Spectrofluorimetric determination of 5-hydroxytryptamine using chlorosulphonylthenoyltrifluoroacetone-europium probe.

A new spectrofluorimetric method was developed for the determination of trace amounts of 5-hydroxytryptamine (5-HT). Using chlorosulphonylthenoyltrifluoroacetone (CTTA)-europium (Eu(3+)) ion as a fluorescent probe in a buffer solution at pH 11.0, 5-HT can remarkably enhance the fluorescence intensity of the CTTA-Eu(3+) complex at lambda = 612 nm; the enhanced fluorescence intensity of Eu(3+) is proportional to the concentration of 5-HT. Optimum conditions for the determination of 5-HT were also investigated. The linear range and detection limit for the determination of 5-HT were 1.0 x 10(-7)-1.2 x 10(-5) mol/L and 8.5 x 10(-8) mol/L, respectively. This method is simple, practical and relatively free of interference from coexisting substances, and can be applied to assess 5-HT in urine samples.     

Studies on the fluorescence reaction between nucleic acid and the complex of cobalt(II) with 5-(3-fluoro-4-chlorophenylazo)-8-sulfonamidoquinoline and its applications

Based on the enhancement of the fluorescence intensity of the complex of 5-(3-fluoro-4-chlorophenylazo)-8-sulfonamidoquinoline (FCPBSQ) with Co(2+) by nucleic acid in the presence of Tween 80 and in the weakly basic medium, a fluorescence method for the determination of nucleic acid was proposed. The calibration graphs for the determination of denatured calf thymus DNA (ct DNA), fish sperm DNA (fs DNA), and yeast RNA (yt RNA) were obtained in concentration ranges 0.050-4.0, 0.10-3.0, and 0.050-3.0 microg/ml with limits of detection of 0.010, 0.030, and 0.020 microg/ml, respectively. The method has been satisfactorily used for the determination of DNA in wheat cell extraction and ct DNA, fs DNA, and yt RNA in synthetic samples. Investigations on the binding mode by the Scatchard plots method suggested that both intercalation and electrostatic binding modes existed in this system.     

Time-resolved immunofluorometric assays with measurement of a europium chelate in solution: application for sensitive determination of fibronectin

A novel detection principle applicable for sensitive measurement of molecules of biological interest by time-resolved fluorescence spectrophotometry is described. Our method is based on the quantification of the Eu3+ chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) in solution in presence of an excess of Eu3+ ions. BCPDA-labeled solid phase complexes obtained by conventional immunoassay procedures are transferred into solution using urea/SDS/Eu3+ as dissociating and fluorescent lanthanide ion reagent. Two 'sandwich-type' assay variants based on the above methodology were realized for the determination of small amounts of fibronectin (FN) in biological fluids. FN is captured from solution by solid phase coated gelatin or a monoclonal antibody, respectively. Rabbit anti-FN antiserum used as second antibody is detected with a biotinylated anti-rabbit IgG antibody. Fluoresence is measured after incubation with streptavidin-BCPDA and dissociation of solid phase complexes as described. Both assays have a detection limit (blank + 3 x SD) of less than 0.5 ng/ml FN, a dynamic range of up to 300 ng/ml, and intraserial coefficients of variation of 4.4 and 6.3%, respectively. Median FN concentrations in saliva of healthy individuals were 104 (gelatin) and 36 ng/ml (double antibody), respectively.     

The sensitive determination of nucleic acids using a fluorescence-quenching method.

Experiments indicated that nucleic acids can quench the fluorescence of the Eu3+ -2-thenoyltrifluoroacetone (TTA)-1,10-phenanthroline (Phen) system. Based on this, a sensitive method for the determination of nucleic acids was proposed. The experiments indicated that under the optimum conditions, the quenched fluorescence intensity was in proportion to the concentration of nucleic acids in the range 1.0 x 10(-11)-1.0 x 10(-6) g/mL for yeast RNA (yRNA), 5.0 x 10(-11)-5.0 x 10(-7) g/mL for fish sperm (fsDNA) and 1.0 x 10(-10)-1.5 x 10(-6) g/mL for calf thymus DNA (ctDNA). Their detection limits were 3.0 x 10(-12), 4.0 x 10(-12) and 5.0 x 10(-11) g/mL, respectively. Therefore, the proposed method is one of the most sensitive methods available. The interaction between nucleic acids and Eu3+ -TTA-Phen is also discussed.     

