A novel flow-injection chemiluminescence system for the determination of guanine.

A novel flow-injection chemiluminescence (CL) method for the determination of guanine was developed. The procedure is based on the CL reaction of guanine with hydrogen peroxide in borax buffer (pH 8.5) with Co2+ as a catalyst. The calibration graph is linear within the range of 3 x 10(-7)-9 x 10(-5) g/mL. A detection limit of 1 x 10(-7) g/mL, along with a relative standard deviation of 2.23% (3 x 10(-7) g/mL guanine, n = 11), were obtained. The present procedure was applied to the measurement of guanine in urine with recoveries of 97.5-107.5%. A possible CL mechanism of the reaction system is proposed.     

Direct chemiluminescence determination of ibuprofen by the enhancement of the KMnO(4)-sulphite reaction.

A simple, rapid, flow-injection chemiluminescence (CL) method is described for the determination of ibuprofen. A strong CL signal was detected when a mixture of the analyte and sulphite was injected into acidic KMnO(4). The CL signal is proportional to the concentration of ibuprofen in the range 0.1-10.0 mg/L. The detection limit is 0.02 mg/L ibuprofen, the relative standard deviation is 1.8% (0.5 mg/L ibuprofen; n = 11) and the sample measurement frequency is 120/h. The proposed method was successfully applied to the determination of ibuprofen in pharmaceutical preparations and in spiked urine samples. The mechanism of the CL reaction is also discussed.     

Investigation on the interaction between dihydroxybenzene and Fe3+- H2O2-Rh6G system based on enhancing chemiluminescence.

A highly sensitive flow-injection chemiluminescence (FI-CL) method has been developed for the determination of dihydroxybenzene, based on the hydroxyl radical reaction. Hydroxyl radical (.OH) produced by the reaction of Fe(3+) and H(2)O(2) oxidize rhodamine 6G to produce weak CL. It was observed that catechol and hydroquinone greatly enhanced the weak CL reaction. However, the proposed CL system is not suitable for determination of resorcinol because the enhancement reaction is very slow. The proposed procedure has a linear range of 0.01-2 mg/L for catechol, with a detection limit of 0.006 mg/L, and 0.008-1 mg/L for hydroquinone, with a detection limit of 0.004 mg/L. The possible mechanism of the CL system is discussed.     

Flow-injection chemiluminescent determination of phenothiazines in pharmaceutical preparations

A flow-injection configuration for the chemiluminescent (CL) determination of chlorpromazine, trimeprazine and trifluoperazine is proposed. The procedure is based on the oxidation of the drugs by Fe(III) perchlorate. The Fe(II) formed is measured by its catalytic effect on the CL luminol reaction in the absence of added oxidant. Linear calibration graphs were obtained between 2.0×10⁻⁶ and 1.0×10⁻⁴ M for all three drugs. The applicability of the method to the determination of these phenothiazines was demonstrated by investigating the effect of potential interferences and by analysing commercial pharmaceutical preparations.     

A flow-injection chemiluminescence method for the determination of human serum albumin, using the reaction of fluorescein-human serum albumin-sodium hypochlorite by the enhancement effect of the cationic surfactant cetyltrimethylammonium bromide.

The interaction between surfactant and fluorescein was studied, using a fluorescence spectroscopy and flow-injection (FI) chemiluminescence (CL) method. It was found that the cationic surfactant cetyltrimethylammonium bromide (CTAB) could cause the structural transformation of fluorescein from the quinone to the spirolactone form, and greatly enhance the CL intensity of the fluorescein-human serum albumin (HSA) complex. Based on this finding, a rapid and sensitive FI-CL method was developed for the determination of HSA. Under the optimum conditions, the proposed method has a linear range of 0.05-24.0 microg/mL, with a detection limit of 0.03 microg/mL for HSA (3sigma). The relative standard deviation (RSD) of 1.2 microg/mL HSA (n = 8) is 0.8%. The method was applied to the determination of protein content in urine samples, with satisfactory results. Density functional theory was used to study the mechanism of surfactant-enhanced CL intensity of the fluorescein-HSA complex.     

Chemiluminescence determination of fluoroquinolone antibiotics using a soluble manganese (IV)-sulphite system.

The reaction of soluble manganese (IV) with sulphite in acidic condition was found to elicit weak chemiluminescence (CL). The CL signal was remarkably enhanced in the presence of three fluoroquinolones, viz. norfloxacin, ofloxacin and ciprofloxacin. Based on these observations, a new flow-injection CL method was developed for the determination of these fluoroquinolones. The method allows determination in the range 5.0 x 10(-8)-1.0 x 10(-6) mol/L for norfloxacin, 1.0 x 10(-7)-8.0 x 10(-6) mol/L for ofloxacin and 1.0 x 10(-7)-3.0 x 10(-5) mol/L for ciprofloxacin, with detection limits of 3 x 10(-8) mol/L, 5 x 10(-8) mol/L and 3 x 10(-8) mol/L, respectively. The method was applied to the determination of fluoroquinolones in pharmaceutical preparations.     

