Journal of Fluorescence (v.19, #5)

Fluorescence/photoisomerization competition in trans-aza-1,2-diarylethenes by S. Ciorba; F. Fontana; G. Ciancaleoni; T. Caronna; U. Mazzucato; A. Spalletti (759-768).
The effect of the presence and position of the nitrogen heteroatom on the photobehaviour of a series of symmetric and asymmetric 1,2-diarylethenes, where one or both the one-, two- or three-membered aryl groups, linked to the ethene bridge, contain(s) nitrogen heteroatoms, has been investigated by fluorimetric and photochemical techniques. The yield of the radiative pathway is generally reduced with respect to the corresponding hydrocarbons, due to a smaller energy barrier for trans→cis (E→Z) photoisomerization in the singlet state, which often can be slightly favoured in the aza-compounds. All compounds investigated exist in solution as mixtures of two or more conformers, which can have different spectral and kinetic properties. Attempts to separate the intrinsic properties by selective photoexcitation, at a wavelength where the light is mainly absorbed by one conformer, allowed in some favourable cases the spectra and decay times of the largely prevalent (quasi-pure) conformer to be obtained. In two cases, the formation of intramolecular (hydrogen-bond-type) interactions between the nitrogen atom and the nearest hydrogen atom of the ethene bridge (proved by NMR measurements and confirmed by theoretical calculations) were found to stabilize a specific conformer thus affecting a selective relaxation of the excited molecule.
Keywords: Aza-1,2-diarylethenes; Fluorescence; Photoisomerization; Heteroatom effects

Fluorophotometric Determination of Hydrogen Peroxide with Fluorescin in the Presence of Cobalt (II) and Reaction Against Other Reactive Oxygen Species by Ryosuke Nakahara; Satomi Kashitani; Kumi Hayakawa; Yuuki Kitani; Takako Yamaguchi; Yoshikazu Fujita (769-775).
A fluorophotometric method for the determination of hydrogen peroxide (H2O2) using fluorescin was developed. This method was based on the oxidative reaction of fluorescin, a colorless, non-fluorescent lactoid fluorescein, by H2O2 to give highly fluorescein fluorescence emission. In the determination of H2O2, the calibration curve exhibited linearity over the H2O2 concentration range of 1.5–310 ng mL−1 at an emission wavelength of 525 nm with an excitation of 500 nm and with relative standard deviations (n = 6) of 2.51%, 2.48%, and 1.31% for 3.1 ng mL−1, 30.8 ng mL−1, and for 308 ng mL−1 of H2O2, respectively. The detection limit for H2O2 was 1.9 ng mL−1 six blank determinations was performed (ρ = 6). This proposed method was applied to detection of other reactive oxygen species and nitrogen species (ROS/RNS) such as singlet oxygen (1O2), hydroxyl radical (OH), peroxynitrite (ONOO) etc., and it was possible to detect them with a high sensitivity. In addition, this proposed method was applied to the recovery tests of H2O2 in calf serum, human saliva, rain water, and wheat noodles; the results were satisfactory.
Keywords: Fluorophotometric determination; Hydrogen peroxide; Reactive oxidant species; Reactive nitrogen species; Fluorescin

Fluorescent Recognition of Potassium and Calcium Ions Using Functionalised CdSe / ZnS Quantum Dots by Narinder Singh; Ray C. Mulrooney; Navneet Kaur; John F. Callan (777-782).
Schiff base receptor 1a has been synthesised and attached to the surface of preformed CdSe/ZnS Quantum Dots (QDs) to form QD-conjugate 2a. While 1a was determined to be selective for Mg2+, 2a demonstrated selectivity for both K+ and Ca2+ when tested against a range of physiologically and environmentally relevant cations by changes in the fluorescence spectra. Thus, the nanoparticle surface functions as a scaffold for the organisation of receptors enabling semi-selective binding. The fluorescence response was shown to be linear between 15–50 μM for K+ and 2–35 μM for Ca2+. It was also demonstrated that 2a could measure both K+ and / or Ca2+ in solutions containing both ions.
Keywords: Fluorescence; Quantum dots; Sensor

