DNA aptamer beacon assay for C-telopeptide and handheld fluorometer to monitor bone resorption

A novel DNA aptamer beacon is described for quantification of a 26-amino acid C-telopeptide (CTx) of human type I bone collagen. One aptamer sequence and its reverse complement dominated the aptamer pool (31.6% of sequenced clones). Secondary structures of these aptamers were examined for potential binding pockets. Three-dimensional computer models which analyzed docking topologies and binding energies were in agreement with empirical fluorescence experiments used to select one candidate loop for beacon assay development. All loop structures from the aptamer finalists were end-labeled with TYE 665 and Iowa Black quencher for comparison of beacon fluorescence levels as a function of CTx concentration. The optimal beacon, designated CTx 2R-2h yielded a low ng/ml limit of detection using a commercially available handheld fluorometer. The CTx aptamer beacon bound full-length 26-amino acid CTx peptide, but not a shorter 8-amino acid segment of CTx peptide which is a common target for commercial CTx ELISA kits. The prototype assay was shown to detect CTx peptide from human urine after creatinine and urea were removed by size-exclusion chromatography to prevent nonspecific denaturing of the aptamer beacon. This work demonstrates the potential of aptamer beacons to be utilized for rapid and sensitive bone health monitoring in a handheld or point-of-care format.     

Application of Functionalized ZnSe Nanoparticles to Determinate Heavy Metal Ions

In this paper, ZnSe nanoparticles, which were modified with mercaptoacetic acid (MAA), worked as novel fluorescence sensors for the quantitative determination of copper(II) and nickel(II). Under the optimal conditions, the fluorescence intensities of functionalized ZnSe nanoparticles were quenched by the addtion of copper(II) or nickel(II) ions, there were linear relationships between the relative fluorescence intensity (logF₀/F) and the concentration in the range of 140–2,000 μg/L for copper(II) (R = 0.9973) and 30–1,000 μg/L for nickel(II) (R = 0.9992), the limits of detection were 50 μg/L and 5 μg/L, respectively.     

Using fluorescence measurement of zinc ions liberated from ZnS nanoparticle labels in bioassay for <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7

In this study, an alternative approach using ZnS nanoparticle biolabels as fluorescence signal transducers is reported for the immunoassay of E. coli O157:H7 in tap water samples. Instead of measuring the fluorescence of ZnS nanoparticles in the assay, the fluorescence signal is generated through the binding of zinc ions released from nanoparticle labels with zinc-ion sensitive fluorescence indicator Fluozin-3. In the assay, ZnS nanoparticles around 50 nm in diameter were synthesized, bioconjugated, and applied for the detection of E. coli O157:H7. The assay shows a detection range over two orders of magnitude and a detection limit around 1000 colony-forming units (cfu) of E. coli O157:H7.     

Simultaneous Determination of Acetylsalicylic Acid and Caffeine in Pharmaceutical Formulation by First Derivative Synchronous Fluorimetric Method

A sensitive, rapid, and specific assay has been developed for the simultaneous determination of acetylsalicylic acid and caffeine in commercial tablets based on their natural fluorescence. The mixture of these drugs was resolved by first derivative synchronous fluorimetric technique using two scans. At Δλ=106 nm, using first derivative synchronous scanning, only acetylsalicylic acid yields a detectable signal at 316 nm (peak to zero method) which is unaffected by caffeine. At Δλ=30 nm, the signal of caffeine at 288 nm (peak to zero method) is not affected by acetylsalicylic acid. The range of application is between 0.021 and 41.62 μg ml⁻¹ (correlation coefficient, R=0.9995) for acetylsalicylic acid and between 0.4486 and 44.86 μg ml⁻¹ (correlation coefficient, R=0.99786) for caffeine. The recovery range of 98.40–102% for acetylsalicylic acid and 90–100.5% for caffeine from their synthetic mixture was reported. Overall recovery of both compounds about 97–99% for acetylsalicylic acid and 97–98% for caffeine was obtained from real sample analysis. The detection limits are 0.0013 μg ml⁻¹ and 0.0306 μg ml⁻¹ for acetylsalicylic acid and caffeine, respectively. The relative standard deviation (n=10) for 20 μg ml⁻¹ of acetylsalicylic acid is 2.75% and for 2.2 μg ml⁻¹of caffeine is 1.7%.     

