Detection of Picomole Levels in Lipid Hydroperoxides by a Chemiluminescence Assay

A highly sensitive and simple chemiluminescent method for the quantitation of lipid hydroperoxides at the picomole level is described. The method is based on detecting the chemiluminescence generated during the oxidation of luminol by the reaction with hydroperoxide and cytochrome c under mild conditions. A semilogarithmic relationship was observed between the hydroperoxide added and the chemiluminescence produced. For lipid hydroperoxides, cytochrome c was a most favorable catalyst for generating the chemiluminescence, rather than cytochrome c heme peptide and horseradish peroxidase. This method had high sensitivity to methyl linoleate hydroperoxide, arachidonic acid hydroperoxide and cholesterol hydroperoxide, but low to /-butyl hydroperoxide, J-butyl perbenzoate, diacyl peroxides (lauroyl peroxode and benzoyl peroxide) and dialkyl peroxides (di-/-butyl peroxide and dicumyl peroxide).     

Detection of chemiluminescence in lipid peroxidation of biological systems and its application to HPLC

A combined system of chemiluminescence detection and high performance liquid chromatography (CL–HPLC) was developed to determine primary peroxidation products in biological tissues, such as phosphatidylcholine hydroperoxide (PCOOH). The CL–HPLC assay consists of separation of lipid classes with HPLC and detection of hydroperoxide-specific chemiluminescence. Hydroperoxides react with heme compounds to produce oxidants as suggested by our early studies on tissue low-level chemiluminescence in which singlet molecular oxygen is generated as one of the excited species in several biological systems involving free radical events. In the CL–HPLC method, a cytochrome c–luminol mixture was used as a hydroperoxide-specific luminescent reagent, and the quantification of hydroperoxide was performed by detecting chemiluminescence due to the luminol oxidation caused by the oxidant produced during the lipid hydroperoxides with heme. The detection limit of PCOOH was 10 pmole hydroperoxide–O₂. PCOOH in normal human blood was found to be 10–500 pmol/ml plasma and significantly higher levels of PCOOH were observed in some hospitalized patients.     

Chemiluminescence of 3‐aminophthalic acid anion–hydrogen peroxide–cobalt (II)

The 3‐aminophthalic acid anion is a light emitter in luminol chemiluminescence. In the present study, the chemiluminescence of the 3‐aminophthalic acid anion itself in the presence of hydrogen peroxide–cobalt (II) was studied. The results indicated that 3‐aminophthalic acid anion is highly chemiluminescent in the typical hydrogen peroxide–cobalt (II) system. The peak wavelength of this chemiluminescence and the kinetic profile of the 3‐aminophthalic acid anion–hydrogen peroxide–cobalt (II) reaction showed similarity with that of luminol, but the chemiluminescence of 3‐aminophthalic acid anion had a much lower background signal. In addition, the chemiluminescence mechanism of 3‐aminophthalic acid anion–hydrogen peroxide–cobalt (II) was also discussed and speculated as the interaction between 3‐aminophthalic acid anion and singlet oxygen.     

Chemiluminescent assay of lipid hydroperoxides

The addition of luminol plus a catalyst such as peroxidase or a heme prosthetic group to a solution containing a small quantity of lipid hydroperoxides results in a flash of chemiluminescence, the intensity of which is a function of the hydroperoxide concentrations. Various protocols for lipid hydroperoxide assays have been described and we have studied conditions to increase their sensitivity and specificity. Plasma lipid hydroperoxide determinations require an extraction, since compounds present in plasma interfere with light emission. Moreover, the sensitivity of the assay is by the presence of hydrogen peroxide in the medium, which causes high background values. Catalase does not act on lipid hydroperoxides and can be used to eliminate hydrogen peroxide from the reaction medium. The determination requires a blank tube in which hydroperoxides are destroyed by incubating the sample with haematin plus ascorbate. The increase in the chemiluminescence of the assay tube caused by the presence of lipid hydroperoxides is then compared to the value obtained for an internal standard.     

