A close look at fluorescence quenching of organic dyes by tryptophan.

Understanding fluorescence quenching processes of organic dyes by biomolecular compounds is of fundamental importance for in-vitro and in-vivo fluorescence studies. It has been reported that the excited singlet state of some oxazine and rhodamine derivatives is efficiently and almost exclusively quenched by the amino acid tryptophan (Trp) and the DNA base guanine via photoinduced electron transfer (PET). We present a detailed analysis of the quenching interactions between the oxazine dye MR121 and Trp in aqueous buffer. Steady-state and time-resolved fluorescence spectroscopy, together with fluorescence correlation spectroscopy (FCS), reveal three contributing quenching mechanisms: 1) diffusion-limited dynamic quenching with a bimolecular quenching rate constant k(d) of 4.0 x 10(9) s(-1) M(-1), 2) static quenching with a bimolecular association constant K(s) of 61 M(-1), and 3) a sphere-of-action contribution to static quenching described by an exponential factor with a quenching constant lambda of 22 M(-1). The latter two are characterized as nonfluorescent complexes, formed with approximately 30 % efficiency upon encounter, that are stable for tens of nanoseconds. The measured binding energy of 20-30 kJ mol(-1) is consistent with previous estimates from molecular dynamics simulations that proposed stacked complexes due to hydrophobic forces. We further evaluate the influence of glycerol and denaturant (guanidine hydrochloride) on the formation and stability of quenched complexes. Comparative measurements performed with two other dyes, ATTO 655 and Rhodamine 6G show similar results and thus demonstrate the general applicability of utilizing PET between organic dyes and Trp for the study of conformational dynamics of biopolymers on sub-nanometer length and nanosecond time-scales.     

Diastereodifferentiation of novel naphthalene dyads by fluorescence quenching and excimer formation.

Four new bichromophoric naphthalene dyads in form of diastereomeric pairs are synthesized and photophysically characterized. For a closely related group of compounds (NAP-NAP, MNAP-NAP, MNAP-MNAP), systematic variation of the urea linker led to the observation of intramolecular fluorescence quenching for tertiary urea, but not for secondary urea. Chiral information contained in the dyads has an impact on the efficiency of this process. Furthermore, for the case of (R,R)-MNAP-MNAP, excimer formation in the pre-organized dyad was noted, while being absent in the corresponding (R,S) diastereomer. These differences in photophysical behavior are ascribed to the geometry of the linker and to the chiral information contained in the diastereomeric compounds. Other dyads, such as NPX-NPX, included naproxen as the chromophore. For these compounds strong excimer emission is obtained, however, only very small diastereodifferentiation is observed for the two investigated stereoisomers.     

Iodide fluorescence quenching of sol-gel immobilized BSA

Several potential applications of sol-gel immobilized proteins may require drying the gel before use. The attendant shrinkage may sterically restrict the penetration of solutes to the entrapped protein. In this study, collisional quenching of intrinsic bovine serum albumin (BSA) fluorescence by dissolved iodide was used to quantitatively compare the accessibility of protein molecules entrapped in hydrated and dried sol-gel monoliths. The results show that iodide penetration to immobilized BSA is sterically restricted by approximately 25% in hydrated gels and 50% in dried gels, relative to the quenching behavior of dissolved BSA. The further decrease upon drying is consistent with the overall degree of monolith shrinkage.     

Studies on the fluorescence quenching of polysilane copolymers by chlorohydrocarbons

The room-temperature solution fluorescence quenching of polysilane copolymers by chlorohydrocarbons such as CCl₄, CHCl₃, C₂Cl₆, and Cl₂CHCHCl₂ was studied. The existence of dynamic quenching was preliminarily demonstrated by the experiment of fluorescence lifetime quenching. The fluorescence quenching data were in conformity with the equation: F₀/F = (1+K_SV[Q])exp(NV[Q]), where F and F₀ are the fluorescence intensity with and without the addition of quencher, K_SV is the Stern-Volmer constant, [Q] is the quencher concentration, N is the Avogadro constant, and V is the volume of the active sphere. The fluorescence quenching by the first three chlorohydrocarbons was attributed to the contemporaneous effect of dynamic quenching and static quenching. There exists, at least mathematically, a critical quencher concentration [Q]_C. When the quencher concentration [Q] < [Q]_C, the fluorescence quenching is dominated by the dynamic quenching part; when [Q] > [Q]_C, it is dominated by the static quenching part. However, the fluorescence quenching by Cl₂CHCHCl₂ was attributed to only static quenching. Furthermore, it was proposed that the dynamic quenching may be related with the electrical positivity of the central carbon nucleus of the quenching molecules while the static quenching may be caused by the “outside heavy atom effect” of the Cl element. © 1996 John Wiley & Sons, Inc.     

