Sensitive Fluorometric Method Research Articles

Autofluorescent Hyperbranched Poly(amide amine) as Effective Fluorescent Probe for Label-free Detection of Copper(II) Ions.

A label-free fluorescent probe based on autofluorescent hyperbranched poly(amide amine) (HPAMAM) for copper ions was designed. HPAMAM is a cationic polymer containing many amino groups, which could bind Cu2+ ions to form cupric amine complexes, leading to a selective quenching of the fluorescence intensity of HPAMAM via inner filter effect. The fluorescence intensity of HPAMAM decreased with increasing concentration of Cu2+ ions and the linear response ranged from 0.05 to 25 μM (R2 = 0.995), with the corresponding detection limit (3σ/k) of 17.15 nM. The HPAMAM fluorescent probe provided a simple, rapid, selective and sensitive fluorometric method for detecting Cu2+ ions, which could be also applied for detection of Cu2+ ions in real water samples.

A fluorescence switch sensor used for D-Penicillamine sensing and logic gate based on the fluorescence recovery of carbon dots

A specific and sensitive fluorometric method based on the Carbon dots (CDs) probe which show unique optical properties is proposed for the determination of D-Penicillamine (D-PA) in human serum samples with satisfactory results. The fluorescence of CDs decreased apparently in the presence of mercury ion (Hg2+), Similarly, the addition of D-Penicillamine (D-PA) resulted in efficient fluorescence recovery of the CDs due to the strong affinity between Hg2+ and D-PA. Herein, this phenomenon has been exploited to design an “AND” logic gate and specific fluorescence switch sensor for sensitively sensing D-PA for the first time. Furthermore, the CDs possesses bright blue fluorescence with the maximum excitation and emission wavelength at 358nm and 442nm, respectively. Thus, after the experimental conditions are optimized, the linear range for detection PA is 2.0–24.0μmolL−1, with correlation coefficient and detection limit is R2=0.998, 0.6μmolL−1, respectively. Moreover, the proposed method is not only expected to be come a potential tool for the fast response of D-PA but also possesses the potential for practical application.

Sensitive fluorometric method for the determination of rutin in combined tablet dosage form.

A sensitive method based on the fluorescence enhancement was developed for the determination of rutin. It was found that the rutin could form a fluorescent complex with ytterium (III), and the fluorescence intensity of which could be enhanced by sodium dodecyl benzene sulfonate. Under the optimum conditions, the enhanced fluorescence intensity was in proportion to the concentration of rutin in the range of 12-1200 ng/ml and the detection limit (S/N=3) was 1.3 ng/ml. This method possessed the properties of simplicity, fast assay process and high sensitivity, and has been satisfactorily applied for the analysis of actual sample. The possible interaction mechanism of this system was also discussed in this manuscript.

A novel fluorescence assay for inorganic pyrophosphatase based on modulated aggregation of graphene quantum dots.

A simple and highly sensitive fluorometric method has been developed for inorganic pyrophosphatase (PPase) activity detection based on the disaggregation and aggregation of graphene quantum dots (GQDs). Copper ions can trigger the severe aggregation of GQDs with rich carboxyl groups, which results in effective fluorescence quenching. While, with the addition of pyrophosphate (PPi), the quenched fluorescence is effectively recovered owing to the strong interaction between PPi and Cu(2+). Furthermore, under the catalytic hydrolysis of PPase, the complex of PPi-Cu(2+)-PPi is rapidly disassembled, and the fluorescence is re-quenched. This method is highly sensitive and selective for PPase detection, with a linear correlation between the fluorescence intensity and the PPase concentration in the range from 1 to 200 mU mL(-1) with a detection limit down to 1 mU mL(-1) (S/N = 3). Additionally, the inhibition effect of NaF on the PPase activity is also studied. Thus, the proposed method may hold a potential application in the diagnosis of PPase-related diseases and screening of PPase inhibitors, to evaluate the function and inhibition of PPase in biological systems.