The binding site of the strongly bound Eu3+ in Eu(3+)-regenerated bacteriorhodopsin

A Scatchard plot for the strongly bound Eu3+ to deionized bacteriorhodopsin (bR) was made using a method based on measuring the concentration of unbound Eu3+ from its fluorescence intensity. The results suggest that the first mole of Eu3+ added to a mole of bR is strongly bound by displacing 2-3 protons. In order to reconcile this result with the previous time-resolved fluorescence studies on Eu(3+)-regenerated bR, which showed the presence of 3 sites of comparable binding constants, one is forced to conclude that the emission from the strongly bound Eu3+ is completely quenched, e.g. by energy transfer to the retinal. For this to take place, the Eu3+ must be within a few A from the retinal, i.e. within the retinal pocket (the active site). The possible importance of this conclusion to the deprotonation mechanism of the protonated Schiff base, the switch of the proton pump in bR, is discussed.     

Study on the interaction between protein and Eu(III)-chlorotetracycline complex and the determination of protein using the fluorimetric method.

Chlorotetracycline (CTC) can react with europium ions Eu3+, and the complex emits the intrinsic fluorescence of Eu3+. The intensity is greatly enhanced by proteins and this forms the basis of a new fluorimetric method for determination of protein. Further research indicates that under optimum conditions, the enhanced intensity of fluorescence is in proportion to the concentration of proteins, in the range 2.0 x 10(-7)-1.0 x 10(-5) g/mL for bovine serum albumin (BSA) (linear equation, I(f) = 34.35933 + 11.54467 x 10(6)C)(r = 0.99895) and 8.0 x 10(-7)-1.0 x 10(-5) g/mL for human serum albumin (HSA) (linear equation, I(f) = 76.58881 + 5.3569 x 10(6)C) (r = 0.99283). Detection limits (S/N = 3) were 8.9 x 10(-9) g/mL for BSA and 3.3 x 10(-8) g/mL for HSA. In an assay for BSA in calf serum, this method gave a value close to that determined by the UV spectrophotometric method.     

Spectrofluorimetric determination of bilirubin in serum samples

A new spectrofluorimetric method was developed for determination of trace amount of bilirubin. Using oxytetracycline–Eu³⁺ as a fluorescent probe, in the buffer solution of pH = 7.3, bilirubin can reduce remarkably the fluorescence intensity of the oxytetracycline–Eu³⁺ complex at λ = 612 nm and the reduced fluorescence intensity was in proportion to the concentration of bilirubin. Optimum conditions for the determination of bilirubin were also investigated. The linear range and limit of detection for the determination of bilirubin were 5.0 × 10^?7, 3.0 × 10^?5 and 7.7 × 10^?8 mol L^?1, respectively. This method is simple, practical and relatively free of interference from coexisting substances and can be successfully applied to assess bilirubin in serum samples and compared with the modified Jendrassik–Grof method in clinical analysis. The quenching mechanism of the fluorescence intensity in the system is also discussed. Copyright © 2010 John Wiley & Sons, Ltd.     

The fluorometric determination of europium ion concentration as used in time-resolved fluoroimmunoassay

A model of the relationships between optimal fluorescence yield, Eu3+ ion concentration, and the concentration of beta-diketone in the determination of Eu3+ ion concentration in aqueous solutions as used in time-resolved fluoroimmunoassays was developed. The model, in addition to optimizing the determination of the metal ion, also presents a new simple and rapid method for the determination of stability constants for the formation of beta-diketone-Eu3+ complexes. It is shown how the method may be extended to determine the formation constants for Eu3+ with other chelates.     

Determination of nucleic acid by [tetra‐(3‐methoxy‐4‐hydroxyphenyl)]–Tb3+ porphyrin as the fluorescence spectral probe in bis(2‐ethylhexyl)sulfosuccinate sodium salt micelle system

A new system for the determination of nucleic acid by rare earth metallic porphyrin of [tetra‐(3‐methoxy‐4‐hydroxyphenyl)]–Tb³⁺ [T(3‐MO‐4HP)–Tb³⁺] porphyrin as fluorescence spectral probe has been developed in this paper. Nucleic acid can enhance the fluorescence intensity of the T(3‐MO‐4HP)–Tb³⁺ porphyrin in the presence of bis(2‐ethylhexyl)sulfosuccinate sodium salt(AOT) micelle. In pH 8.00 Tris–HCl buffer solution, under optimum conditions, the enhanced fluorescence intensity is in proportion to the concentration of nucleic acids in the range of 0.05–3.00 µg mL^?1 for calf thymus DNA (ct DNA) and 0.03–4.80 µg mL^?1 for fish sperm DNA(fs DNA). Their detection limits are 0.03 and 0.01 µg mL^?1, respectively. In addition, the binding interaction mechanism between T(3‐MO‐4HP)–Tb³⁺ porphyrin and ct DNA is also investigated by resonance scattering and fluorescence spectra. The maximum binding number is calculated by molar ratio method. The new system can be used for the determination of nucleic acid in pig liver, yielding satisfactory results. Copyright © 2009 John Wiley & Sons, Ltd.     

