Sensitization of surfactants on the chemiluminescence reaction of fluorescein isothiocyanate labeled proteins

The effect of surfactants on fluorescein isothiocyanate (FITC)-bovine serum albumin (BSA)-hypochlorite (ClO(-)), FITC-human serum albumin (HSA)-ClO(-), FITC-ovoconalbumin (OVA)-ClO(-), FITC-hemoglobin (Hb)-ClO(-) systems were investigated with chemiluminescence method by the reversed phase flow injection. It was found that the chemiluminescence (CL) intensity of each system was increased greatly in the presence of cationic surfactants. Cethyltrimethylammonium bromide (CTAB) is the optimal surfactant of these systems. The optimal conditions of the CL reaction and the optimal concentration of CTAB were examined and the function of cationic surfactant CTAB on the CL reaction was also discussed.     

Sensitization of surfactants on the chemiluminescence reaction of fluorescein isothiocyanate labeled proteins

The effect of surfactants on fluorescein isothiocyanate (FITC)–bovine serum albumin (BSA)–hypochlorite (ClO⁻), FITC–human serum albumin (HSA)–ClO⁻, FITC–ovoconalbumin (OVA)–ClO⁻, FITC–hemoglobin (Hb)–ClO⁻ systems were investigated with chemiluminescence method by the reversed phase flow injection. It was found that the chemiluminescence (CL) intensity of each system was increased greatly in the presence of cationic surfactants. Cethyltrimethylammonium bromide (CTAB) is the optimal surfactant of these systems. The optimal conditions of the CL reaction and the optimal concentration of CTAB were examined and the function of cationic surfactant CTAB on the CL reaction was also discussed.     

Reverse micelle-based chemiluminescence system for quinine sulphate.

A new chemiluminescence (CL) method is proposed for the determination of quinine sulphate, which is based on the dichloromethane solvent extraction of the ion-pair complex of tetrachloroaurate (III) with quinine sulphate, and luminol CL detection in a reversed micellar medium formed by the cation surfactant cetyltrimethylammonium bromide in a dichloromethane-cyclohexane (3:7, v/v)-water (0.35 mol/L Na2CO3 buffer solution, pH 11.5). The ion-pair complex of tetrachloroaurate (III) with quinine sulphate produced an analytical CL signal when it entered the reversed micellar water pool. In optimum conditions, CL intensities are proportional to the concentrations of the studied drug over the range 0.015-10 microg/mL, with a detection limit of 1.5 ng/mL. The relative standard deviation (RSD) is 1.38% for 2.5 microg/mL quinine sulphate (n=11). The method has been applied to the determination of the studied drug in biological fluids, with satisfactory results.     

Flow injection analysis-Rayleigh light scattering detection for online determination of protein in human serum sample

A flow injection analysis (FIA) system combined with Rayleigh light scattering (RLS) detection is developed for the sensitive and rapid determination of protein concentration in human serum sample. This method is based on the weak intensity of RLS of Eriochrome Black T (EBT, 2-hydroxy-1-(1-hydroxy-2-naphthylazo)-6-nitronaphthalene-4-sulfonic acid sodium salt), which can be enhanced by the addition of protein in weakly acidic solution. The effects of pH and interfering species on the determination of protein were examined. Calibrations for protein, based on RLS intensity, were linear in the concentration ranges of 7-36 microg/ml for human serum album (HSA) and 8-44 microg/ml for bovine serum album (BSA). The detection limits of the method were found to be 0.882 and 2.507 microg/ml for HSA and BSA, respectively. A relative standard deviation of 0.76% (n=5) was obtained with 20 microg/ml HSA standard solution. The FIA-RLS method was more stable than the general RLS method, and the average RSD value of FIA-RLS was less than that of the general RLS. The sample rate was determined to be 90 samples per hour.     

Interactions between 1-benzoyl-4-p-chlorophenyl thiosemicarbazide and serum albumin: investigation by fluorescence spectroscopy

The interactions between 1-benzoyl-4-p-chlorphenyl thiosemicarbazide (BCPT) and bovine serum albumin (BSA) or human serum albumin (HSA) have been studied by fluorescence spectroscopy. By the analysis of fluorescence spectrum and fluorescence intensity, it was showed that BCPT has a strong ability to quench the intrinsic fluorescence of both bovine serum albumin and human serum albumin through a static quenching procedure. The binding constants of BCPT with BSA or HSA were determined at different temperatures based on the fluorescence quenching results. The binding sites were obtained and the binding force were suggested to be mainly hydrophobic. The effect of common ions on the binding constants was also investigated. A new fluorescence spectroscopy assay of the proteins is presented. The linear range is 5.36-67.0 microg mL(-1) with recovery of 101.1% for BSA, and the linear range is 8.28-144.9 microg mL(-1) with recovery of 102.6% for HSA. Determination of the proteins in bovine serum or in human serum by this method gives results which are very close to those obtained by using Coomassie Brilliant Blue G-250 colorimetry. A practical method was proposed for the determination of BCPT in human serum samples.     