A Study of the Interaction Between Malachite Green and Lysozyme by Steady-State Fluorescence by Fei Ding; Wei Liu; Feng Liu; Zhi-Yuan Li; Ying Sun (783-791).
The interaction of a N-methylated diaminotriphenylmethane dye, malachite green, with lysozyme was investigated by fluorescence spectroscopic techniques under physiological conditions. The binding parameters have been evaluated by fluorescence quenching methods. The results revealed that malachite green caused the fluorescence quenching of lysozyme through a static quenching procedure. The thermodynamic parameters like ΔH and ΔS were calculated to be −15.33 kJ mol−1 and 19.47 J mol−1 K−1 according to van’t Hoff equation, respectively, which proves main interaction between malachite green and lysozyme is hydrophobic forces and hydrogen bond contact. The distance r between donor (lysozyme) and acceptor (malachite green) was obtained to be 3.82 nm according to Fӧrster’s theory. The results of synchronous fluorescence, UV/vis and three-dimensional fluorescence spectra showed that binding of malachite green with lysozyme can induce conformational changes in lysozyme. In addition, the effects of common ions on the constants of lysozyme-malachite green complex were also discussed.
Keywords: Malachite green; Lysozyme; Fluorescence quenching; Thermodynamic parameter

The preparation of a novel type of low-molecular-weight amphiphilic organogelator bearing three long 14-alkyl chains and hydrophilic oligo(oxyethylene) groups was described. Ultra-violet absorption and fluorescence spectra give evidence of the energy transfer between organic ligands to lanthanide ions. Characteristic green, blue and red luminescence of the organogels were obtained and interesting emission properties of terbium, dysprosium and europium ions were unexpectedly observed at the first time during the order-disorder phase transition point (29 °C).
Keywords: Lanthanide; Luminescence; Organo-gels; Micro-meter

Study on the Binding Behavior of Lysozyme with Cephalosporin Analogues by Fluorescence Spectroscopy by Zhuming Wang; Xijuan Tan; Donghua Chen; Qiaoli Yue; Zhenghua Song (801-808).
It was first found that the intrinsic fluorescence of lysozyme at 340 nm can be quenched by cephalosporin analogues through the static quenching and non-radiative energy transferring procedure. In the acetate buffer solution with pH 7.0 and 298 K, the quenching fluorescence intensity was in a good linearity over the concentration of drugs in the range of 1–100 μmol L−1, 0.1–100 μmol L−1, 0.5–100 μmol L−1 and 0.05–100 μmol L−1 for cefradine, cefuroxime, cefotaxime and ceftriaxone, respectively. The quenching ability or the binding ability of the studied drugs followed the pattern: ceftriaxone > cefotaxime > cefuroxime > cefradine, which was close to the order of their antibacterial ability. The binding parameters including the association constant and the number of binding potential point were calculated at different temperatures (288, 298 and 308 K), and thermodynamic parameters ΔH°, ΔS° and ΔG° were given. The binding mode of lysozyme with cephalosporins showed that the hydrophobic effect might play a major role. The binding distance between cephalosporin and tryptophan residue in lysozyme was obtained. The results provided the quantitative information for the binding of cephalosporin to lysozyme, and it was suggested that the drugs probably bound to the active site near Trp62 in lysozyme.
Keywords: Lysozyme; Cefradine; Cefuroxime; Cefotaxime; Ceftriaxone; Hydrophobic effect

Study and Application of the Interaction Between Asymmetrical Porphyrin and Ascorbic Acid by Shuting Sun; Dan Wu; Qin Wei; Yanyan Han; Xin Chen; Yunxia Shen; Peihua Zhu; Bin Du (809-815).
The interaction between novel asymmetrical porphyrin 5,10,15-triphenyl-20-pyridyl porphyrin and ascorbic acid has been studied by fluorophotometry. It has been found experimentally that in the presence of CTMAB micro-emulsion, the absorption and fluorescence intensity of the porphyrin was greatly enhanced. The addition of ascorbic acid caused the decrease of the intensity, and there was a linear relationship between the fluorescence quenching value and the concentration of ascorbic acid in certain extent. Based on this phenomenon, a new fluorescence quenching method for the determination of ascorbic acid was established, and utilized in the determination of commercial tablets with satisfactory results. The quenching mechanism between porphyrin and ascorbic acid was preliminary studied, and the quenching was belonging to static quenching.
Keywords: 5,10,15-triphenyl-20-pyridyl porphyrin; Ascorbic acid; Fluorescence quenching; Static quenching