A chemiluminescent immunosensor based on antibody immobilized carboxylic resin beads coupled with micro-bubble accelerated immunoreaction for fast flow-injection immunoassay

A novel immunoaffinity column used as an immunosensor for flow-injection chemiluminescent (CL) immunoassay was prepared by immobilizing antibody on carboxylic resin beads. The immunosensor could fast recognize and trap the immunocomplex of horseradish peroxidase (HRP)-labeled antibody and antigen, which was firstly formed with a micro-bubble accelerated pre-incubation process, to produce a sandwich immunocomplex. The HRP introduced in the immunoaffinity column could catalyze the CL reaction to produce enzyme-enhanced emission. With alpha-fetoprotein (AFP) as a mode, a flow-injection CL immunoassay was proposed. The whole assay for one sample, including the pre-incubation and the regeneration of immunoaffinity column, could be performed within 16min. The linear range was 1.0-80ng/ml with a correlation coefficient of 0.998 and a detection limit of 0.1ng/ml at a signal/noise ratio of 3. The intra- and inter-assay coefficients of variation at 20ng/ml AFP were 1.2% and 8.5%, respectively. The storage stability of the immunoaffinity column and the accuracy for sample detection were acceptable. This flexible, sensitive, low-cost, and rapid method is valuable for clinical immunoassay.     

Reagentless and regenerable immunosensor for monitoring of immunoglobulin G based on non-separation immunoassay

Based on the enhancement of fluorescein isothiocyanate (FITC) fluorescence caused by reactions between proteins, we developed a reagentless, regenerable and rapid immunosensing system to determine immunoglobulin G (IgG). Fluorescence intensity of the immobilized FITC depends on IgG concentration, ranging from 10 to 50 microg/ml, specifically, even with co-existing proteins. The response time is 30 min during steady-state measurement and is less than a minute during transient measurement. When the FITC-labeled protein A binds to IgG, the surrounding atmosphere of FITC becomes hydrophobic. Since the fluorescence intensity of fluorescent substances generally increases at a hydrophobic environment, FITC fluorescence intensity increases with the concentration of protein A bonding to IgG. This system is regenerable because the fluorescence enhancement repeatedly occurs every time the immobilized fluorescent reagent is immersed in sample solutions.     

An integrated approach for thermal stabilization of a mesophilic adenylate kinase

Thermally stable proteins are desirable for research and industrial purposes, but redesigning proteins for higher thermal stability can be challenging. A number of different techniques have been used to improve the thermal stability of proteins, but the extents of stability enhancement were sometimes unpredictable and not significant. Here, we systematically tested the effects of multiple stabilization techniques including a bioinformatic method and structure‐guided mutagenesis on a single protein, thereby providing an integrated approach to protein thermal stabilization. Using a mesophilic adenylate kinase (AK) as a model, we identified stabilizing mutations based on various stabilization techniques, and generated a series of AK variants by introducing mutations both individually and collectively. The redesigned proteins displayed a range of increased thermal stabilities, the most stable of which was comparable to a naturally evolved thermophilic homologue with more than a 25° increase in its thermal denaturation midpoint. We also solved crystal structures of three representative variants including the most stable variant, to confirm the structural basis for their increased stabilities. These results provide a unique opportunity for systematically analyzing the effectiveness and additivity of various stabilization mechanisms, and they represent a useful approach for improving protein stability by integrating the reduction of local structural entropy and the optimization of global noncovalent interactions such as hydrophobic contact and ion pairs. Proteins 2014; 82:1947–1959. © 2014 Wiley Periodicals, Inc.     

A study on the micelle-sensitized Ce(IV)-Na2S2O3-norfloxacin chemiluminescence system and its applications.

A new chemiluminescence (CL) flow-injection method was developed for the determination of norfloxacin. The method is based on the CL reaction of norfloxacin with sodium thiosulphate and Ce(IV) in sulphuric acid medium sensitized by sodium dodecylsulphate. Under optimum conditions, the CL intensity is proportional to the concentration of the norfloxacin in the range 3.89 x 10(-8)-7.18 x 10(-6) g/mL. The detection limit (3 s/k) was 2.21 x 10(-9) g/mL for norfloxacin. The method has been applied successfully to the determination of norfloxacin in pharmaceutical formulations and human urine. The mechanism for this chemiluminescence system is discussed.     