Development of a ubiquitin transfer assay for high throughput screening by fluorescence resonance energy transfer

An assay based on fluorescence resonance energy transfer (FRET) has been developed to screen for ubiquitination inhibitors. The assay measures the transfer of ubiquitin from Ubc4 to HECT protein Rsc 1083. Secondary reagents (streptavidin and antibody to glutathione-S-transferase [GST]), pre-labeled with fluorophores (europium chelate, Eu(3+), and allophycocyanin [APC]), are noncovalently attached via tags (biotin and GST) to the reactants (ubiquitin and Rsc). When Rsc is ubiquitinated, Eu(3+) and APC are brought into close proximity, permitting energy transfer between the two fluorescent labels. FRET was measured as time-resolved fluorescence at the emission wavelength of APC, almost entirely free of nonspecific fluorescence from Eu(3+) and APC. The FRET assay generated a lower ratio of signal to background (8 vs. 31) than an assay for the same ubiquitination step that was developed as a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). However, compared to the DELFIA method, use of FRET resulted in higher precision (4% vs. 11% intraplate coefficient of variation). Quenching of fluorescence was minimal when compounds were screened at 10 microg/ml using FRET. Employing a quick and simple homogeneous method, the FRET assay for ubiquitin transfer is ideally suited for high throughput screening.     

Characterization of (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum by laser-excited europium luminescence

The molecular environment of Ca2+ translocating sites of skeletal muscle sarcoplasmic reticulum (SR) (Ca2+ + Mg2+)-ATPase has been studied by pulsed-laser excited luminescence of Eu3+ used as a Ca2+ analogue. Interaction of Eu3+ with SR was characterized by investigating its effect on partial reactions of the Ca2+ transport cycle. In native SR vesicles, Eu3+ was found to inhibit Ca2+ binding, phosphoenzyme formation, ATP hydrolysis activity and Ca2+ uptake in parallel fashion. The non-specific binding of Eu3+ to acidic phospholipids associated with the enzyme was prevented by purifying (Ca2+ + Mg2+)-ATPase and exchanging the endogenous lipids with a neutral phospholipid, dioleoylglycerophosphocholine. The results demonstrate that the observed inhibition of Ca2+ transport by Eu3+ is due to its binding to Ca2+ translocating sites. The 7F0----5D0 transition of Eu3+ bound to these sites was monitored. The non-Lorentzian nature of the excitation profile and a double-exponential fluorescence decay revealed the heterogeneity of the two sites. Measurement of fluorescence decay rates in H2O/D2O mixture buffers further distinguished the sites. The number of water molecules in the first co-ordination sphere of Eu3+ bound at transport sites were found to be 4 and 1.5. Addition of ATP reduced these numbers to zero and 0.6. These data show that the calcium ions in translocating sites are well enclosed by protein ligands and are further occluded down to zero or one water molecule of solvation during the transport process.     

Spectrofluorimetric determination of folic acid in tablets and urine samples using 1,10‐phenanthroline‐terbium probe

A new spectrofluorimetric method was reported for the determination of folic acid (FA), based on its quenching effect on the fluorescence intensity of Tb³⁺–1,10‐phenanthroline complex as a fluorescent probe. The quenched fluorescence intensity at an emission wavelength of 545 nm was proportional to the concentration of FA in Tris–HCl buffer solution of pH 6.2. The effects of pH, time, order of addition of reagents, temperature and concentrations of Tb³⁺, buffer and 1,10‐phenanthroline were investigated and optimized. The linear range for the determination of FA was 0.01–1.1 mg/L. The detection limit was 0.003 mg/L and the relative standard deviation for replicated determination of 1 mg/L of folic acid was 1.2%. This method was simple, practical and relatively free from interference effects. It was successfully applied to assess FA in pharmaceutical tablets and urine samples. Copyright © 2010 John Wiley & Sons, Ltd.     