Simultaneous Determination of Labetalol and Furosemide by First-Derivative Synchronous Spectrofluorimetry by D. R. El-Wasseef; D. T. El-Sherbiny; M. A. Abu-El-Enein; S. M. El-Ashry (817-828).
A rapid, simple and highly sensitive first derivative synchronous spectrofluorimetric method was developed for the simultaneous analysis of a binary mixture of labetalol HCl (LBT) and furosemide (FUR) without prior separation. The method was based upon measuring the first derivative of synchronous fluorescence spectra of the two drugs at Δλ =130 nm in aqueous ethanol (55% V/V). The different experimental parameters affecting the synchronous fluorescence of the studied drugs were carefully studied and optimized. The first derivative amplitude-concentration plots were rectilinear over the range of 0.10 to 1.00 μg/mL and 0.05–0.50 μg/mL with lower detection limits of 0.0149 and 7×10−3 μg/mL and quantification limits of 0.045 and 0.021 μg/mL for LBT and FUR, respectively. The proposed method was successfully applied for the determination of the studied drugs in synthetic mixtures. The results obtained were in good agreement with those obtained by the reference methods.
Keywords: Labetalol; Furosemide; First derivative synchronous spectrofluorimetry

3,6-diaryl-2,5-dihydro-1,4-diketopyrrolo[3,4-c]pyrrole-1,4-dione (DPPD) derivatives are used commercially as red pigments, because of their color strength, brightness, and very low solubility in most common solvents. These products, although highly colored, appear to be of limited value as dyes, since they show a tendency to form an aggregate when incorporated into a solvent. Monoalkyl and dialkyl derivatives of DPPD were synthesized and then characterized with IR, 1HNMR, UV-Vis absorption and emission spectroscopy. Molar absorption coefficient, singlet energy level and Stokes’ shift values of DPPD derivatives were declared. Monoalkyl and dialkyl derivatives of DPPD’s solubilities were measured from saturated solutions of dimethylformamide (DMF), tetrahydrofuran (THF), dichloromethane (DCM), acetonitrile (ACN) and toluene and they are compared with each other.
Keywords: Diketopyrrolopyrrole; Fluorescence spectroscopy; Photocharacterization; Solubility calculation; Dye; Synthesis

The extended Förster theory (EFT) of electronic energy transport accounts for translational and rotational dynamics, which are neglected by the classical Förster theory (FT). EFT has been developed for electronic energy transfer within donor-acceptor pairs [Isaksson, et al, Phys. Chem. Chem. Phys., 9, 1941(2007)] and donor-donor pairs [Johansson, et al, J. Chem. Phys., 105, 10896 (1996); Norlin, et al, Phys. Chem. Chem. Phys., 10, 6962(2008)]. For donors that exhibit different or identical non-exponential fluorescence relaxation within a donor-donor pair, the process of reverberating energy migration is reversible to a higher or lower degree. Here the impact of the EFT has been studied with respect to its influence on fluorescence quantum yields, fluorescence lifetimes as well as depolarisation experiments. The FT predicts relative fluorescence quantum yields which usually agree with the EFT within experimental accuracy, however, substantial deviations occurs in the steady-state and in particular the time-resolved depolarisation data.
Keywords: Electronic energy migration; Homotransfer; Donor-Donor Energy Migration (DDEM); Partial Donor-Donor Energy Migration (PDDEM)

A novel 6-ethoxy chromone-3-carbaldehyde benzoyl hydrazone (L) and its Ln(III) complexes, [Ln = Sm (1), Eu (2), Gd (3), Tb (4)], have been synthesized and characterized. The fluorescence properties of the Eu(III) and Sm(III) complexes in solid state and Eu(III) complex in different solutions (DMF, DMSO, methanol and acetonitrile) were investigated. At the same time, the DNA-binding properties of the two complexes are investigated using UV-Vis absorption spectroscopy, fluorescence spectroscopy, viscosity measurement. All the experimental evidences indicate that the two complexes can bind to CT-DNA via an intercalation mechanism. Furthermore, antioxidant activity tests in vitro showed that the complexes have significant antioxidative activity against hydroxyl free radicals from the Fenton reaction.
Keywords: 6-Ethoxy chromone-3-carbaldehyde benzoyl hydrazone; Rare earth complexes; DNA binding

