同步荧光分析法的应用及其新进展

同步荧光技术是解决多组分荧光物质同时测定的良好手段之一.本文从恒波长同步荧光法、恒能量同步荧光法、可变角同步荧光法、恒基体同步荧光法以及它们与导数技术、低温技术、化学计量学方法的联用等方面对同步荧光技术领域出现的新技术及应用作一评述.     

Water-soluble fluorescent boronic acid compounds for saccharide sensing: substituent effects on their fluorescence properties

Four new naphthalene-based boronic acid compounds (1-4) were synthesized. The effect of various carbohydrates on their fluorescence properties has been studied in aqueous phosphate buffer at pH 7.4. Different substitutions on the aniline group of the naphthalene ring resulted in significant differences in fluorescence properties for these four compounds. Compound 1 shows ratiometric fluorescence changes upon addition of a sugar. Compounds 2 and 3 do not show ratiometric fluorescence changes but show very large fluorescence intensity changes (about 70-fold fluorescence intensity increase). In addition to the quantifiable fluorescence property changes upon sugar addition, the fluorescence color changes of 1-3 are also visible to the naked eye. However, amidation of the aniline nitrogen atom significantly diminishes the fluorescence intensity of compound 4. The crystal structure of one boronic acid provided some insight into the structural features that are important for the fluorescence properties of these compounds.     

Meso-四对甲氧基苯基卟啉荧光被Hg(Ⅱ)猝灭后荧光恢复的研究

通过 meso-四对甲氧基卟啉荧光被Hg2 猝灭后荧光恢复这一现象证明了卟啉的自组装行为.meso-四对甲氧基苯基卟啉丙酮溶液荧光加入Hg2 后猝灭,少量水的引入使卟啉通过氢键发生自组装,这时荧光恢复.Hg2 加入后立即发生猝灭,但是恢复却是比较缓慢的,连续加入Hg2 ,荧光呈周期性的猝灭和恢复.并且通过荧光光谱和紫外光谱论证了这一现象.     

Microanatomical and biochemical origins of normal and precancerous cervical autofluorescence using laser-scanning fluorescence confocal microscopy

Clinical studies have shown that in vivo fluorescence spectroscopy can improve the diagnosis of cervical precancer. Recent work suggests that epithelial fluorescence increases, whereas stromal fluorescence decreases, with precancer. However, the microanatomic and biochemical sources of fluorescence in living cervical tissue have not yet been established. This study aims to characterize the origins of living normal and precancerous cervical fluorescence at microscopic levels using laser-scanning fluorescence confocal microscopy. Ten pairs of colposcopically normal and abnormal biopsies were obtained; transverse, 200 microm thick, short-term tissue cultures were prepared and imaged when viable with UV (351-364 nm) and 488 nm excitation before and after addition of the vital dye, Mitotracker Orange. In normal epithelium basal epithelial cells showed cytoplasmic fluorescence; parabasal, intermediate and superficial cells showed fluorescence only at the periphery of the cell. In low-grade precancers cytoplasmic fluorescence was visible in the bottom one-third of the epithelium; in high-grade precancers cytoplasmic fluorescence was visible throughout the lower two-thirds of the epithelium. Cytoplasmic fluorescence was colocalized with the MitoTracker probe and is attributed to mitochondrial reduced form of nicotinamide adenine dinucleotide at UV excitation and mitochondrial flavin adenine dinucleotide at 488 nm excitation. Stromal fluorescence originated from matrix fibers; with the development of precancer the density and fluorescence intensity of matrix fibers decrease. Autofluorescence properties of precancerous cervix reflect an increased number of metabolically active mitochondria in epithelial cells and a reduced stromal fluorescence, which can be an indicator for altered communication between precancerous epithelium and stroma. These changes can explain differences in in vivo fluorescence spectra of normal and precancerous cervical tissue.     

Spectrum Correction in Study of Solvation Dynamics by Fluorescence Non-collinear Optical Parametric Ampli cation Spectroscopy

Femtosecond time-resolved fluorescence non-collinear optical parametric amplification spectroscopy can extract the curve of spectral gain from its parametric superfluorescence. This unique spectrum correction method enables fluorescence non-collinear optical parametric amplification spectroscopy acquiring the genuine transient fluorescence spectrum of the studied system. In this work we employ fluorescence non-collinear optical parametric amplification spectroscopy technique to study the solvation dynamics of DCM dye in ethanol solution, and confirm that genuine solvation correlation function and shift of peak frequency can be derived from transient fluorescence spectra after the spectral gain correction. It demonstrates that fluorescence non-collinear optical parametric amplification spectroscopy can benefit the research fields, which focuses on both fluorescence intensity dynamics and fluorescence spectral shape evolution.     