A novel [Ag(NH3)2]+ probe for chemiluminescent imaging detection of proteins after polyacrylamide gel electrophoresis

The development of a novel [Ag(NH3)2]+ probe chemiluminescence (CL)-based imaging method for the detection of various proteins after PAGE is described. The detection is based upon the probe [Ag(NH3)2]+ catalyzing the CL reaction of the luminol-potassium persulfate system. The proposed method detects various proteins labeled by [Ag(NH3)2]+ and expands the application scope to SDS gels. It also detects proteins directly in polyacrylamide gels, without tedious transferring procedures. Furthermore, successful identification of proteins by peptide mass profiling using ionization MS was easily performed, and no pretreatments of gel prior to digestion are needed. Detection limits for standard marker proteins match CBB-R250 staining and the linear dynamic range is superior to CBB-R250 staining and silver staining. The CL imaging conditions, including luminescent reagents, silver ion concentration, the ammonia-controlled system and the washing reagents parameters have also been optimized.     

GnRH-like proteins in cows: concentrations during corpora lutea development and selective localization in granulosal cells

Gonadotropin-releasing hormone (GnRH)-like proteins with anti-gonadotropic properties were recently discovered in the ovaries of several species, including humans. Since neither GnRH receptors nor GnRH are in bovine ovarian tissue, we examined, in the present studies, whether concentrations of GnRH-like proteins varied during development of the corpus luteum (CL) and whether GnRH-like proteins were selectively localized in ovarian cells of cows. For these studies, GnRH-like proteins were extracted from various ovarian and nonovarian tissues and fluids and fractionated for hydrophobic interaction chromatography. A highly specific and sensitive radioreceptor assay (RRA) was used to quantify concentrations of GnRH-like proteins. The major findings of these studies demonstrated that 1) the amount of GnRH-like proteins in the corpus luteum (CL) was proportional to the weight of the CL; 2) the concentration of GnRH-like proteins in luteal tissue decreased during development of the CL; 3) GnRH-like proteins were in ovarian and numerous nonovarian tissues, but were not in the heart, plasma, or follicular fluid; 4) the retention time for GnRH-like proteins following high-pressure liquid chromatography (HPLC) varied with the tissue source; and 5) compared with all other tissues, the greatest concentration of GnRH-like proteins was in granulosal cells. We concluded that the concentration of GnRH-like proteins in luteal cells decreased during development of the CL, and that a specific GnRH-like protein was selectively localized in bovine granulosal cells.     

A rapid and high‐throughput method for the determination of serum uric acid based on microarray technology and nanomaterial

Serum uric acid (SUA) is a new therapeutic target for non‐alcoholic fatty liver disease (NAFLD). In this study, we introduced a chemiluminescence (CL) method combined with microarray technology and a simple fabrication procedure to obtain a highly sensitive SUA probe based on a mesoporous metal oxide nanomaterial. The high‐throughput method was based on the generation of H₂O₂ from SUA by immobilized uricase and its measurement by a CL reaction catalyzed by mesoporous metal oxide nanomaterials. The CL probe was designed for SUA The linear range of the uric acid concentration was 0.6–9 μM and the detection limit was 0.1 μM. In comparison with the other SUA detection techniques, this method has the advantages of a low detection limit, high sensitivity and simplicity. A new sensitive high‐throughput approach was obtained for the determination of SUA.     

Flow‐based analysis using microfluidics–chemiluminescence systems

This review will discuss various approaches and techniques in which analysis using microfluidics–chemiluminescence systems (MF–CL) has been reported. A variety of applications is examined, including environmental, pharmaceutical, biological, food and herbal analysis. Reported uses of CL reagents, sample introduction techniques, sample pretreatment methods, CL signal enhancement and detection systems are discussed. A hydrodynamic pumping system is predominately used for these applications. However, several reports are available in which electro‐osmotic (EO) pumping has been implemented. Various sample pretreatment methods have been used, including liquid–liquid extraction, solid‐phase extraction and molecularly imprinted polymers. A wide range of innovative techniques has been reported for CL signal enhancement. Most of these techniques are based on enhancement of the mixing process in the microfluidics channels, which leads to enhancement of the CL signal. However, other techniques are also reported, such as mirror reaction, liquid core waveguide, on‐line pre‐derivatization and the use of an opaque white chip with a thin transparent seal. Photodetectors are the most commonly used detectors; however, other detection systems have also been used, including integrated electrochemiluminescence (ECL) and organic photodiodes (OPDs). Copyright © 2012 John Wiley & Sons, Ltd.     