A chemiluminescence method to detect hydroquinone with water‐soluble sulphonato‐(salen)manganese(III) complex as catalyst

A water‐soluble sulphonato‐(salen)manganese(III) complex with excellent catalytic properties was synthesized and demonstrated to greatly enhance the chemiluminescence signal of the hydrogen peroxide ? luminol reaction. Coupled with flow‐injection technique, a simple and sensitive chemiluminescence method was first developed to detect hydroquinone based on the chemiluminescence system of the hydrogen peroxide–luminol–sulphonato‐(salen)manganese(III) complex. Under optimal conditions, the assay exhibited a wide linear range from 0.1 to 10 ng mL^–1 with a detection limit of 0.05 ng mL^–1 for hydroquinone. The method was applied successfully to detect hydroquinone in tap‐water and mineral‐water, with a sampling frequency of 120 times per hour. The relative standard deviation for determination of hydroquinone was less than 5.6%, and the recoveries ranged from 96.8 to 103.0%. The ultraviolet spectra, chemiluminescence spectra, and the reaction kinetics for the peroxide–luminol–sulphonato‐(salen)manganese(III) complex system were employed to study the possible chemiluminescence mechanism. The proposed chemiluminescence analysis technique is rapid and sensitive, with low cost, and could be easily extended and applied to other compounds. Copyright © 2015 John Wiley & Sons, Ltd.     

Electroenzymatic Synthesis of Chiral Sulfoxides

Chloroperoxidase (CPO) from Caldariomyces fumago (E.C. 1.11.1.10) is able to enantioselectively oxidize various sulfides to the corresponding (R)‐enantiomer of the sulfoxides. For these oxidations the enzyme requires an oxidant. Most commonly, tert‐butyl hydroperoxide (TBHP) and hydrogen peroxide are used. As it is known that these oxidants inactivate the enzyme, the enzymatic reaction was combined with the electrochemical in situ generation of hydrogen peroxide. As substrates for this combination of an enzymatic and an electrochemical reaction methyl p‐tolyl sulfide, 1‐methoxy‐4‐(methylthio)benzene and N‐MOC‐L ‐methionine methyl ester were used to carry out batch experiments.     

A highly sensitive and temporal visualization system for gaseous ethanol with chemiluminescence enhancer

A two‐dimensional gaseous ethanol visualization system has been developed and demonstrated using a horseradish peroxidase–luminol–hydrogen peroxide system with high‐purity luminol solution and a chemiluminescence (CL) enhancer. This system measures ethanol concentrations as intensities of CL via the luminol reaction. CL was emitted when the gaseous ethanol was injected onto an enzyme‐immobilized membrane, which was employed as a screen for two‐dimensional gas visualization. The average intensity of CL on the substrate was linearly related to the concentration of standard ethanol gas. These results were compared with the CL intensity of the CCD camera recording image in the visualization system. This system is available for gas components not only for spatial but also for temporal analysis in real time. A high‐purity sodium salt HG solution (L‐HG) instead of standard luminol solution and an enhancer, eosin Y (EY) solution, were adapted for improvement of CL intensity of the system. The visualization of gaseous ethanol was achieved at a detection limit of 3 ppm at optimized concentrations of L‐HG solution and EY. Copyright © 2011 John Wiley & Sons, Ltd.     

Luminol oxidation catalyzed by royal palm leaf peroxidase

We optimized the conditions for oxidation of luminol by hydrogen peroxide in the presence of peroxidase (EC 1.11.1.7) from royal palm leaves (Roystonea regia). The pH range (8.3–8.6) corresponding to maximum chemiluminescence was similar for palm tree peroxidase and horseradish peroxidase. Variations in the concentration of the Tris buffer were accompanied by changes in chemiluminescence. Note that maximum chemiluminescence was observed in the 30 mM Tris solution. The detection limit of the enzyme assay during luminol oxidation by hydrogen peroxide was 1 pM. The specific feature of palm tree peroxidase was the generation of a long-term chemiluminescent signal. In combination with the data on the high stability of palm tree peroxidase, our results indicate that this enzyme is promising for its use in analytical studies.     

Determination of parathion residues in rice samples using a flow injection chemiluminescence method

A sensitive ?ow injection chemiluminescence method is described for the determination of parathion pesticide, based upon its direct chemiluminescence reaction with luminol and hydrogen peroxide in the presence of non‐ionic surfactant polyethylene glycol 400. Under the selected experimental conditions, the concentration of parathion is proportional to the CL intensity in the range 0.02–1.0 mg/L. The detection limit was 0.008 mg/L and the relative standard deviation was 2.8% for 0.2 mg/L parathion solution (n = 11). This method was successfully applied to the determination of parathion residue in rice samples. Copyright © 2003 John Wiley & Sons, Ltd.     