On the Coherent Description of Diffusion‐Influenced Fluorescence Quenching Experiments II: Early Events

In a previous article we showed how to perform and analyze steady‐state and nanosecond time‐resolved experiments on fluorescence quenching by electron transfer in a coherent manner. Now, by making use of a superior time resolution, we explore the first stages of this kind of reaction. The novel information gained enables us to merge the results on the viscosity and the driving‐force dependencies of the reaction rate. A unique set of parameters for a single reaction channel suffices to describe all the results in the frame of differential encounter theory for diffusion‐influenced, bimolecular, remote electron‐transfer reactions. The inclusion of the solvent structure is crucial for the understanding of the reaction kinetics. To the authors’ best knowledge, this is the first time that such a comprehensive set of data has been successfully and jointly explained in the field, with physically sound parameters for electron‐transfer reactions.     

能量转移荧光猝灭法测定加替沙星

在λ_(ex)/λ_(em)=470/566 nm、BR缓冲溶液(pH=5.72)、十二烷基苯磺酸钠介质中,吖啶橙(AO)与罗丹明6G(R6G)间能发生有效的能量转移,使R6G的荧光强度显著增强;加替沙星的加入,使R6G的荧光发生猝灭.应用AO-R6G能量转移荧光猝灭法测定加替沙星含量,提高了测定的灵敏度和选择性.加替沙星的浓度在0.6～9.0 μmol·L~(-1)范围内与R6G荧光猝灭程度呈线性关系;方法检出限为0.52 μmol·L~(-1);平行6次测定样品相对标准偏差为0.62%～0.84%;回收率为90.0%～105%.常见金属离子及药物敷料对测定无干扰,不经分离直接用于药物中加替沙星的测定.     

Rate kernel theory for pseudo-first-order kinetics of diffusion-influenced reactions and application to fluorescence quenching kinetics

Theoretical foundation of rate kernel equation approaches for diffusion-influenced chemical reactions is presented and applied to explain the kinetics of fluorescence quenching reactions. A many-body master equation is constructed by introducing stochastic terms, which characterize the rates of chemical reactions, into the many-body Smoluchowski equation. A Langevin-type of memory equation for the density fields of reactants evolving under the influence of time-independent perturbation is derived. This equation should be useful in predicting the time evolution of reactant concentrations approaching the steady state attained by the perturbation as well as the steady-state concentrations. The dynamics of fluctuation occurring in equilibrium state can be predicted by the memory equation by turning the perturbation off and consequently may be useful in obtaining the linear response to a time-dependent perturbation. It is found that unimolecular decay processes including the time-independent perturbation can be incorporated into bimolecular reaction kinetics as a Laplace transform variable. As a result, a theory for bimolecular reactions along with the unimolecular process turned off is sufficient to predict overall reaction kinetics including the effects of unimolecular reactions and perturbation. As the present formulation is applied to steady-state kinetics of fluorescence quenching reactions, the exact relation between fluorophore concentrations and the intensity of excitation light is derived.     

Harnessing a ratiometric fluorescence output from a sensor array

Ratiometric fluorescence-based sensors are widely sought after because they can effectively convert even relatively small changes in optical output into a strong and easy-to-read signal. However, ratiometric sensor molecules are usually difficult to make. We present a proof-of-principle experiment that shows that efficient ratiometric sensing may be achieved by an array of two chromophores, one providing an on-to-off response and the second yielding an off-to-on response in a complementary fashion. In the case that both chromophores emit light of different color, the result is a switching of colors that may be utilized in the same way as from a true ratiometric probe. The chromophore array comprises two sensor elements: i) a polyurethane membrane with embedded N-anthracen-9-yl-methyl-N-7-nitrobenzoxa-[1,2,5]diazo-4-yl-N',N'-dimethylethylenediamine hydrochloride and ii) a membrane with N,N-dimethyl-N'-(9-methylanthracenyl)ethylenediamine. A combination of photoinduced electron transfer (PET) and fluorescence resonance energy transfer (FRET) allows for green-to-blue emission switching in the presence of Zn(II) ions. The sensing experiments carried out with different Zn(II) salts at controlled pH revealed that the degree of color switching in the individual sensor elements depends on both the presence of Zn(II) ions and the counter anion. These results suggest that sensing of both cations and anions may perhaps be extended to different cation-anion pairs.     