Determination of propylthiouracil and methylthiouracil in human serum using high-performance liquid chromatography with chemiluminescence detection

Based on the sensitizing effect of formaldehyde on the chemiluminescence (CL) reaction of propylthiouracil (PTU) and methylthiouracil (MTU) with acidic potassium permanganate and the combination technique of high-performance liquid chromatography (HPLC), a sensitive, selective and simple post-column CL detection method for determining PTU and MTU is described. The optimal conditions for the CL detection and HPLC separation were carried out. The linear ranges were 0.1-20 microg mL(-1) for MTU and 0.1-10 microg mL(-1) for PTU, the detection limits were 0.03 microg mL(-1) for PTU, 0.03 microg mL(-1) for MTU and the quantification limits were 0.1 microg mL(-1) for PTU, 0.1 microg mL(-1) for MTU. The method has been satisfactorily applied for the determination of MTU and PTU in human serum samples.     

A novel HPLC-UV/nano-TiO2-chemiluminescence system for the determination of selenocystine and selenomethionine

Active oxygen species from the photocatalytic reaction in aqueous solution react with luminol to emit strong chemiluminescence (CL), and this can be inhibited by the UV decomposed-products of selenocystine (SeCys) or selenomethionine (SeMet). Based on this phenomenon, a novel hyphenated technique, HPLC-UV/nano-TiO(2)-CL, was established for the determination of SeCys and SeMet. The effects of pH, the UV irradiation time, the TiO(2) coated on the inner surface of the reaction tubing, and the Co(2+) catalyst concentration on the CL intensity and/or chromatographic resolution were systematically investigated. Under these optimized conditions, the inhibited CL intensity has a good linear relationship with the concentration of SeCys in the range of 0.04-10.6 microg mL(-1) or SeMet in the range of 0.05-12.4 microg mL(-1), with a limit of detection (S/N=3) of 6.4 microg L(-1) for SeCys or 12 microg L(-1) for SeMet. As an example, the method was preliminarily applied to the determination of the selenoamino acids in garlic and rabbit serum, with a recovery of 88-104%.     

Flow-injection resonance light scattering detection of proteins at the nanogram level.

A resonance light scattering (RLS) detection method for protein was developed, using a flow-injection system based on the enhancement of RLS signals from Biebrich scarlet (BS) by protein. The enhanced RLS intensities at 286.0 nm, in acidic aqueous medium, were proportional to the protein concentration over the range 0.005-18 microg/mL and 0.008-16 microg/mL for human serum albumin (HSA) and bovine serum albumin (BSA), respectively, with corresponding limits of detection (3sigma) of 5.00 ng/mL for HSA, and 7.80 ng/mL for BSA. The method was successfully applied to the quantification of total proteins in human serum samples.     

Aleppo tannin: structural analysis and salivary amylase inhibition

The effectiveness and specificity of a tannin inhibition on human salivary amylase (HSA) catalyzed hydrolysis was studied using 2-chloro-4-nitrophenyl 4-O-beta-D-galactopyranosyl-alpha-maltoside (GalG(2)-CNP) and amylose substrates. Aleppo tannin was isolated from the gall nut of Aleppo oak. This tannin is a gallotannin, in which glucose is esterified with gallic acids. This is the first kinetic report, which details the inhibitory effects of this compound on HSA. A mixed non-competitive type inhibition has been observed on both substrates. The extent of inhibition is markedly dependent on the substrate-type. Kinetic constants were calculated from Lineweaver-Burk secondary plots for GalG(2)-CNP (K(EI) 0.82 microg mL(-1), K(ESI) 3.3 microg mL(-1)). This indicates a 1:1 binding ratio of inhibitor-enzyme and/or inhibitor-enzyme-substrate complex. When amylose was the substrate the binding ratio of inhibitor to enzyme-substrate complex was found to be 2:1, with the binding constants of K(EI) 17.4 microg mL(-1), K(ESI) 14.9 microg mL(-1), K(ESI(2)) 9.6 microg mL(-1). Presumably, the tannin inhibitor can bind not only to HSA, but to the amylose substrate, as well. Kinetic data suggest that Aleppo tannin is a more efficient amylase inhibitor than the recently studied other tannin with quinic acid core (GalG(2)-CNP: K(EI) 9.0 microg mL(-1), K(ESI) 47.9 microg mL(-1)).     