Rate of Convergence Towards the Hartree–von Neumann Limit in the Mean-Field Regime

In the mean-field regime we prove convergence, with explicit bounds, of N-particle density matrices satisfying the time-dependent von Neumann equation with factorized initial data to a product of one particle density matrices satisfying the Hartree–von Neumann equation. To prove explicit bounds we generalize techniques developed by Pickl (in A simple derivation of mean field limits for quantum systems. ArXiv:0907.4464, 2009) and Knowles–Pickl (in Commun. Math. Phys. 298(1):101–138, 2010).     

Determination of Tetracaine Hydrochloride by Fluorescence Quenching Method with Some Aromatic Amino Acids as Probes

A novel fluorescence quenching method for the determination of tetracaine hydrochloride (TA·HCl) concentration with some aromatic amino acids as fluorescence probe has been developed. In pH 6.3 acidic medium, tryptophane (Trp), tyrosine (Tyr) or phenylalanine (Phe) can react with tetracaine hydrochloride to form an ion-association complex by electrostatic attraction, aromatic stacking interaction and Van der Waals’ force, which lead to fluorescence quenching of above amino acids. The maximum fluorescence excitation and emission wavelengths of them are located at 278, 274, 258 nm and 354, 306, 285 nm, respectively. The relative fluorescence intensity (F ₀/F) is proportional to the TA·HCl concentration in certain range. The linear ranges and detection limits are 1.2–5.0 μg/mL and 0.37 μg/mL for Tyr-TA·HCl system, 1.3–6.0 μg/mL and 0.38 μg/mL for Trp-TA·HCl system, and 1.4–6.0 μg/mL and 0.41 μg/mL for Phe-TA·HCl system. The optimum reaction conditions, influencing factors and the effect of coexisting substances are investigated. And the results show the method has a good selectivity. Judging from the effect of temperature, the Stern-Volmer plots and fluorescence lifetime determination, the quenching of fluorescence of amino acids by TA·HCl is a static quenching process.     

A Coumarin-Based Fluorescent Chemosensor for Zn<Superscript>2+</Superscript> in Aqueous Ethanol Media

A coumarin-based fluorescent chemosensor 1 for Zn²⁺ was designed and synthesized. Compound 1 exhibits lower background fluorescence due to intramolecular photoinduced electron transfer. However, upon mixing with Zn²⁺ in 30% (v/v) aqueous ethanol, a “turn-on” fluorescence emission is observed. The fluorescence emission increases linearly with Zn²⁺ concentration in the range 0.5–10 μmol L⁻¹ with a detection limit of 0.29 μmol L⁻¹. No remarkable emission enhancement was, however, observed for other metal ions. The proposed chemosensor was applied to the determination of Zn²⁺ in water samples with satisfactory results.     

Fluorescence lifetime characterization of bacteria using total lifetime distribution analysis with the maximum entropy method

Total lifetime distribution analysis was employed to obtain fluorescence lifetime profiles of the intrinsic fluorescence ofPseudomonas fluorescens, Escherichia coli, Bacillus subtilis, andStaphylococcus epidermidis. The lifetimes were measured using a multiharmonic Fourier transform phase-modulation fluorometer which can simultaneously measure the phase shift and demodulation at many modulation frequencies. The 364-nm line from an argon-ion laser and the 325- and 442-nm lines from a helium-cadmium laser were used for sample excitation. Broad emission windows were used to capture as much of the bacterial emission as possible for the lifetime measurements. The maximum entropy method was used to recover lifetime profiles from the multifrequency phasemodulation data. At all three excitation wavelengths, the bacteria exhibited three lifetime components, in the ranges of 0.5-1, 2–3, and 4–8 ns. Using 325-nm excitation, a fourth component, in the range of 9–14 ns, was recovered in all of the bacteria; using 364-nm excitation, the fourth component was resolved only in the two Gram-negative bacteria (P. fluorescens andE. coli). Excitation at 364 nm provided the most reproducible lifetime profiles and showed some differences among the four bacteria.     

Spectrofluorimetric Determination of Etodolac, Moxepril HCl and Fexofenadine HCl Using Europium Sensitized Fluorescence in Bulk and Pharmaceutical Preparations

A simple, selective and sensitive luminescence method has been developed for the assay of etodolac (I), moxepril HCl (II) and fexofenadine HCl (III) in bulk drug and pharmaceutical formulations. The method is based on the luminescence sensitization of europium (Eu³⁺) by complexation with the studied drugs. The fluorescence intensities of the products were measured at 667 nm for (I) and at 615 for (II) and (III) while exciting at 276 for all the studied drugs. The fluorescence intensity was directly proportional to the concentration over the range (20–280), (40–240) and (30–80) ng/ml with limits of detection (LOD) = 0.93, 0.92 and 0.95 μg/ml for drugs I, II and III respectively. Optimum conditions for the formation of the complex in methanol were carefully studied. The proposed method was successfully applied for the assay of the studied drugs in pharmaceutical formulations with excellent recovery.     

