Hind III, Msp1, Sau 3A1 were used to cut the plasmid (pUC19) at specific sequences in addition to the non-specific endonuclease DNase I. The effects of these types of damage on repeated melting and annealing of dsDNA were observed in real time using several fluorescence indicator dyes. Low concentrations of dsDNA (between 10 and 100 ng/ml) and small volumes (20 μl) were required for this assay. Repeated measures yielded a coefficient of variation of 2% (CV%). In addition to measuring various DNA damaging agents, the potential application of this assay to study the efficiency of various sun blocking agents against UV-induced DNA damage is discussed.     

光谱法研究Cu~(2+)诱导的不同时间的LDL体外氧化

氧化低密度脂蛋白(oxidized low density lipoprotein,oxLDI)被认为是动脉硬化的关键致病因素.文章综合运用紫外吸收光谱、荧光光谱、同步荧光光谱及圆二色(circular diclaro‘lsm.CD)光谱研究了(好'诱导不同时间的U)IJ体外氧化过程.随着氧化时间的增加.生成的丙二醛(malonaldehyde,MDA)的量逐渐增加,其相对应的430 nm 处的荧光强度也随之加强,氧化过程中生成了新的氧化产物.氧化低密度脂蛋白紫外吸收增强;表征氨基酸残基信息的特征荧光强度和同步荧光强度降低;载脂蛋白B-100(apolipoproteinB-100,apoB-100)α-螺旋含量减少.实验结果表明,LDL氧化过程中Cu2+不仅诱导了氧化产物的生成,而且改变了apoB-100的构象进而导致了生色基团的微环境的变化.     

Sensitized Fluorescence of Silver Nanoparticles in the Presence of Pyrene

The fluorescence of pyrene adsorbed onto the surface of the cetyltrimethylammonium-coated silver nanoparticles was studied. Pyrene molecules adsorbed on freshly prepared silver particles were found to be in close proximity to silver surface thus providing the possibility of energy transfer from excited pyrene to silver cores of the particles. In that case along with the expected fluorescence of pyrene we observed the fluorescence of the silver nanoparticles induced by the excited pyrene molecules. In due course the restructuring of the cetyltrimethylammonium layer resulted in moving of pyrene molecules away from silver surface and simultaneous disappearance of the silver nanoparticles fluorescence band. These data strongly support the recent hypothesis of fluorophore-plasmon coupled emission.     

对NFDA1和芘荧光猝灭性质的比较研究

对Bei和芘在电子给体化合物,N,N－二甲苯胺（DMA）和电子受体化合物对苯二甲酸二甲酯（DMTP）作用下的荧光猝灭进行了研究。结果表明芘与DMA和DMTP在室温下均可形成激基化合物（exciplex),而Ben仅仅与DMA形成exciplex。荧光猝灭的数据符合Stern-Volmer方程：F0／F＝1＋KSV[Q],芘的Stern-Volmer常数KSV大于Bei.Bei和芘在荧光猝灭方面的差别是由于Bei分子并不是一个典型的大共轭体系,分子的共平面性不如芘那样典型。     

Light scattering and fluorescence probe studies on micellar properties of Triton X-100 in KCl solutions

The effect of KCl on micelle formation and structure of Triton X-100 (TX-100) was investigated by using combined static and dynamic light scattering measurements, together with the fluorescence probe technique. An analysis of the light scattering data, including hydrodynamic radius and micellar aggregation number, accounted for both micelle growth and hydration. Fluorescence studies using pyrene as a probe were carried out to determine the critical micelle concentration (CMC) as a function of solution composition. In addition, with the aim of gaining information on the possible changes in the micro-environmental properties of TX-100 micelles, fluorescence probe studies, including intermolecular pyrene excimer formation and fluorescence polarization of coumarin 6 associated with micelles, were carried out. It was found that the addition of electrolyte induces a decrease in the CMC and an increase in both aggregation number and hydration. However, complementary data of partial specific volume and cloud point of the surfactant suggested that the main contribution to micellar hydration is due to water mechanically trapped in the micelle. Fluorescence measurements do not indicate changes in the micellar micropolarity, probably due to modifications of the solubilization site of the probe caused by the micellar growth. Both pyrene excimer formation and fluorescence polarization of coumarin 6 revealed an increase in microviscosity with electrolyte addition, which is consistent with increased micellar hydration.     

External Heavy Atom Quenching of the Fluorescence of Pyrene via Charge-Transfer Complexes.

The third harmonic (355 nm) of a pulsed, Nd-YAG laser has been used to induce pyrene fluorescence in cyclohexane solution. The rate constants of fluorescence quenching of pyrene by alkyl and aryl bromides have been measured. The increase of k_q with increasing the electron affinity of the quencher is attributed to CT interactions between the fluorophore and the heavy atom quencher involving a CT encounter complex, in which the quencher acts as an electron acceptor.     

Multi-spectroscopic techniques to evaluate the toxicity of alloyed CdSeS quantum dots

Alloyed CdSeS quantum dots (QDs) were successfully synthesized in aqueous phase using microwave irradiation. UV–vis spectroscopy, fluorescence spectroscopy, transmission electron microscopy (TEM) and X-ray diffraction (XRD) techniques proved that the prepared alloyed QDs are composed of a CdSe-rich core and thick CdS shell with homogeneous size distributions. In order to study its biological toxicity, multi-spectroscopic techniques were adopted to investigate their conjugation with BSA. Fluorescence quenching methods indicated the prepared CdSeS QDs strongly quenched the fluorescence of BSA due to the formation of non-fluorescence ground-state complex. UV–vis absorbance spectra, synchronous fluorescence spectra and CD spectra further confirmed the alloyed CdSeS QDs binded with BSA and destroyed their hydrogen bonding networks, which induced the conformation changes of this macromolecule.     

Complexation of pyrene and anthracene with human blood plasma

We have studied the interaction between polycyclic aromatic hydrocarbons (pyrene and anthracene) with human serum albumin (HSA) and human blood plasma. We have shown that the increase in the fluorescence intensity and the decrease in the polarity index of pyrene on going from an aqueous solution to a pH 7.4 buffer solution of HSA suggests that polycyclic aromatic hydrocarbons are localized in the hydrophobic microphase of the proteins. The increase in the fluorescence intensity for anthracene and pyrene, and also the decrease in the polarity index of pyrene on going from HSA to blood plasma is connected with the fact that polycyclic aromatic hydrocarbons can bind both to plasma proteins and to plasma lipids. When sodium dodecyl sulfate (SDS) is added to the blood plasma in a concentration greater than the critical micelle concentration, we observe an increase in the fluorescence intensity and the polarity index of pyrene. We hypothesize that this is connected with localization of pyrene near the interface between the hydrophobic and hydrophilic phases of the protein-SDS system. We have established that SDS leads to a change in the structure of blood plasma proteins and promotes escape of polycyclic aromatic hydrocarbons from the protein globules. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 75, No. 3, pp. 379–382, May–June, 2008.