Synthesis, Characterization, DNA-Binding and Antiproliferative Activity of Nd(III) Complexes With N-(Nitrogen Heterocyclic) Norcantharidin Acylamide Acid by Wen-Zhong Zhu; Qiu-Yue Lin; Mei Lu; Rui-Ding Hu; Xiao-Liang Zheng; Jian-Ping Cheng; Yun-Yun Wang (857-866).
Three novel complexes [Nd(L)(NO3)(H2O)2]·NO3·2H2O (HL1 = N-pyrimidine norcantharidin acylamide acid, C12H13N3O4; HL2 = N-pyridine norcantharidin acylamide acid, C13H14N2O4; HL3 = N-phenyl norcantharidin acylamide acid, C14H15NO4) were synthesized. HL1, HL2 and HL3 are the ligand of complex(1), complex(2) and complex(3), respectively. Their structures were characterized by elemental analysis, conductivity measurement, infrared spectra and thermogravimetric analysis. The DNA-binding properties of the complexes have been investigated by fluorescence spectroscopy and viscosity measurements. The results suggest that the complexes can bind to DNA by partial intercalation. The liner Stern-Volmer quenching constant Ksq values are 3.3(±0.21)(1), 1.7(±0.19)(2) and 0.9(±0.04)(3), respectively. Complex (1) and (2) have been found to cleave pBR322 plasmid DNA at physiological pH and temperature. The test of antiproliferation activity indicates that complex(1) has strong antiproliferative ability against the SMMC7721 (IC50 = 131.7 ± 23.4 μmol·L−1) and A549 (IC50 = 128.4 ± 19.9 μmol·L−1) cell lines. The inhibition rates of complex(2) (IC50 = 86.3 ± 11.3 μmol·L−1) are much higher than that of NCTD (IC50 = 115.5 ± 9.5 μmol·L−1) and HL2 (111.0 ± 5.7 μmol·L−1) against SMMC7721 cell lines.
Keywords: Norcantharidin; Nd(III) complexes; Pyrimidine; Pyridine; DNA binding; Antiproliferative activity

The reaction of acidic potassium permanganate with a wide range of compounds is known to produce a broad red emission, and there is strong evidence for an excited manganese(II) emitting species. Nevertheless, numerous researchers have proposed other emitters for reactions with acidic potassium permanganate, particularly for systems where fluorescent compounds were present, either as enhancers or reaction products. We have examined many reactions of this type and found that, in most cases, the same red emission was produced. There were, however, some exceptions, including the oxidation of dihydralazine, certain thiols and sulphite (each in the presence of an enhancer).
Keywords: Chemiluminescence spectra; Fluorescence spectra; Emitting species; Energy transfer; Potassium permanganate

The fluorescence photobleaching recovery method with pulse-position modulation (PPM) of bleaching/probing irradiation is presented. This approach is modification of previously announced photobleaching recovery under decaying photobleaching (FRDP) approach, which employs the amplitude control of irradiation intensity. Underlying idea was to solve the problem of setup nonlinearity by methodical modification that was successfully done. The irradiation intensity is series of equal bleach-probe pulses with increasing distance. The more sophisticated series was proposed as a convolution of subseries, which one characterizing its own time scale. It allows to detect the fluorescence kinetics in the wide range of time and of different nature simultaneously. The method was applied on model systems as FITC-HSA in the water/glycerol mixture and FITC-DMPE in the POPC multibilayers. The method allowed to detect and investigate diffusion processes, presence immobile fraction, effect of limited area of liposomes and reversible fluorescence kinetics.
Keywords: Photobleaching; FITC; Albumin; POPC; Diffusion

Luminescent Donor-Acceptor β-Diketones: Modulation of Emission by Solvent Polarity and Group II Metal Binding by Guoqing Zhang; Shin Han Kim; Ruffin E. Evans; Byeong Hyo Kim; J. N. Demas; Cassandra L. Fraser (881-889).
A class of aryl trifluoromethyl-containing β-diketones were synthesized via one step Claisen condensation. These π-conjugated diketones exhibit strong solvatochromism from intramolecular donor-acceptor charge transfer (CT). In addition, fluorescence quantum yields (ϕf) and lifetimes (τf) were measured in different solvents. Diketones exhibit bathochromic shifts in emission spectra with increasing solvent polarity. Fluorescence changes upon Group II metal binding were also studied. Despite the relatively simple structure, the anthracene-CF3 diketone, atm, has strong binding affinity for Mg2+. A 70 nm blue shift and sixfold increase in intensity were observed upon addition of only one equivalent MgCl2 in ethanol solution. It also shows selectivity for Mg2+ binding even in the presence of excess Ca2+. Association constant (Ka) calculations suggest atm has two orders of magnitude stronger chelation for divalent magnesium than for calcium. These findings make atm an attractive starting point for molecular probe and light emitting material design.
Keywords: Fluorescent β-diketones; Anthracene; Naphthalene; Solvatochromism; Mg2+ and Ca2+ binding