空间分辨荧光分析技术

空间分辨荧光分析技术突破了传统荧光分析的局限,为获得空间定位信息提供了技术保障。系统地综述了构成该技术的共焦荧光法、全内反射荧光法、多光子荧光法以及近场荧光法等4种方法的原理、特点、发展及其应用,并且强调了其在单分子测定中的作用。引用文献64篇。     

三维同步荧光法研究喜树生物碱衍生物的荧光光谱性质

本文用三维同步荧光法详细研究了喜树生物碱衍生物的共性和特性,以及它们之间荧光光谱的变化过程,说明结构、取代基和溶剂在荧光光谱的变化中所起的作用,并用同步荧光法不经分离同时测定实际样品中的喜树碱和羟基喜树碱。     

External electric field effects on fluorescence of pyrene butyric acid in a polymer film: concentration dependence and temperature dependence

Fluorescence spectra and electrofluorescence spectra (plots of the electric field-induced change in fluorescence intensity as a function of wavelength) have been measured at different temperatures for pyrene butyric acid (PBA) in a PMMA film at different concentrations. At a low concentration of 0.5 mol % where fluorescence emitted from the locally excited state of PBA (LE fluorescence) is dominant, LE fluorescence spectra show only the Stark shift in the presence of an electric field (F), which results from the difference in molecular polarizability between the ground and emitting states. At a high concentration of 10 mol % where the so-called sandwich-type excimer fluorescence (EX(1)) is dominant, both EX(1) and LE fluorescence are quenched by F. Another fluorescence assigned to a partially overlapped excimer (EX(2)) also exists at room temperature, and this emission is enhanced by F. As the temperature decreases, three fluorescence emissions whose electric field effects are different from each other become clear besides EX(1) and LE fluorescence, indicating that at least five fluorescence components exist at high concentrations at low temperatures. At a medium concentration of 5 mol % where EX(1) is comparable in intensity to the LE fluorescence, the intensity of EX(1) is not affected by F at any temperature, but LE fluorescence and EX(2) are markedly influenced by F at room temperature, and four fluorescence emissions are confirmed at low temperatures.     

酪氨酸的分频荧光光谱研究

酪氨酸在300nm处产生一个荧光峰,在600nm处产生一个1／2分频荧光峰,在900nm处产生一个1／3分频荧光峰,此三峰具有相似的荧光特性。根据非线性光学分频荧光原理探讨了酪氨酸分频荧光峰产生的原因。     

Study on the Interaction of Pentachlorophenol with Urease in Aqueous Solution by Multiple Spectroscopic Techniques

The interactions between pentachlorophenol (PCP) and jack bean urease were studied using UV/vis absorption, CD, fluorescence, synchronous fluorescence, and three-dimensional fluorescence spectroscopic techniques. The fluorescence data showed that the fluorescence quenching of urease by PCP the results of the formation of a PCP–urease complex involving a hydrophobic interaction. The distance r between the donor (urease) and acceptor (PCP) was obtained from the fluorescence resonance energy transfer. The effect of PCP on the conformation of urease was analyzed using UV/vis absorption, synchronous fluorescence and three-dimensional fluorescence spectroscopic techniques. The result showed that PCP can enter into the hydrophobic pocket at the interface of urease and that the micro environments around the tyrosine and tryptophan residues were changed.     

Highly fluorescent chameleon nanoparticles and polymer films: multicomponent organic systems that combine FRET and photochromic switching

We describe the preparation of highly efficient stimulus-responsive fluorescence color-tuning in self-assembled supramolecular scaffold systems. The systems consisted of a photochromic compound (BP-BTE) in combination with unique luminescent organic materials (CN-MBE, TPS-CNMBE, TPA-2CNMBE) that exhibited intense fluorescence in the solid state. The emission spectrum was tuned by introducing fluorescence resonance energy transfer and photochromic switching capabilities into the system. The materials were used to successfully demonstrate novel fluorescence patterns that were responsive to multiple stimuli, displayed reversible fluorescence switching, and provided a nondestructive readout of the fluorescence signal.     