Determination of ethylenediaminetetraacetic acid base on the reversion of fluorescence quenching of 2-pyridinecarbaldehyde-p-phenyldihydrazone by ferric iron

A fast and sensitive fluorometric method for determination of an emerging pollutant, ethylenediaminetetraacetic acid (EDTA), was proposed based on the reversion of fluorescence quenching of 2-pyridinecarbaldehyde-p-phenyldihydrazone by ferric iron. It was found that Fe3+ could selectively quench the fluorescence of 2PC-PPH, while EDTA could recover it due to its strong coordination ability with Fe3+. Based on this, we established an “Off and On” fluorescence method for determination of EDTA. Under the optimum conditions, the linear range and detection limit (3S/N) for EDTA determination were 2.0×10−6∼1.4×10−5molL−1 and 1.5×10−7molL−1, respectively. The relative standard deviation for the determination of 7.0×10−6molL−1 EDTA was 3.7% (n=11). The method was successfully applied to determination of EDTA in tap water, lake water and investigating degradation of EDTA by TiO2/UV process.

Highly Sensitive Fluorometric Assay Method for Acetylcholinesterase Inhibitor Based on Nile Red‐Adsorbed Gold Nanoparticles

AbstractA new sensitive fluorometric assay method for acetylcholinesterase (AChE) and its inhibitor was developed using a fluorescent dye, nile red (NR). Due to the fluorescence resonance energy transfer between the NR and the gold nanoparticle (AuNPs), the fluorescence was quenched. AChE can break down acetylthiocholine to produce a thiol‐bearing compound, thiocholine. In the presence of thiocholine, the nile red is replaced from the AuNPs surfaces and simultaneously transformed to a derivative of nile red. The fluorescence intensity of the derivative is much stronger than that of the native nile red with the same concentration and its maximum emission wavelength has a blue shift so that the sensor achieves a good signal‐to‐background ratio. In addition, when organophosphate pesticide (OPs) exists, the activity of AChE can be inhibited, the generation of thiocholine will be prevented and no fluorescence enhancement occurs. The results show that the method is sensitive to AChE and paraoxon with the detection limits of 0.2 mU/mL and 0.05 ng/mL, respectively.

Enzymatic detection of ethanol based on H2O2-sensitive quantum dots

A rapid and sensitive fluorometric method for the enzymatic detection of ethanol using CdSe/ZnS quantum dots (QDs) is proposed. The photoluminescence of QDs is sensitive to H2O2. This finding leads to a novel approach for the determination of ethanol using alcohol oxidase (AO x ) which, on oxidation of ethanol, produces H2O2. The method has higher sensitivity, wider analytical range (0.1–8 mmol/L), and a lower detection limit (0.05 mmol/L). The relationship between quenching of the photoluminescence of the QDs and the concentration of ethanol is linear.

A fast and sensitive method for the determination of nitrite in human plasma by capillary electrophoresis with fluorescence detection

Analysis of nitrite, the indicator of nitric oxide (NO) generation in vivo, provides a useful tool to study NO synthesis in vivo. A fast and sensitive fluorometric CE method was developed for determination of nitrite in human plasma through its derivatization with 2,3-diaminonaphthalene (DAN). Nitrite in human plasma was easily reacted with DAN under acid conditions to yield the highly fluorescent 2,3-naphthotriazole (NAT). Fluorescence detection was optimized to achieve subnanomolar detection which allows a direct analysis of plasma samples unlike most CE–UV methods using sample stacking. Acetonitrile was used to remove the protein. Short-end injection and a high voltage (−30kV) were used to shorten the analysis time. The good separation was achieved with 20mM borate buffer at pH 9.23. The separation of NAT was obtained within 1.4min. The deproteinized plasma sample was injected hydrodynamically for 5s at −50mbar into a 60cm×75μm internal diameter uncoated fused-silica capillary. Excitation wavelength was selected with a broad-band filter (240–400nm), and the emitted light was measured at 418nm by the use of a cutoff filter. A good linearity (R2=0.9975) was obtained in the range from 2 to 500nM. The detection limit of nitrite was 0.6nM in original plasma samples, which is 750 times lower than our previous CE–UV method. The developed fluorometric CE method offers the advantages of more simple system and lower cost compared with the current fluorometric HPLC methods without losing sensitivity. The detected mean nitrite concentration in human plasma by this method was consistent with the most frequently reported values.