A rapid, simple, and highly sensitive second-derivative synchronous fluorimetric (SDSF) method has been developed for the simultaneous analysis of binary mixtures of fluphenazine hydrochloride (FLZ) and nortriptyline hydrochloride (NTP) in their co-formulated tablets. The method is based upon measurement of the native fluorescence of these drugs at constant wavelength difference (Δλ) = 120 nm in acetic acid. The different experimental parameters affecting the fluorescence intensity of the studied drugs were carefully studied and optimized. The fluorescence-concentration plots were rectilinear over the range of 0.25–3.0 and 1–10 μg/ml for FLZ and NTP respectively, with lower detection limits (LOD) of 0.05 and 0.18 μg/ml and quantitation limits of 0.15 and 0.53 μg/ml for FLZ and NTP respectively. The proposed method was successfully applied for the determination of the studied compounds in their synthetic mixtures and in commercial co-formulated tablets. The results obtained were in good agreement with those obtained by the reference methods.
Keywords: Second derivative synchronous spectrofluorimetry; Fluphenazine; Nortriptyline; Simultaneous determination

Automatic discrimination of plant species is required for precision farming and for advanced environmental protection. For this task, reflected sunlight has already been tested whereas fluorescence emission has been only scarcely considered. Here, we investigated the discriminative potential of chlorophyll fluorescence imaging in a case study using three closely related plant species of the family Lamiaceae. We compared discriminative potential of eight classifiers and four feature selection methods to identify the fluorescence parameters that can yield the highest contrast between the species. Three plant species: Ocimum basilicum, Origanum majorana and Origanum vulgare were grown separately as well as in pots where all three species were mixed. First, eight statistical classifiers were applied and tested in simulated species discrimination. The performance of the Quadratic Discriminant Classifier was found to be the most efficient. This classifier was further applied in combination with four different methods of feature selection. The Sequential Forward Floating Selection was found as the most efficient method for selecting the best performing subset of fluorescence images. The ability of the combinatorial statistical techniques for discriminating the species was also compared to the resolving power of conventional fluorescence parameters and found to be more efficient.
Keywords: Classification; Feature selectionl; Chlorophyll fluorescence transient; Precision farming; Statistical classifier

A Photostable, pH-Invariant Fluorescein Derivative for Single-Molecule Microscopy by Baoxu Liu; Steven Fletcher; Miriam Avadisian; Patrick T. Gunning; Claudiu C. Gradinaru (915-920).
We herein report the comprehensive characterization of the spectral and single-photon fluorescence properties of a recently synthesized fluorescein derivative and its biotinylated analog. The fluorophore displays significant increases in photostability compared to the known fluorescein label fluorescein isothiocyanate (FITC), as well as superb pH independence. This fluorescein variant has two readily accessible functional groups (aniline NH2 and phenol OH) that can be activated or blocked independently and can serve, for instance, as a fluorescent bridge between two different recognition motifs. Excellent single-photon counting fluorescence data demonstrates that it is also a particularly appropriate probe for single-molecule studies of biological interactions.
Keywords: Fluorescence spectroscopy; Photostability; Single-molecule biophysics; FRET

In this paper are presented absorption and fluorescence emission properties of 3-styrylindoles viz. 3-(2-phenylethenyl-E)-NH-indole (1), 3-[2-(4-nitrophenyl)ethenyl-E)-NH-indole (2), 3-[2-(4-cyanophenyl)ethenyl-E]-N-ethylindole (3) and 3-[2-(4-cyanophenyl)ethenyl-E]-NH-indole (4) in organic solvents, 1,4-dioxane-water binary mixtures and micelles (SDS, CTAB and Triton-X-100). The fluorescence properties of 2-4 have been utilized to probe the microenvironment (binding constant, CMC, micropolarity and solubilization site) of the micelles.
Keywords: Styrylindoles; Fluorescence probe; Quenching; Micelle; Charge transfer

We present a detailed study on fluorescence enhancement by ‘stroboscopic’ illumination with light pulses having duration ranging from few milliseconds to sub-picoseconds. We show how a delicate balance between pulse width and pulse repetition rate can result in an unprecedented fluorescence enhancement that has immediate applications in fluorescence imaging.
Keywords: Fluorescence enhancement; Reduced photo-bleaching; Photo-damage