A novel chemiluminescence method for the determination of orciprenaline based on ferricyanide-rhodamine 6G.

A novel flow injection chemiluminescence method for the determination of orciprenaline was developed. The method is based on the chemiluminescence (CL) reaction of orciprenaline with potassium ferricyanide in sodium hydroxide medium, sensitized by the fluorescent dye rhodamine 6G. The proposed procedure allows quantitation of orciprenaline in the concentration range 0.01-1.2 microg/mL, with a detection limit of 7.2 x 10(-3) microg/mL. The relative standard deviation (RSD) is 2.7% for 0.1 microg/mL orciprenaline (n = 9). The sampling frequency was calculated at approximately 120/h. The method was successfully applied to the determination of orciprenaline in pharmaceutical preparations. A brief discussion on the possible CL reaction mechanism is presented.     

Studies on binding interactions between clenbuterol hydrochloride and two serum albumins by multispectroscopic approaches in vitro

In this study, binding properties of clenbuterol hydrochloride (CL) with human serum albumin (HSA) and bovine serum albumin (BSA) were examined using constant protein concentrations and various CL contents under physiological conditions. The binding parameters were confirmed using fluorescence quenching spectroscopy at various temperatures. The experimental results confirmed that the quenching mechanisms of CL and HSA/BSA were both static quenching processes. The thermodynamic parameters, namely, enthalpy change (ΔH) and entropy change (ΔS), were calculated according to the van't Hoff equation, which suggested that the electrostatic interactions were the predominant intermolecular forces in stabilizing the CL–HSA complex, and hydrogen bonds and van der Waals force were the predominant intermolecular forces in stabilizing the CL–BSA complex. Furthermore, the conformational changes of HSA/BSA in the presence of CL were determined using the data obtained from three‐dimensional fluorescence spectroscopy, ultraviolet‐visible absorption spectroscopy and circular dichroism spectroscopy. Copyright © 2013 John Wiley & Sons, Ltd.     

Chemiluminescence determination of gemifloxacin based on diperiodatoargentate (III)‐sulphuric acid reaction in a micellar medium

A novel flow‐injection chemiluminescence (FI‐CL) analysis method for the determination of gemifloxacin in the presence of cetyltrimethylammonium bromide (CTAB) surfactant micelles is described. Strong CL signal was generated during the reaction of gemifloxacin with diperiodatoargentate (III) in a sulfuric acid medium sensitized by CTAB. Under optimum experimental conditions, the CL intensity was linearly related to the concentration of gemifloxacin from 1.0 × 10^‐9 to 3.0 × 10^‐7 g/mL and the detection limit was 7.3 × 10^‐10 g/mL (3σ). The relative standard deviation (RSD) was 1.7 % for a 3.0 × 10^‐8 g/mL gemifloxacin solution (11 repeated measurements). The proposed method was successfully applied to the determination of gemifloxacin in pharmaceutical preparations and biological fluids. The possible mechanism of the CL reaction is also discussed briefly. Copyright © 2012 John Wiley & Sons, Ltd.     

Quantitative determination of bilirubin by inhibition of chemiluminescence from lucigenin.

Bilirubin is a metabolic breakdown product of blood haem, of great biological and diagnostic importance. A new chemiluminescence (CL) method has been developed for the quantification of bilirubin. The method is combined with the flow injection analysis (FIA) technique and based on the inhibition effect of bilirubin on the CL from the lucigenin-hydrogen peroxide system in an alkaline medium. Under the optimum conditions, the decreased CL intensity was proportional to the concentration of bilirubin, in the range 0.0585-58.47 microg/mL. The detection limit estimated from the calibration graph was about 7.8826 ng/mL. The relative standard deviation (RSD) of 10 parallel measurements (1 x 10(-4) mol/L bilirubin) was 2.5%. Recoveries of bilirubin were found to fall in the range 94-97.5% using control sera. The method is interference-free, fast and easy to carry out.     

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.     

Flow injection analysis of riboflavin with chemiluminescence detection using a N-halo compounds-luminol system.