Spectrofluorimetric Determination of 3-hidroxy-2-naphthoic Acid by Use of Its Ternary Complex with Zirconium (IV) and Beta-Cyclodextrin: Application to Determination in River Water

A spectrofluorimetric method has been developed for the determination of 3-hydroxy-2-naphthoic acid (3H2NA) by formation of a ternary complex with zirconium (IV) and β-cyclodextrin (β-CD). It has been observed that the fluorescence intensity of 3H2NA is greatly enhanced when the ternary complex is formed and is accompanied with shifts in the excitation and emission wavelengths. The conditions for the formation of the ternary complex have been optimized and the stoichiometry has been calculated, resulting a 1:2:1 complex (3H2NA:Zr: β-CD). The linear range was 20–2000 ng mL⁻¹ and the detection and quantification limits calculated were 17 and 58 ng mL⁻¹, respectively. The proposed method was applied to the determination of 3H2NA in river water. To eliminate interferences an off-line solid phase extraction (SPE) procedure using C18 cartridges was used. The extraction procedure was optimized and good recoveries were obtained (around 100%) with relative standard deviations (RSDs) of less than 5%.     

New Classes of Toda Soliton Solutions

We provide a detailed investigation of limits of N–soliton solutions of the Toda lattice as N tends to infinity. Our principal results yield new classes of Toda solutions including, in particular, new kinds of soliton–like (i.e., reflectionless) solutions. As a byproduct we solve an inverse spectral problem for one–dimensional Jacobi operators and explicitly construct tri–diagonal matrices that yield a purely absolutely continuous spectrum in (-1,1) and give rise to an eigenvalue spectrum that includes any prescribed countable and bounded subset of . Received: 16 October 1995/Accepted: 23 July 1996     

An Indirect Competitive Fluorescence Immunoassay for Determination of Dicyclohexyl Phthalate in Water Samples

A sensitive and specific indirect competitive fluorescence immunoassays (FIA) has been developed for the quantitative determination of dicyclohexyl phthalate (DCHP) using an antigen-coated plate format. The polyclonal antibodies raised against dicyclohexyl 4-amino phthalate conjugated to bovine serum albumin (BSA) by the amino diazotization linkage method. Antiserum with a sufficiently high titer was generated in rabbits and fluorescein isothiocyanate (FITC) was used as sensitive labels to construct the fluorescence immunoassay (FIA) for measurement of targeted compounds. Under optimized FIA condition, the quantitative working range was from 0.1 to 200 μg L⁻¹ with a limit of detection of 0.05 μg L⁻¹. Other similar phthalate compounds do not interfere significantly in the analysis using this immunoassay technique, and the cross-reactivity rates were less than 10%. Four kinds of water samples (tap water, lake water, river water and leachate) had been detected in this assay, the recovery was 91.3–107.8%. The proposed fluorescence immunoassay turned out to be a powerful tool for monitoring of dicyclohexyl phthalate in water samples at trace level.     

A Simple and Sensitive Spectrofluorimetric Method for Analysis of Some Nitrofuran Drugs in Pharmaceutical Preparations

A simple, rapid, selective and sensitive spectrofluorimetric method was described for the analysis of three nitrofuran drugs, namely, nifuroxazide (NX), nitrofurantoin (NT) and nitrofurazone (NZ). The method involved the alkaline hydrolysis of the studied drugs by warming with 0.1 M sodium hydroxide solution then dilution with distilled water for NX or 2-propanol for NT and NZ. The formed fluorophores were measured at 465 nm (λ _Ex 265 nm), 458 nm (λ _Ex 245 nm) and 445 nm (λ _Ex 245 nm) for NX, NT and NZ, respectively. The reaction pathway was discussed and the structures of the fluorescent products were proposed. The different experimental parameters were studied and optimized. Regression analysis showed good correlation between fluorescence intensity and concentration over the ranges 0.08–1.00, 0.02–0.24 and 0.004–0.050 μg ml⁻¹ for NX, NT and NZ, respectively. The limits of detection of the method were 8.0, 1.9 and 0.3 ng ml⁻¹ for NX, NT and NZ, respectively. The proposed method was validated in terms of accuracy, precision and specificity, and it was successfully applied for the assay of the three nitrofurans in their different dosage forms. No interference was observed from common pharmaceutical adjuvants. The results were favorably compared with those obtained by reference spectrophotometric methods.     