Fluorimetric determination of febuxostat in dosage forms and in real human plasma via Förster resonance energy transfer

A rapid, simple, selective and precise fluorimetric method was developed and validated for determination of a selective xanthine oxidase inhibitor; febuxostat (FBX) in pharmaceutical formulations and in human plasma. The proposed method is based on quenching effect of FBX on the fluorescence intensity of terbium (Tb³⁺) through fluorescence resonance energy transfer (FRET) from Tb³⁺ to FBX. The formed complex was measured at λ_ex. 320 nm/λ_em. 490 nm against a reagent blank. Fluorescence intensity of Tb³⁺ was diminished when FBX was added. A linear relationship between the fluorescence quenching value of the formed complex and the concentration of FBX was investigated. The reaction conditions and the fluorescence spectral properties of the complex have been studied. The linearity range of the developed method was 1.0–16.0 μg/ml. The suggested method was applied successfully for the estimation of FBX in bulk powder, dosage forms and spiked plasma samples with excellent recoveries (96.79–98.89%). In addition, the developed method has been successfully applied for determination of FBX in real plasma samples collected from healthy volunteers with good recoveries (82.06–85.65%). All obtained results of the developed method were statistically analyzed and validated according to ICH (International Conference on Harmonization) guidelines.     

Development of novel fluorescent probe 3-perylene diphenylphosphine for determination of lipid hydroperoxide with fluorescent image analysis

A novel fluorescent probe 3-perylene diphenylphosphine (3-PeDPP) was synthesized for the direct analysis of lipid hydroperoxides. The structure of 3-PeDPP was identified by the spectroscopic data, FAB-MS, (1)H NMR, and (13)C NMR. The reactivities of 3-PeDPP with lipid hydroperoxides were investigated in chloroform/MeOH homogeneous solutions and PC liposome model systems oxidized by either 2,2'-azobis(2-amidinopropane)dihydrochloride and photosensitized oxidation. The fluorescence intensity derived from 3-perylene diphenylphosphineoxide (3-PeDPPO) increased proportionally with amount of hydroperoxides produced in homogeneous solutions and liposome model systems. 3-PeDPP was easily incorporated into mouse myeloma SP2 cells and thin tissue section for dynamic membrane lipid peroxidation studies. Linear correlations between fluorescence intensity and amount of hydroperoxides in the cell membrane and tissue sections were obtained. The fluorescence intensity from 2-dimensional image analysis was also well correlated with lipid hydroperoxide level in these models. Thus, the novel probe 3-PeDPP is useful for the direct determination of lipid hydroperoxides in biological materials.     

Binding of Eu3+ to cardiac sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase-laser excited Eu3+ spectroscopic studies.

The binding of Eu3+ with Ca2+-stimulated, Mg2+-dependent adenosine triphosphatase ([Ca2+ + Mg2+]-ATPase) of cardiac sarcoplasmic reticulum (SR) has been investigated using direct laser excited Eu3+ luminescence. Eu3+ is found to inhibit both Ca2+-dependent ATPase activity and Ca2+-uptake in a parallel manner. This is attributed to the binding of Eu3+ to the high affinity Ca2+-binding sites. The Ki for Ca2+-dependent ATPase is approximately 50 nM. The 7F0----5D0 excitation spectrum of Eu3+ in cardiac SR shows a peak at 579.3 nm, as compared to 578.8 nm in potassium-morpholino propane sulfonic acid (K-MOPS) pH 6.8. Upon binding with cardiac SR, Eu3+ shows an increase in fluorescence intensity as well as in lifetime values. The fluorescence decay of bound Eu3+ exhibits a double-exponential curve. The apparent number of water molecules in the first coordination sphere of Eu3+ in SR is 2.8 for the short component and 1.0 for the long component. In the presence of ATP, a further increase in fluorescence lifetimes is observed, and the number of water molecules in the first coordination sphere of Eu3+ is reduced further to 1.3 and 0.5. The double exponential nature of the decay curve and the different number of water molecules coordinated to Eu3+ for both decay components suggest that Eu3+ binds to two sites and that these are heterogeneous. The reduction in the number of H2O ligands in the presence of ATP shows a change in the molecular environment of the Eu3+-binding sites upon phosphoenzyme formation, with a movement of Eu3+ to an occluded site on the enzyme.     

Sensitive time-resolved fluoroimmunoassay for simultaneous detection of serum thyroid-stimulating hormone and total thyroxin with Eu and Sm as labels