Development and Validation of a Terbium-Sensitized LuminescenceAnalytical Method for Deferiprone.

A sensitive fluorometric method for the determination of deferiprone (DFP) based on the formation of a luminescent complex with Tb(3+) ions in aqueous solutions is reported. The maximum excitation and emission wavelengths were 295 and 545 nm, respectively. The effects of various factors on the luminescence intensity of the system were investigated and optimized, then under the optimum conditions, the method was validated. The method validation results indicated that the relative intensity at 545 nm has a linear relationship with the concentration of DFP in aqueous solutions at the range of 7.2 × 10(-9) to 1.4 × 10(-5) M, the detection and quantification limits were calculated respectively as 6.3 × 10(-9) and 2.1 × 10(-8) M, precision and accuracy of the method were lower than 5% and the recovery was between 100.1% and 102.3%. The results indicated that this method was simple, time saving, specific, accurate and precise for the determination of DFP in aqueous solutions. After optimization and validation, the method successfully applied for determination of DFP in tablet dosage forms. The stoichiometry of the Tb(3+)-DFP complex was found as 1:3 and the complex formation constant was 1.6 × 10(16).

エキシマー蛍光誘導体化法による河川水中のビスフェノールＡの液体クロマトグラフ分析

Gas chromatography/mass spectrometry (GC/MS) is generally used for analysis of bisphenols in field environments. However, there are the cases that the sensitivity is not enough to determine their concentration. In this work, a liquid-chromatographic determination based on intramolecular excimer-forming fluorescence derivatization with a pyrene reagent, 4-(1-pyrene)butyryl chloride was examined for the analysis of bisphenol A in river waters. This method is highly selective and sensitive fluorometric method for biogenic related compounds, polyamines, polyphenols and dicarboxylic acids though it has never been applied for analysis of bisphenols in field environments. With the method, we determined concentrations of bisphenol A in waters sampled form 6 rivers in Shizuoka Prefecture. As a result, the values determined were ranged from 0.0016-0.17μg/L. They agreed with those determined by conventional method with GC/MS.

A rapid and sensitive fluorometric method for the quantitative analysis of snake venom metalloproteases and their inhibitors

Metalloproteases are responsible for the hemorrhagic effects of many snake venoms and contribute to other pathways that lead to local tissue damage. Methods that quantify snake venom metalloproteases (SVMP) are therefore valuable tools in research on the clinical, physiological, and biochemical effects of envenomation. Comparative analysis of individual, population, and species differences requires screening of large numbers of samples and treatments, and therefore require a method of quantifying SVMP activity that is simple, rapid, and sensitive. This paper demonstrates the properties of a new fluorometric assay of SVMP activity that can provide a measure of metalloprotease activity in 1 h. The assay is reliable, with variation among replicates sufficiently small to reliably detect differences in between species (F19,60 = 2924, p < 0.001), even for those venoms with low overall activity. It is also sensitive enough to detect differences among venoms using <2 ng of whole venom protein. We provide an example use of this assay to detect the presence of natural SVMP inhibitors in minute samples of blood plasma from rock squirrels (S. variegatus), a natural prey species for North American rattlesnakes. We propose this assay is a useful addition to the set of tools used to characterize venoms, as well as high-throughput screening of natural or synthetic inhibitors, or other novel therapeutic agents against SVMP effects.