吖啶橙-罗丹明B能量转移荧光猝灭法测定叶酸

研究了吖啶橙(AO)与罗丹明B(RB)间发生能量转移的最佳条件,在pH=6.80的Britton-Robinson(B-R)缓冲溶液,十二烷基苯磺酸钠的介质中,AO-RB间发生有效能量转移,使RB荧光大大增强,叶酸(FA)的加入使能量转移体系的RB的荧光强度降低,即发生猝灭.以此建立了利用AO-RB能量转移荧光猝灭法测...     

Theoretical description of solvent effects on fluorescence spectra of bulky charge transfer compound DMA-DMPP

Several theoretical models are compared to reproduce the spectroscopic fluorescence shift of 4-(4′-N,N-dimethylaminophenyl)-3,5-dimethyl-1,7-diphenyl-bis-pyrazolo-[3,4-b;4′,3′e]-pyridine (DMA-DMPP) in different solvents. DMA-DMPP is used as a model compound because it shows a large shift in emission energy for solvents of various polarities and dual fluorescence in polar protic solvents. Although the simple Onsager model is not able to reproduce the experimental results, the self-consistent reaction field (SCRF) model with extension to excited states based on the AM1 Hamiltonian yields excellent agreement. According to the latter model, the red-shifted emission band can be related to a highly polar charge transfer state without geometrical rearrangements, whereas the normal (short wavelength) emission is attributed to emission from an excited state with increased conjugation in a flattened geometry. A supramolecular approach with six molecules of water surrounding the solute can explain satisfactorily the two distinct fluorescence bands. In protic solvents, the emitting CT state shows additional stabilization of the locally excited state with a planar conformation. © 1998 John Wiley & Sons, Inc. J Comput Chem 19: 1584–1595, 1998     

Fluorescence quenching of flavins by reductive agents

The fluorescence behaviour of the flavins riboflavin, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and lumiflavin in aqueous solution at pH 8 in the presence of the reducing agents β-mercaptoethanol (β-ME), dithiothreitol (DTT), and sodium nitrite (NaNO₂) is studied under aerobic conditions. The fluorescence quantum yields and fluorescence lifetimes are determined as a function of the reducing agent concentration. For all three reducing agents diffusion controlled dynamic fluorescence quenching is observed which is thought to be due to photo-induced reductive electron transfer. For DTT additionally static fluorescence quenching occurs.     

Electron-transfer fluorescence quenching processes in coaggregates between excited N-alkylcarbazoles as electron donors and 2, 4-dinitrophenyl carboxylates or pentafluorophenyl carboxylates as acceptors

Electron-transfer processes facilitated by hydrophobic-lipophilic interaction ( HLI) between excited N-alkylcar-bazoles (1-n, n=4, 8, 12, 16) as electron donors and 2,4-dintrophenyl carboxylates (2-n, n = 4, 8, 12, 16) or pentafluorophenyl carboxylates (3-n, n = 4, 8, 12, 16) as electron acceptors have been investigated by means of fluorescence spectroscopy in aqueous or aquiorgano binary mixtures. The fluorescence quenching of -n * by 2-n or -n indicates that preassociation precedes the electron transfer. The extent of HLI-driven coaggregation of the acceptor and the donor may be assessed from the B value of the equation I0/I = A B [Q]. The chain-length effect and possibly also a chain-fold-ability effect, as well as the solvent aggregating power (SAgP) effect have been observed. Comparison of the quenching constants (B) for 1-n * /2-n combinations and 1-n * / 3-n combinations shows that the order of increasing R values for the quenching processes is 3-n < 2-n.     