Fluorescence study of protein-lipid complexes with a new symmetric squarylium probe

The novel symmetric squarylium derivative SQ-1 has been synthesized and tested for its sensitivity to the formation of protein-lipid complexes. SQ-1 binding to the model membranes composed of zwitterionic lipid phosphatidylcholine (PC) and its mixtures with anionic lipid cardiolipin (CL) in different molar ratios was found to be controlled mainly by hydrophobic interactions. Lysozyme (Lz) and ribonuclease A (RNase) exerted an influence on the probe association with lipid vesicles resulting presumably from the competition between SQ-1 and the proteins for bilayer free volume and modification of its properties. The magnitude of this effect was much higher for lysozyme which may stem from the amphipathy of protein alpha-helix involved in the membrane binding. Varying membrane composition provides evidence for the dye sensitivity to both hydrophobic and electrostatic protein-lipid interactions. Fluorescence anisotropy studies uncovered the restriction of SQ-1 rotational mobility in lipid environment in the presence of Lz and RNase being indicative of the incorporation of the proteins into bilayer interior. The results of binding, fluorescence quenching and kinetic experiments suggested lysozyme-induced local lipid demixing upon protein association with negatively charged membranes with threshold concentration of CL for the lipid demixing being 10 mol%.     

Nickel oxide hollow microsphere for the chemiluminescence determination of tuberculostatic drug isoniazid

In this article, nickel(II) oxide (NiO) hollow microspheres (HMSs) were fabricated and used to catalyze chemiluminescence (CL) reaction. The studied CL reaction is the luminol-oxygen reaction that was used as a sensitive analytical tool for measuring tuberculostatic drug isoniazid (IND) in pharmaceutical formulations and water samples. The CL method was established based on the suppression impact of IND on the CL reaction. The NiO HMSs were produced by a simple hydrothermal method and characterized by several spectroscopic techniques. The result of essential parameters on the analytical performance of the CL method, including concentrations of sodium hydroxide (NaOH), luminol, and NiO HMSs were investigated. At the optimum conditions, the calibration curve for IND was linear in the range of 8.00 × 10⁻⁷ to 1.00 × 10⁻⁴ mol L⁻¹ (R² = 0.99). A detection limit (3S) of 2.00 × 10⁻⁷ mol L⁻¹ was obtained for this method. The acceptable relative standard deviation (RSD) was obtained for the proposed CL method (2.63%, n = 10) for a 5.00 × 10⁻⁶ mol L⁻¹ IND solution. The mechanism of the CL reaction was also discussed.     

Potential of the luminol reaction in the sensitive detection of pesticide residues by flow injection analysis.

This study presents the first analytical application of the luminol chemiluminescence (CL) reaction for the sensitive detection of carbamate residues. Some experiments have been carried out to check the influence of the presence of traces of a N-methylcarbamate (carbaryl) on the CL emission produced from the oxidation of luminol using different oxidants, showing a significant enhancing effect on the CL emission when the oxidation of luminol is produced by potassium permanganate in alkaline medium, this enhancement being proportional to the carbaryl concentration. This fact has permitted the establishment of a sensitive chemiluminescence flow-injection (CL-FIA) method for the direct determination of carbaryl. The optimization of instrumental and chemical variables influencing the CL response has been carried out by applying experimental designs. Under the optimal conditions, the CL intensity was linear for a carbaryl concentration over the range 5-100 ng/mL with a detection limit of 4.9 ng/mL. This luminol-KMnO4-based FIA-CL system in basic medium shows an easy, fast and cheap alternative detection mode for the analysis of carbaryl residues in environmental water samples.     

Surface properties of Staphylococcus aureus affecting chemiluminescence response of human phagocytes

To assess the surface properties of Staphylococcus aureus affecting the response of human phagocytes, the effects of the organisms with different surface properties on the chemiluminescence (CL) response of human phagocytes were examined. The magnitude of the phagocytic CL response to hydrophobic strains was significantly greater than that to hydrophilic strains, while no significant difference in the CL response was seen between protein A-deficient strains and their parent strains. The CL response to the hydrophilic organisms prepared from a hydrophobic strain by trypsin treatment decreased significantly. These results suggest that the phagocytic CL response to staphylococci depends on the hydrophobicity of the surface, but not on the presence of protein A. Two protein A-deficient strains which were isolated from protein A-positive strains showed identical hydrophobicity with their parent strains. All of the hydrophilic strains isolated from hydrophobic strains possessed protein A identical to that of their parent strains. Moreover, a hydrophilic strain could be isolated from a protein A-deficient, hydrophobic strain. These results strongly suggest that protein A is not solely responsible for the surface hydrophobicity of S. aureus.