The Impact of Standard and Autohemochemotherapy for Catecholamine Levels in the Spleen and Thymus of Rats with Sarcoma-45

The aim of this study was to detect specific features of the effects of the blood-drug complex on catecholaminergic activity of the thymus and spleen. Catecholamine content was quantitatively measured by modified highly sensitive fluorometric method. Standard cyclophosphamide therapy led to tissue "adrenalization" against the background of reduced concentrations of norepinephrine (in the thymus) and dopamine (in both organs). Administration of cyclophosphamide by the autohemochemotherapy method stabilized catecholamine levels in the thymus and spleen of rats. Hence, cyclophosphamide autohemochemotherapy is more effective due to limitation of the destructive "stress" effect of the cytostatic on organs of the immune system.

Abstract 2759: Validated liquid chromatography-mass spectrometry assay for determination of busulfan: Application to clinical pharmacokinetics studies

Abstract Busulfan (BU), an alkylating agent, also known to have anti-leukemic efficacy, is commonly used in preparative regimens for allogenic and autologous stem cell marrow transplantation. The efficacy of BU is related to systemic levels and the area under concentration-time curve (AUC), and hence, a timely pharmacokinetic (PK) report is critical for subsequent dose adjustment. We developed two highly sensitive methods: a rapid, accurate, and sensitive liquid chromatography-mass spectrometry (LC-MS) method using direct inject tandem mass spectrometry with SIM mode, and an alternate equally sensitive high performance liquid chromatographic fluorometric (HPLC-FL) method for the quantitation of BU in human plasma. Method: BU concentrations were quantified in 50 μL of plasma spiked with [2H8]-busulfan, followed by liquid-liquid extraction with ethyl acetate. The solvent extract was dried under nitrogen and the residue was dissolved in mobile phase and analyzed using LC-MS. The mobile was 10mmol/L ammonium acetate and 10mL/L acetic acid in water and acetonitrile, (70:30) and flow rate was 0.5ml/min. Separation of BU was performed on a Gemini C18 column (4.6 × 150 mm, 3μm) at 30°C. BU and IS were detected as ammonium adducts in selected-ion monitoring mode at m/z 264.2 and 272.2 at the retention time of 5.8 min. When plasma samples were analyzed by HPLC-FL method, 200μL plasma spiked with 1,6 (methanesulfonyloxy) octane was extracted in ethyl acetate and dried under air at 45°C. The residue was dissolved in 200μL ethanol and derivatized with 2-naphthelenethiol according to Nara S., et al (Analytical Sci. 2000,16, 287). The samples were analyzed on a HPLC system with fluorescence detector set at excitation wavelength 255 nm and emission wavelength 370 nm. The mobile was a mixture of methanol: acetonitrile: sodium acetate buffer (0.1M, pH 7.0) using gradient elution, and flow rate was 1.0 mL/min. Results: Both assay methods are comparable in precision, accuracy, linearity, and sensitivity. The calibration curve for LC-MS assay was linear at 31.25-1000ng/ml. The LOD of assay was 7.8ng/ml and the LOQ was 31.25ng/ml. The correlation coefficient for calibration curve was 0.997±0.003 (n =4). The intra and inter-assay precision was less than 3.0% and the accuracy ranged from 95.4% to 101.7%. Busulfan AUClast calculation comparison with HPLC-FL derivatization method showed an average difference between the assays of 7.7%. Conclusion: The method LC-MS is highly accurate, reproducible, and requires less specimen, sample preparation, and analysis time over the HPLC-FL derivitization method. The new LC-MS assay is rapid and sensitive and provides an appropriate method for quantification of BU in human plasma, making therapeutic drug monitoring of BU faster and easier in clinical practice. Acknowledgement: The study is supported by funds from Century for the Cure and U01 grant from NCI. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2759.