Appearance of pure fluorocarbon micelles surveyed by fluorescence quenching of amphiphilic quinoline derivatives in fluorocarbon and hydrocarbon surfactant mixtures

A halide-sensitive fluorescence probe was utilized to evaluate the miscibility of fluorocarbon and hydrocarbon surfactants in aqueous micellar systems. The fluorescence of 6-methoxy-N-1,1,2,2-tetrahydroheptadecafluorodecylquinolinium chloride, FC10MQ, was quenched by halide ions dissociated from the surfactant. The fluorescence in micellar solutions showed an initially rapid decay. This suggests that halide ions effectively quench FC10MQ fluorescence at the micellar surface. The subsequent slow decay corresponds to the quenching of FC10MQ fluorescence in the aqueous bulk phase by the free counterions. The Stern-Volmer plots for fluorescence quenching gave a distinct break at the critical micelle concentration of the cationic surfactants. The abrupt increase in fluorescence quenching is attributed to the solubilization of the probe in the micelles. The fluorescence quenching behavior provides direct information about the immiscibility of fluorocarbon and hydrocarbon species in micelles, and the results indicate that almost pure fluorocarbon micelles appear in surfactants mixtures.     

Characterization of the interaction between farrerol and bovine serum albumin by fluorescence and circular dichroism

The binding of farrerol to bovine serum albumin (BSA) in aqueous solution was investigated by fluorescence quenching spectra, synchronous fluorescence spectra, circular dichroism (CD) and the three-dimensional (3D) fluorescence spectra at pH 7.40. The results of fluorescence titration indicated that farrerol could quench the intrinsic fluorescence of BSA in a static quenching way. The cause of showing upward curvy patterns in Stern-Volmer plots was analyzed. The binding sites number n and binding constant K using fluorescence quenching equation at 310 K were calculated. The binding distance and the energy transfer efficiency between farrerol and BSA were also obtained according to the theory of F?rster's non-radiation energy transfer. The effect of some metal ions on the binding constant of farrerol with BSA was also studied. The effect of farrerol on the conformation of BSA was analyzed using CD, synchronous fluorescence spectra and three-dimensional (3D) fluorescence spectra under experimental conditions. Furthermore, the fluorescence displacement experiments indicated that farrerol could bind to the site I of BSA.     

Fluorescence properties of 1-naphthol, 2-naphthol and 1,2,3,4-tetrahydronaphthol in aqueous alcohol solvents with and without beta-cyclodextrin

The fluorescence properties of 1-naphthol, 2-naphthol and 1,2,3,4-tetrahydronaphthol were obtained in binary aqueous-alcohol solvents with and without beta-cyclodextrin. The fluorescence of both the molecular and anionic forms of 1-naphthol and 2-naphthol were observed in the binary solvents without beta-cyclodextrin. Only the fluorescence of the molecular form of 2-naphthol appeared in the binary solvents with beta-cyclodextrin present, and its fluorescence was quenched with increasing amounts of beta-cyclodextrin. However, the fluorescence intensity of the molecular form of 1-naphthol increased with an increasing amount of beta-cyclodextrin in the binary solvents. The fluorescence intensity of 1,2,3,4-tetrahydronaphthol decreased with an increase in the amount of beta-cyclodextrin. The fluorescence results were interpreted with the Stern-Volmer equation and a modified Stern-Volmer equation.     

Fluorescence of indocyanine green in blood: intensity dependence on concentration and stabilization with sodium polyaspartate.

Indocyanine green (ICG) has been widely used in cardiovascular, hepatic, and ophthalmologic studies. Application of this fluorescent dye has been handicapped by its poor stability in solution and by the complex dependence of its fluorescence intensity on concentration. Noncovalent interactions between ICG and sodium polyaspartate (PASP) stabilize ICG fluorescence in aqueous solution, but the effect of PASP on ICG fluorescence in blood has not been described. The current study had two main goals: first, to characterize in vitro in blood the relationship between fluorescence intensity and concentration of ICG-PASP (ICG) and the stability of this relationship over time; second, to test a new phenomenological model describing the dependence of ICG fluorescence on concentration. Freshly-prepared ICG and ICG-PASP solutions produced the same fluorescence intensity over a wide range of concentrations (0.0005-0.1271 mg/ml). The peak fluorescence of ICG was reduced by 11% after 10 h and by 72% at 7 days. In contrast, the peak fluorescence intensity of ICG-PASP solutions was nearly unchanged for up to 14 days. The dependence of the fluorescence intensity on concentration was accurately represented by our model that accounted for the generation of fluorescence following light absorption, and for the reabsorption of the emitted fluorescence by ICG.     