Chemiluminescence intensities and spectra of luminol oxidation by sodium hypochlorite in the presence of hydrogen peroxide

Hydrogen peroxide amplifies the chemiluminescence in the oxidation of luminol by sodium hypochlorite. A linear relationship between concentration of hydrogen peroxide and light intensity was found in the concentration range 5 × 10^?8?7.5 × 10^?6 mol/l. At 7.5 × 10^?6 mol/l H₂O₂ the chemiluminescence is amplified 550—fold. The chemiluminescence spectra of these reactions have a wavelength maximum at 431 nm independent of the concentration of hydrogen peroxide. The results indicate that hydrogen peroxide is a necessary component in the chemiluminescent oxidation of the luminol by sodium hypochlorite.     

Luminol-enhanced chemiluminescence of rabbit polymorphonuclear leukocytes: The nature of oxidants that directly cause luminol oxidation

In this study, we investigated the pathways (including the formation of hydroxyl radicals and chloramines) leading to luminol chemiluminescence induced by hypochlorite generated in a suspension of stimulated rabbit polymorphonuclear leukocytes. Chemiluminescence of leukocytes stimulated by phorbol myristate acetate, which was enhanced by luminol (0.02 mM), did not change in the presence of dimethyl sulfoxide at moderate concentrations (0.02–2.6 mM), under which the latter should manifest the specific ability to scavenge hydroxyl radicals. This indicates that stimulation of polymorphonuclear leukocytes is not accompanied by the generation of hydroxyl radicals with the involvement of superoxide anion and hypochlorite synthesized by myeloperoxidase. At high concentrations of dimethyl sulfoxide (260 mM), chemiluminescence markedly declined because dimethyl sulfoxide directly reacts with hypochlorite. The luminol emission intensity considerably increased after its addition to a suspension of leukocytes that were preliminarily stimulated for 10 min. This effect was caused by the accumulation of hydrogen peroxide rather than chloramines. Exogenous amino acids and taurine at high concentrations (3–15 mM) quench chemiluminescence. All these data indicate that chemiluminescence in the system studied is largely determined by the direct initial reaction of hypochlorite with luminol, the emission intensity increasing as a result of oxidation of luminol transformation products by hydrogen peroxide.     

Enhanced chemiluminescence: A sensitive analytical system for detection of sweet potato peroxidase

3-(10'-Phenothiazinyl)propane-1-sulfonate (SPTZ) was shown to be a potent enhancer of anionic sweet potato peroxidase (aSPP)-induced chemiluminescence. The optimal conditions for aSPP-catalyzed oxidation of luminol were investigated by varying the concentrations of luminol, hydrogen peroxide, Tris, and SPTZ as well as the pH values of the reaction mixture. Addition of 4-morpholinopyridine (MORP) to the reaction mixture markedly increased the light intensity. Using SPTZ and MORP together enhanced the effect 265 times. The lower detection limit (LDL) of SPP was 0.09 pM, approximately in 10 times lower than that for the cationic isozyme c of horseradish peroxidase/4-iodophenol system. It was shown that aSPP in the presence of SPTZ produced a longer lasting chemiluminescent signal.     

Hypohalites and related oxidants as chemiluminescence reagents: a review

This review concerns the use of hypochlorite, hypobromite and related oxidants (such as N‐bromosuccinimide and 1,3‐dibromo‐5,5‐dimethylhydantoin) as chemiluminescence reagents and includes references to 249 papers that were published prior to mid‐2003. Particular emphasis has been placed on proposed emitting species and the mechanisms of the light‐producing pathways. The analytical applications of this chemistry have been summarized in three tables: (1) quanti?cation of hypohalites and related compounds (including halides, which are initially oxidized); (2) enhancement or inhibition of luminol chemiluminescence; and (3) direct chemiluminescence reactions with hypohalite reagents. Copyright © 2004 John Wiley & Sons, Ltd.     

Flow‐injection chemiluminescence method for the determination of chloramphenicol based on luminol–sodium periodate order‐transform second‐chemiluminescence reaction

A new chemiluminescence (CL) reaction was observed when chloramphenicol solution was injected into the mixture after the end of the reaction of alkaline luminol and sodium periodate or sodium periodate was injected into the reaction mixture of chloramphenicol and alkaline luminol. This reaction is described as an order‐transform second‐chemiluminescence (OTSCL) reaction. The OTSCL method combined with a flow‐injection technique was applied to the determination of chloramphenicol. The optimum conditions for the order‐transform second‐chemiluminescence emission were investigated. A mechanism for OTSCL has been proposed on the basis of the chemiluminescence kinetic characteristics, the UV‐visible spectra and the chemiluminescent spectra. Under optimal experimental conditions, the CL response is proportional to the concentration of chloramphenicol over the range 5.0 × 10^?7–5.0 × 10^?5 mol/L with a correlation coefficient of 0.9969 and a detection limit of 6.0 × 10^?8 mol/L (3σ). The relative standard deviation (RSD) for 11 repeated determinations of 5.0 × 10^?6 mol/L chloramphenicol is 1.7%. The method has been applied to the determination of chloramphenicol in pharmaceutical samples with satisfactory results. Copyright © 2011 John Wiley & Sons, Ltd.     