Based on a novel cocoating strategy and dissociation enhancement lanthanide fluorescence immunoassay technique, a sensitive time-resolved fluoroimmunoassay (TRFIA) has been developed for simultaneous quantification of human serum thyroid-stimulating hormone (TSH) and thyroxin (T4) in a one-and-the-same assay procedure. The new cocoating strategy for preparing highly active surface anti-TSH and anti-T4 monoclonal antibodies (McAbs) was performed by a three-step protocol. Namely, anti-TSH McAb at high concentration (10 micro g/ml) and extensively biotinylated bovine serum albumin (BSA) at low concentration (0.5 micro g/ml) were coated on microwells by passive adsorption, then streptavidin was captured by the surface BSA-biotin, and finally biotinylated anti-T4 McAb was immobilized by the remnant binding sites of the bound streptavidin. In the present TSH/T4 TRFIA, both sandwich- and competitive-type configurations were involved, and Eu(3+) and Sm(3+) were used as labels for TSH and T4 detection, respectively. The method showed rapid kinetics; the equilibrium was reached within 30min at 37 degrees C due to the use of high concentrations of reaction reagents, rapid agitation, and small reaction volume. The lower limits of detection of the method were 0.028 mIU/L for TSH and 4.1 nmol/L for T4 with 20 micro L of sample volume. The assay ranges for TSH and T4 were 0.21-80.00 mIU/L and 20-300 nmol/L, respectively. The correlation between the TSH/T4 values obtained by the present TSH/T4 TRFIA and those obtained by commercial chemiluminescence immunoassay was satisfactory.     

Simultaneous determination of amlodipine and metoprolol in their combined dosage form using a synchronous fluorescence spectrofluorimetric method

Highly sensitive, rapid, accurate and precise synchronous fluorescence spectrofluorimetric method has been developed for simultaneous analysis of a mixture of amlodipine (AMD) and metoprolol (MET). The method relies on measuring the relative synchronous fluorescence intensity of both drugs at Δλ of 90 nm in acetate buffer solution at pH 5. The experimental parameters influencing the developed method were investigated and optimized. The method was linear over the ranges 0.2–2 μg/ml and 0.5–10 μg/ml for AMD and MET, respectively. The limits of detection were 50 ng/ml for AMD and 130 ng/ml for MET while the limits of quantitation were 150 ng/ml for AMD and 390 ng/ml for MET. The developed method was applied successfully for the determination of the two drugs in their co‐formulated tablet. The mean percent recoveries were found to be 100.51 and 99.57 for AMD and MET, respectively.     

The determination of the Mg2+.ATP dissociation constant by competition with Eu3+ ion using laser-induced Eu3+ ion luminescence spectroscopy

A direct spectroscopic method for the determination of the submicromolar dissociation constant of Eu3+. ATP using laser-induced Eu3+ ion luminescence spectroscopy is described. The dissociation constant of Mg2+.ATP is then determined by the competition of Mg2+ with Eu3+ for the binding of ATP. The experiments were performed in 2H2O to mitigate the significant quenching of the Eu3+ luminescence that occurs in 1H2O. Values for the effective dissociation constants of the 1:1 ATP metal ion complexes of 1.2 +/- 0.3 X 10(-7) and 2.7 +/- 0.7 X 10(-4) M are obtained for Eu3+ and Mg2+, respectively, at p2H 5.8.     

Fluorescence probe enhanced spectrofluorimetric method for the determination of sparfloxacin in tablets and biological fluids

Yttrium‐sensitized fluorescence was used to develop a sensitive and simple spectrofluorimetric method for the determination of sparfloxacin. The method is based on the strong fluorescence of sparfloxacin after adding the fluorescence probe yttrium in buffer solution (pH = 8), and various factors influencing fluorescence were investigated. Under optimum conditions, the enhanced fluorescence intensity of the system showed a good linear relationship with the concentration of sparfloxacin over the range 8 × 10^?7 to 1.4 × 10^?5 mol L^?1 with a correlation coefficient of 0.9997. The detection limit (S/N = 3) was determined as 9.01 × 10^?8 mol L^?1. The mechanism of the sensitizing effect of probe was discussed. This method has been successfully applied for the determination of sparfloxacin in pharmaceuticals, human urine and serum samples; the result obtained was satisfactory. Copyright © 2010 John Wiley & Sons, Ltd.     

Fluorescence enhancement effect of the morin-Al3+ -sodium dodecyl benzene sulphonate-protein system and the determination of proteins.

The fluorescence intensity of the morin-Al(3+) complex was greatly enhanced by proteins in the presence of sodium dodecyl benzene sulphonate (SDBS). Based on this, a new fluorimetric method for the determination of protein was developed. Under optimum conditions, the enhanced intensity of fluorescence was in proportion to the concentration of proteins in the range 1.0 x 10(-8)-1.3 x 10(-5) g/mL for bovine serum albumin (BSA), 4.0 x 10(-8)-1.2 x 10(-5) g/mL for egg albumin (EA) and 5.0 x 10(-8)-1.2 x 10(-5) g/mL for human serum albumin (HSA). Their detection limits (S:N = 3) were 5.0 x 10(-9), 1.8 x 10(-8) and 1.6 x 10(-8) g/mL, respectively. The interaction mechanism was also studied.