Autofluorescence spectroscopy of normal and malignant human breast cell lines

The fluorescence of tryptophan, reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) were characterized in normal human breast cells as well as in malignant human breast cells of similar and dissimilar genetic origins. Fluorescence measurements of each cell line were made over a wide range of cell concentrations, and the fluorescence per cell was determined from the slope in the linear range of the fluorescence intensity vs cell concentration plot. All of the malignant cells showed a statistically significant decrease in the tryptophan fluorescence per cell relative to that of the normal cells. No statistically significant differences were observed in the NAD(P)H or FAD fluorescence per cell between the normal and any of the malignant cell types. NAD(P)H fluorescence was also imaged from monolayers of the normal and malignant cells (of similar genetic origin) using two-photon fluorescence microscopy. A statistically significant decrease in the NAD(P)H fluorescence with malignancy was observed, suggesting that fluorescence imaging of single cells or the cell monolayer preparation may provide more contrast than volume-averaged fluorescence measurements of cells in suspension. In conclusion, the differences in normal and malignant human breast tissue fluorescence spectra may be attributed in part to differences in the intrinsic cellular fluorescence of normal and malignant breast epithelial cells.     

Photobleaching of Arterial Fluorescent Compounds: Characterization of Elastin, Collagen and Cholesterol Time-resolved Spectra during Prolonged Ultraviolet Irradiation

To study the photobleaching of the main fluorescent compounds of the arterial wall, we repeatedly measured the time-resolved fluorescence of elastin, collagen and cholesterol during 560 s of excitation with nitrogen laser pulses. Three fluence rate levels were used: 0.72, 7.25 and 21.75 microW/mm2. The irradiation-related changes of the fluorescence intensity and of the time-resolved fluorescence decay constants were characterized for the emission at 390, 430 and 470 nm. The fluorescence intensity at 390 nm decreased by 25-35% when the fluence delivered was 4 mJ/mm2, a common value in fluorescence studies of the arterial wall. Cholesterol fluorescence photobleached the most, and elastin fluorescence photobleached the least. Photobleaching was most intense at 390 nm and least intense at 470 nm such that the emission spectra of the three compounds were markedly distorted by photobleaching. The time-resolved decay constants and the fluorescence lifetime were not altered by irradiation when the fluence was below 4 mJ/mm2. The spectral distortions associated with photobleaching complicate the interpretation of arterial wall fluorescence in terms of tissue content in elastin, collagen and cholesterol. Use of the time-dependent features of the emission that are not altered by photobleaching should increase the accuracy of arterial wall analysis by fluorescence spectroscopy.     

同步荧光光谱分析中几个问题的探讨

通过实验阐明了同步荧光光谱与普通荧光光谱之间的关系，在此基础上讨论了溶剂散射光对同步荧光分析的影响以及同步荧光分析的灵敏度。     

Solvent effect in an “isolated” solvated molecule in a free jet: Intramolecular charge-transfer state of solvated 9,9'-bianthryl

The fluorescence spectra of bare and solvated 9,9'-bianthryl were recorded in a free Jet. In the case of bare bianthryl, only the fluorescence from the locally excited (LE) state was observed, even on excitation to a high vibronic state. In contrast, the excitation of the 0-0 band of 9,9'-bianthryl solvated by acetone gave fluorescence from the charge-transfer (CT) state. The ratio of the intensity of the CT fluorescence to that of the LE fluorescence increases with increasing degree of solvation. Solvation by cyclohexane did not give CT fluorescence.     

对几种氨基甲酸酯类农药的荧光特性研究

研究了有机物分子结构与荧光产生之间的关系。利用这一理论分析了西维因、克百威、残杀威等几种常用的氨基甲酸酯类农药的分子结构并揭示了它们的荧光光学特性,从而确定了氨基甲酸酯类农药产生荧光的特性,为进一步利用荧光分析法对氨基甲酸酯类农药进行检测提供理论依据。并且利用稳态荧光光谱仪对西维因和克百威标准溶液进行了荧光光谱实验,结果表明,氨基甲酸酯类农药在一定的溶剂条件下是能够受激发荧光的,是可以利用荧光光谱分析法来对它们进行检测．