Luminescence of biologically active 24-epicastasterone and a model compound

Biologically active brassinosteroid 24-epicastasterone, ring B of which contains a C=O group and has the nπ*-configuration for a low-lying electronic excited state, exhibits rapid fluorescence. The wavelengths of the fluorescence maxima of the steroid dissolved in hexane and acetonitrile are equal to 332 and 394 nm, respectively. The fluorescence lifetime of the steroid dissolved in acetonitrile is τ = 9.9 nsec. Solutions of 24-epibrassinolide do not luminesce. The long-wavelength electronic absorption band λ_max^abs = 340 nm in the absorption spectrum of an ethanol solution of model compound 2, ring D of which contains a C=O group π*-conjugated with the C=C double bond of ring C, like in the spectrum of the steroid, has a low extinction coefficient. An ethanol solution of 2 does not fluoresce. 24-Epicastasterone at 77 K in ethanol solution exhibits phosphorescence with λ_max^phos = 447 nm. The phosphorescence decay is exponential with τ = 0.79 msec. Compound 2 also phosphoresces. The phosphorescence spectrum of its ethanol solution has a maximum at 490 nm. The phosphorescence decay is nonexponential in the early stage. The phosphorescence lifetime is 25 msec in the exponential decay region. __________ Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 75, No. 2, pp. 182–186, March–April, 2008.     

Francisella tularensis detection using magnetic labels and a magnetic biosensor based on frequency mixing

A biosensor that uses resonant coils with a special frequency-mixing technique and magnetic beads as detectable labels has been established for the detection of Francisella tularensis, the causative agent for tularemia. The detection principle is based on a sandwich immunoassay using an anti-Ft antibody for immunofiltration immobilized to ABICAP^® polyethylene filters, and biotinylated with streptavidin-coated magnetic beads as labels. The linear detection range of this biosensor was found to be 10⁴–10⁶ cfu F. tularensis lipopolysaccharide (LPS) per ml. Tested sample matrices were physiological PBS buffer and rabbit serum.     

Fluorescence Enhancement Effect for the Determination of Polychlorinated Biphenyls with Bovine Serum Albumin

A sensitive and selective method for the trace determination of 3, 3’, 4, 4’-tetrachlorobiphenyl (PCB77) by using bovine serum albumin (BSA) as a fluorescence probe was introduced. Under optimum conditions, the enhanced fluorescence intensity was proportional to the concentration of polychlorinated biphenyls in the range of 8.9 × 10⁻⁸–5.0 × 10⁻⁶ mol L⁻¹ for PCB77, and 5.0 × 10⁻⁷–5.0 × 10⁻⁶ mol L⁻¹ for 2, 2’, 5, 5’-tetrachlorbiphenyl (PCB52). The detection limits (S/N = 3) of PCB77 and PCB52 were 2.6 × 10⁻⁸ mol L⁻¹ and 2.9 × 10⁻⁷ mol L⁻¹, respectively. Furthermore, the fluorescence enhancement mechanism was discussed in detail. Results indicated that fluorescence enhancement of the system originated from the formation of BSA-PCBs complexes. In addition, PCBs were mainly bound to the tyrosine residues in BSA molecules.     

Push-Pull Fluorophores Based on Imidazole-4,5-dicarbonitrile: A Comparison of Spectral Properties in Solution and Polymer Matrices

Spectral properties of novel type of fluorophores consist of a π-conjugated system end-capped with an electron-donating N,N-dimethylaminophenyl group and an electron-withdrawing imidazole-4,5-dicarbonitrile moiety were examined. An additional π-linker separating these two structural units comprises simple bond (B1P), phenyl (B2B), styryl (B3S) and ethynylphenyl (B4A) moieties. The absorption and fluorescence spectra were taken in cyclohexane, chloroform, acetonitrile, methanol and in polymer matrices such as polystyrene, poly(methyl methacrylate) and poly(vinylchloride). The longest-wavelength absorption band was observed in the range of 300 to 400 nm. Intense fluorescence with quantum yields of 0.2 to 1.0 was observed in cyclohexane, chloroform and in polymer matrices within the range of 380 to 500 nm. The fluorescence was strongly quenched in neat acetonitrile and methanol. The fluorescence lifetimes are in the range of 1–4 ns for all measured fluorophores. The large Stokes shift (4,000 to 8,000 cm⁻¹) indicates a large difference in the spatial arrangement of the chromophore in the absorbing and the emitting states. The observed fluorescence of all fluorophores in chloroform was quenched by 1-oxo-2,2,6,6-tetramethyl-4-hydroxy piperidine by the diffusion-controlled bimolecular rate (cca 2 × 10¹⁰ L mol⁻¹ s⁻¹). Polar solvents such as acetonitrile and methanol quenched the fluorescence as well but probably via a different mechanism.     