Fluorometric determination of hydrogen peroxide in milk by using a Fenton reaction system

A simple and highly sensitive fluorometric method is proposed for the determination of H 2O 2 in milk samples. In this method, non-fluorescent coumarin is oxidised to highly fluorescent 7-hydroxycoumarin by hydroxyl radicals ( OH) generated in a Fenton reaction, and the oxidation product has strong fluorescence with a maximum intensity at 456 nm and can be used as a fluorescence probe for H 2O 2. Under the optimal conditions (2.5 × 10 −4 mol L −1 iron(II) ions, 4.0 × 10 −4 mol L −1 coumarin, solution pH 3.0, reaction time 9 min, and excitation at 346 nm), the proposed method presents wide linear responses between the fluorescence intensity and H 2O 2 concentration in a wide range from 2.0 × 10 −8 to 2.0 × 10 −5 mol L −1, with a detection limit ( S/ N = 3) of 5.0 × 10 −9 mol L −1. After possible interferences are evaluated for a series of chemical substances, the present method has been applied to the determination of hydrogen peroxide in milk with satisfactory results.

Fluorometric determination of nitrite with 2,3-diaminonaphthalene by reverse phase HPLC under alkaline conditions

Introduction In this study, a simple and sensitive fluorometric HPLC method was described for the determination of nitrite, an indicator of nitric oxide production in biological samples. Methods Nitrite was reacted with 2,3-diaminonaphthalene (DAN) to form fluorescent 2,3-naphthotriazole (NAT). Because NAT has higher fluorescence intensity at alkaline conditions, a reverse phase HPLC method employing a polymer-based column was applied to separate NAT using an alkaline mobile phase system. Results NAT was well separated from DAN and other fluorescent substances with increased detection sensitivity under alkaline conditions. A linear relationship ( r = 0.999) between the fluorescence intensity of NAT and the concentration of nitrite was observed in the range from 0 to 800 nM with the detection limit of about 25 nM. Discussion The present HPLC method appears suitable for routine analysis of nitrite in crude biological samples, since the polymer-based column can withstand rigorous cleaning with either acid or base.

Selective Determination of Tryptophan-containing Peptides through Precolumn Derivatization and Liquid Chromatography Using Intramolecular Fluorescence Resonance Energy Transfer Detection

A selective and sensitive fluorometric determination method for native fluorescent peptides has been developed. This method is based on intramolecular fluorescence resonance energy transfer (FRET) detection in a liquid chromatography (LC) system following precolumn derivatization of the amino groups of tryptophan (Trp)-containing peptides. In this detection process, we monitored the FRET from the native fluorescent Trp moieties (donor) to the derivatized fluorophore (acceptor). From a screening study involving 10 fluorescent reagents, we found that o-phthalaldehyde (OPA) generated FRET most effectively. The OPA derivatives of the native fluorescent peptides emitted OPA fluorescence (445 nm) through an intramolecular FRET process when they were excited at the excitation maximum wavelength of the Trp-containing peptides (280 nm). The generation of FRET was confirmed through comparison with the analysis of a non-fluorescent peptide (C-reactive protein fragment (77 - 82)) performed using LC and a three-dimensional fluorescence detection system. We were able to separate the OPA derivatives of the Trp-containing peptides when performing LC on a reversed-phase column. The detection limits (signal-to-noise ratio = 3) for the Trp-containing peptides, at a 20-microL injection volume, were 41 - 180 fmol. The sensitivity of the intramolecular FRET-forming derivatization method is higher than that of the system that takes advantage of the conventional detection of OPA derivatives. Moreover, native non-fluorescent amines and peptides in the sample monitored at FRET detection are weaker than those of conventional fluorescence detection.