牛血红蛋白催化荧光猝灭法测定痕量甲巯咪唑

在NH3·H2O-NH4Cl缓冲溶液中,牛血红蛋白催化H202氧化L-酪氨酸产生荧光,甲巯咪唑对该荧光体系具有较强的猝灭作用.据此建立了酶催化荧光猝灭法测定痕量甲巯咪唑的方法.结果表明,甲巯咪唑含量在0.986～14.8 μg/L范围内与荧光强度变化值呈线性关系,其线性回归方程为△F405nm=50.923p+191....     

Time-resolved fluorescence analysis of the mobile flavin cofactor in p -hydroxybenzoate hydroxylase

Conformational heterogeneity of the FAD cofactor in p-hydroxybenzoate hydroxylase (PHBH) was investigated with time-resolved polarized flavin fluorescence. For binary enzyme/substrate (analogue) complexes of wild-type PHBH and Tyr222 mutants, crystallographic studies have revealed two distinct flavin conformations; the ‘in’ conformation with the isoalloxazine ring located in the active site, and the ‘out’ conformation with the isoalloxazine ring disposed towards the protein surface. Fluorescence-lifetime analysis of these complexes revealed similar lifetime distributions for the ‘in’ and ‘out’ conformations. The reason for this is twofold. First, the active site of PHBH contains various potential fluorescence-quenching sites close to the flavin. Fluorescence analysis of uncomplexed PHBH Y222V and Y222A showed that Tyr222 is responsible for picosecond fluorescence quenching free enzyme. In addition, other potential quenching sites, including a tryptophan and two tyrosines involved in substrate binding, are located nearby. Since the shortest distance between these quenching sites and the isoalloxazine ring differs only little on average, these aromatic residues are likely to contribute to fluorescence quenching. Second, the effect of flavin conformation on the fluorescence lifetime distribution is blurred by binding of the aromatic substrates: saturation with aromatic substrates induces highly efficient fluorescence quenching. The flavin conformation is therefore only reflected in the small relative contributions of the longer lifetimes.     

Using single-molecule fluorescence spectroscopy to study electron transfer.

Techniques in single-molecule fluorescence spectroscopy now allow sophisticated studies of photophysical processes in single molecules. As interest grows in the possibilities of molecular electronics, researchers have begun to turn these techniques to the study of electron transfer. Electron-transfer reactions have now been detected and measured at the single-molecule level in a variety of systems and on a variety of timescales by adapting techniques from previous single-molecule fluorescence studies.     

Unique fluorescence behavior of perylenetetracarboxydiimides in polyimide systems

Perylenetetracarboxydiimide (PEDI) molecularly dispersed in polyamic acid (PAA) and polyimide (PI) films has unique fluorescence properties. An originally strong fluorescence of PEDI is efficiently quenched in the PAA films. The systematic variation of the chain structure of the PAA matrices revealed that the aromatic amide groups in the PAA chains function as a quencher. When a PAA derived from 3,4,3′4′-biphenyltetracarboxylic dianhydride (BPDA) and p-phenylenediamine (PDA), BPDA/PDA, was used as a matrix polymer, the fluorescence of the dye dispersed in the film increased abruptly as imidization of the matrix proceeds. But annealing at temperatures higher than 320°C in the step-heating process caused a gradual decrease in the fluorescence intensity. The decreased intensity results from the dye–PDA units interactions intensified by the denser molecular packing of the matrix polymer chains. PEDI shows significant dependence of the fluorescence intensity on the chain structure of the PI matrices. In the various PI films containing a fixed diamine component, the dye fluorescence intensity reduces linearly with an increase in the intramolecular charge transfer ability of the PI matrices. From the result, we propose a fluorescence quenching mechanism through multistep electron transfer processes. The BPDA/PDA polyimide matrix leads to a strong PEDI fluorescence whereas the pyromellitic dianhydride (PMDA)-based PI matrices do not. For the blends composed of these PIs, the fluorescence of PEDI bound into the main chains provides a valuable indicator of the miscibility on the molecular level. © 1998 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 36: 827–840, 1998     

荧光反猝灭法测定药剂中卡托普利

建立荧光反猝灭法测定卡托普利(CAT)的体系.碘(I-3)与罗丹明B(RhB)缔合作用使罗丹明B荧光信号强度减弱直至荧光猝灭,而卡托普利可将I-3还原为I-,使体系荧光信号再现.在2～20μmol·L-1范围内荧光强度再现值ΔF与卡托普利浓度有线性关系:ΔF=-8.438+10.774c(r=0.9986),检出限为3...