Stress-Induced Alteration of Chlorophyll Fluorescence Polarization and Spectrum in Leaves of <Emphasis Type="Italic">Alocasia macrorrhiza</Emphasis> L. Schott

The value of intrinsic chlorophyll fluorescence polarization, and the intensity in emission spectrum were investigated in leaf segments of Alocasia macrorrhiza under several stress conditions including different temperatures (25–50°C), various concentrations of NaCl (0–250 mM), methyl viologen (MV, 0–25 μM), SDS (0–1.0%) and NaHSO₃ (0–80 μM). Fluorescence emission spectrum of leaves at wavelength regions of 500–800 nm was monitored by excitation at 436 nm. The value of fluorescence polarization (P value), as result of energy transfer and mutual orientation between chlorophyll molecules, was determined by excitation at 436 nm and emission at 685 nm. The results showed that elevated temperature and concentrations of salt (NaCl), photooxidant (MV), surfactant (SDS) and simulated SO₂ (NaHSO₃) treatments all induced a reduction of fluorescence polarization to various degrees. However, alteration of the fluorescence spectrum and emission intensity of F₆₈₅ and F₇₃₁ depended on the individual treatment. Increase in temperature and concentration of NaHSO₃ enhanced fluorescence intensity mainly at F₆₈₅, while an increase in MV concentration led to a decrease at both F₆₈₅ and F₇₃₁. On the contrary, NaCl and SDS did not cause remarkable change in fluorescence spectrum. Among different treatments, the negative correlation between polarization and fluorescence intensity was found with NaHSO₃ treatments only. We concluded that P value being measured with intrinsic chlorophyll fluorescence as probe in leaves is a susceptible indicator responding to changes in environmental conditions. The alteration of P value and fluorescence intensity might not always be shown a functional relation pattern. The possible reasons of differed response to various treatments were discussed.     

Steady-State and Time Resolved Fluorescence Analysis on Tyrosine–Histidine Model Compounds

Four model compounds, for a tyrosine–histidine covalent bonding, 2-(5-imidazolyl)-4-methylphenol (C–C bonding in ortho-position at the phenyl group); 2′-(1-imidazolyl)-4-methylphenol (C–N bonding in ortho′-position at the phenyl group); 2-(5-imidazolyl)-4-H-phenol and 2-(5-imidazolyl)-4-H-phenol, at physiological pH have been studied by UV-Vis absorption, steady-state and time resolved fluorescence spectroscopy. Their absorption and emission properties are presented and discussed. The photophysical properties depend on the para-substituted phenyl group as well as on C–C/C–N bonding in the Phenol–Imidazole linkage. The N position, N1–N3/N1–N4, in the imidazole group was found to be relevant. The results are discussed with relevance to the redox processes of tyrosine and to better understand the role of a tyrosine–histidine covalent linkage as found in cytochrome c oxidase.     

First Derivative Synchronous Fluorescence Spectroscopy for the Simultaneous Determination of Sulpiride and Mebeverine Hydrochloride in Their Combined Tablets and Application to Real Human Plasma

A rapid, simple and highly sensitive first derivative synchronous fluorometric method has been developed for the simultaneous analysis of binary mixture of sulpiride (SUL) and mebeverine hydrochloride (MEB). The method is based upon measurement of the synchronous fluorescence intensity of these drugs at ∆λ = 100 nm in water. The different experimental parameters affecting the fluorescence of the two drugs were carefully studied and optimized. The fluorescence-concentration plots were rectilinear over the range of 0.05–1 μg/mL and 0.2–3.2 μg/mL for SUL and MEB respectively with lower detection limits (LOD) of 0.006 and 0.01 μg/mL and quantification limits (LOQ) of 0.0.02 and 0.05 μg/mL for SUL and MEB, respectively. The proposed method was successfully applied for the determination of the two compounds in synthetic mixtures and in commercial tablets. The high sensitivity attained by the proposed method allowed the determination of both of SUL and MEB metabolite (veratic acid) in real human plasma samples applying second derivative synchronous fluorometric technique. The mean% recoveries (n = 3) for both MEB metabolite (veratic acid) and SUL were 99.82 ± 2.53 and 98.84 ± 6.20 for spiked human plasma respectively, while for real human plasma, the mean% recoveries (n = 3) were 91.49 ± 4.25 and 91.36 ± 8.46 respectively.