Spectrofluorometric Determination of Ketorolac Tromethamine Via Its Oxidation with Cerium(IV) in Pharmaceutical Preparations and Biological Fluids

A simple and sensitive fluorometric method for determination of ketorolac tromethamine was studied. The method depends on oxidation of the drug with cerium(IV) and subsequent monitoring of the fluorescence of the induced cerium(III) at lambda(em) 365 nm after excitation at 255 nm. Different variables affecting the reaction conditions, such as the concentrations of cerium(IV), sulfuric acid concentration, reaction time, and temperature, were carefully studied and optimized. Under the optimum conditions, a linear relationship was found between the relative fluorescence intensity and the concentration of the investigated drug in the range of 0.1-0.8 microg/mL. No interferences could be observed from the excipients commonly present in dosage forms. The proposed method was successfully applied to the analysis of the investigated drug in its pure form, pharmaceutical preparations, and biological fluids with good accuracy and precision. The recoveries for pharmaceutical formulations ranged from 99.8-101.0 +/- 0.6% for tablets, 98.5-101.0 +/- 1.0% for ampoules, and 99.0-100.5 +/- 0.7% for eye drops. The results obtained by the proposed method were satisfactory compared with those obtained by the official method. The recoveries for biological fluids were 99.1-100.4 +/- 0.7 and 99.0-100.0 +/- 0.5% for plasma and urine, respectively.

Development of Novel Reagent for Hantzsch Reaction for the Determination of Formaldehyde by Spectrophotometry and Fluorometry

A novel reagent, acetoacetanilide (AAA), was introduced to the determination of formaldehyde based on Hantzsch reaction. A simple and highly sensitive fluorometric method was achieved by using AAA. The main advantages in the use of this reagent are: the reaction is carried out at room temperature without any heating system, the cyclization product based on Hantzsch reaction is soluble in water, and the product can be detected by spectrophotometry and fluorometry. The maximum absorption wavelength of the product occurs at 368 nm, and the maximum excitation and emission wavelengths are found at 370 and 470 nm, respectively. Several important experimental variables of the procedures were examined; particularly, the reaction temperature, reaction time, concentrations of reagents, and pH of the reagent solution were optimized for improving the detecting sensitivity. The calibration graph was linear in the range of 1 x 10(-7) - 1 x 10(-6) M or much higher concentrations. The limit of detection (LOD), based on three times of the standard deviation of the reagent blank, was 2.0 x 10(-8) M. The proposed method was applied to the determination of formaldehyde in environmental water samples. Many foreign species commonly existing in water samples did not interfere with the determination of formaldehyde in the proposed method.

Determination of Nucleic Acids Using Fluorescence Probe Sensitized by Microemulsion

Because the fluorescence of azur A can be quenched by adding nucleic acid, a sensitive fluorometric method for determination of nucleic acids at nanogram levels was established. Using optimal conditions, the calibration curves were linear in the range of 0-6.0 microg/mL for calf thymus deoxyribonucleic acid (ct DNA) and 0-7.0 microg/mL for herring sperm DNA (hs DNA). The limits of determination were 3.5 and 3.8 ng/mL, respectively, which shows the high sensitivity of this method. Triton X-100 microemulsion was applied as a sensitive media to enhance the sensitivity. The binding mode concerning the interactions of azur A with nucleic acids was also studied and the association constant with different binding numbers was obtained. The method has been applied to the determination of nucleic acid in both synthetic and real samples, such as cauliflower and pork liver, with satisfactory results.

Fluorometric determination of DNA using nano-SiO2 particles as an effective dispersant and stabilizer for acridine orange

A sensitive fluorometric method for the determination of ctDNA (calf thymus DNA) is presented. It has good selectivity and sensitivity and uses nano-SiO2 particles as an effective dispersant and stabilizer for acridine orange (AO). Compared to resonance light scattering (RLS) and the conventional method that uses organic dyes as fluorescence probe, the new method is more tolerant towards coexisting foreign substances and also more stable. With 20 mg nano-SiO2 particles, 10 µmol L−1 AO, at pH 8.01 and an ionic strength of 0.02 mol L−1, the interaction of AO with nano-SiO2 and ctDNA results in fluorescent signal enhancement. The extent of enhancement was in good proportion to the concentration of ctDNA at excitation/emission wavelengths of 490/523 nm, respectively. The calibration curve was linear over 0.66–55.60 µg mL−1. The determination limit (3σ) was 15 ng mL−1. The method was applied to the determination of ctDNA in synthetic samples with satisfactory results.