A chemiluminescent method for the determination of riboflavin is described. The method is based on the chemiluminescence (CL) generated during the oxidation of luminol by N-bromosuccinimide (NBS) and N-chlorosuccinimide (NCS) in alkaline medium. It was found that riboflavin could greatly enhance this CL intensity when present in the luminol solution. Based on this observation, a new flow-injection CL method for the determination of riboflavin is proposed in this paper. The detection limits were 7.5 ng/mL and 3.5 ng/mL of riboflavin for the NBS- and NCS-luminol CL systems, respectively. The relative CL intensity was linear, with riboflavin concentration in the range 19-600 ng/mL and 600-2000 ng/mL for the NBS-luminol CL system, and 12-200 ng/mL and 200-2000 ng/mL for the NCS-luminol CL system. The results obtained for the assay of pharmaceutical preparations compared well with those obtained by the official method and demonstrated good accuracy and precision.     

Flow-injection determination of carbaryl and carbofuran based on KMnO(4)-Na(2)SO(3) chemiluminescence detection.

A flow-injection method is described for the determination of carbaryl and carbofuran. It was found that a strong chemiluminescence (CL) signal was generated when these pesticides were mixed with Na(2)SO(3) and KMnO(4) in acidic medium. Under the optimum experimental conditions, the enhanced CL intensity was linear, with the concentrations in the range 0.1-2.0 microg/mL (r(2) = 0.9996 and 0.9993, n = 6) with relative standard deviation (n = 4) in the range 1.0-2.3%. The limits of detection (3sigma blank) were 10 and 50 ng/mL, respectively, with a sample throughput of 180/h. The proposed method was applied to determine carbaryl and carbofuran in freshwaters with satisfactory results. Most metal and non-metal ions and some pesticides, such as carbophenothion and aldicarb, do not interfere with the determination. Dinoseb, diazinon and malathion calibration graphs (in the range 0.2-2.0 microg/mL, r(2) = 0.9966-0.9988, n = 6) were also established with relative standard deviations (n = 4) in the range 1.2-2.0% with limits of detection (3sigma blank) in the range 100-300 ng/mL.     

Sensitive determination of synephrine by flow-injection chemiluminescence.

It was found that light emission produced by the oxidation of luminol by potassium ferricyanide in basic medium was enhanced by synephrine, an anti-obesity drug. The optimum conditions for this chemiluminescent reaction were studied in detail in a flow injection system and employed in a new, simple and rapid method for the determination of synephrine. A mechanism for this reaction is proposed, based on the chemiluminescence reaction spectra. In the optimum conditions, CL intensity is proportional to concentration of synephrine in the 0.008-1 microg/mL range. The limit of detection is 1.6 ng/mL for synephrine (3sigma), and the relative standard deviation (n = 11) is 2.6% for 0.5 microg/mL synephrine. The method was applied to the determination of synephrine in herbal products, citrus fruit and biological fluids. The recoveries were satisfactory (90-102%). The results given by the proposed method are in good agreement with those given by HPLC-UV.     

A new and simple resonance Rayleigh scattering method for human serum albumin using graphite oxide as probe

Graphite oxide (GO) was prepared by the Hummer procedure, and can be dispersed to stable colloid solution by ultrasonic wave. The GO exhibited an absorption peak at 313 nm, and a resonance Rayleigh scattering (RRS) peak at 490 nm. In pH 4.6 HAc‐NaAc buffer solution, human serum albumin (HSA) combined with GO probe to form large HSA‐GO particles that caused the RRS peak increasing at 490 nm. The increased RRS intensity was linear to HSA concentration in the range 0.50–200 µg/mL. Thus, a new and simple RRS method was proposed for the determination of HSA in samples, with a recovery of 98.1–104%. Copyright © 2012 John Wiley & Sons, Ltd.     

Improvement of the acridine orange-protein-surfactant system for protein estimation based on aromatic ring stacking effect of sodium dodecyl benzene sulphonate.

The fluorescence of acridine orange (AO) is greatly quenched by the anionic surfactant sodium dodecyl benzene sulphonate (SDBS), but when protein is added into the AO-SDBS system, the fluorescence intensity of the latter is enhanced again. Based on this, a new fluorimetric method of determination of protein was developed. Under optimum conditions, the enhanced intensity of fluorescence is in proportion to the concentration of protein, such as bovine serum albumin (BSA), human serum albumin (HSA) and egg albumin (EA), over a wide range with detection limits at the 10(-9) g/mL level. This method has been satisfactorily used for the determination of protein in samples. We compared results using 280 nm and 490 nm excitation wavelengths and the mechanism of the assay.     

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.