Luminol oxidation by hydrogen peroxide with chemiluminescent signal formation catalyzed by peroxygenase from the fungus Agrocybe aegerita V.Brig.

Conditions of luminol oxidation by hydrogen peroxide in the presence of peroxygenase from the mushroom Agrocybe aegerita V.Brig. have been optimized. The pH value (8.8) at which fungal peroxygenase produces a maximum chemiluminescent signal has been shown to be similar to the pH optimum value of horseradish peroxidase. Luminescence intensity changed when the concentration of Tris-buffer was varied; maximum intensity of chemiluminescence was observed in 40 mM solution. It has been shown that enhancer (p-iodophenol) addition to the substrate mixture containing A. aegerita peroxygenase exerted almost no influence on the intensity of the chemiluminescent signal, similarly to soybean, palm, and sweet potato peroxidases. Detection limit of the enzyme in the reaction of luminol oxidation by hydrogen peroxide was 0.8 pM. High stability combined with high sensitivity make this enzyme a promising analytical reagent.     

Optimization of peroxynitrite–luminol chemiluminescence system for detecting peroxynitrite in cell culture solution exposed to carbon disulphide

We established a peroxynitrite–luminol chemiluminescence system for detecting peroxynitrite in cell culture solution exposed to carbon disulphide (CS₂). Three factors, including exposure time to ozone (Factor A), volume of peroxynitrite (ONOO^?) solution (Factor B) and luminol concentrations (Factor C) at three levels were selected and the combinations were in accordance with orthogonal design L₉ (3⁴). Peroxynitrite was generated from the reaction of ozone and 0.01 mol/L sodium azide (NaN₃) dissolved in carbonic acid buffer solution (pH 11), and it was reacted with luminol to yield chemiluminescence. The peak value, peak time and kinetic curve of the light emission were observed. The selected combination conditions were 50 s ozone, 800 µL peroxynitrite and 0.001 mol/L luminol solution. Cell culture solution with CS₂ enhanced the emission intensity of chemiluminescence (F = 8.38, p = 0.018) and shortened the peak time to chemiluminescence (F = 139.00, p = 0.0001). The data demonstrated that this luminol chemiluminescence system is suitable for detecting peroxynitrite in cell culture solutions for evaluating the effect of CS₂ on endothelial cells. Copyright © 2003 John Wiley & Sons, Ltd.     

Long‐term chemiluminescence signal is produced in the course of luminol oxidation catalyzed by enhancer‐independent peroxidase purified from Jatropha curcas leaves

Isoenzyme c of horseradish peroxidase (HRP‐C) is widely used in enzyme immunoassay combined with chemiluminescence (CL) detection. For this application, HRP‐C activity measurement is usually based on luminol oxidation in the presence of hydrogen peroxide (H₂O₂). However, this catalysis reaction was enhancer dependent. In this study, we demonstrated that Jatropha curcas peroxidase (JcGP1) showed high efficiency in catalyzing luminol oxidation in the presence of H₂O₂. Compared with HRP‐C, the JcGP1‐induced reaction was enhancer independent, which made the enzyme‐linked immunosorbent assay (ELISA) simpler. In addition, the JcGP1 catalyzed reaction showed a long‐term stable CL signal. We optimized the conditions for JcGP1 catalysis and determined the favorable conditions as follows: 50 mM Tris buffer (pH 8.2) containing 10 mM H₂O₂, 14 mM luminol and 0.75 M NaCl. The optimum catalysis temperature was 30°C. The detection limit of JcGP1 under optimum condition was 0.2 pM. Long‐term stable CL signal combined with enhancer‐independent property indicated that JcGP1 might be a valuable candidate peroxidase for clinical diagnosis and enzyme immunoassay with CL detection. Copyright © 2014 John Wiley & Sons, Ltd.     

Luminol-hydrogen peroxide chemiluminescence produced by sweet potato peroxidase.

Anionic sweet potato peroxidase (SPP; Ipomoea batatas) was shown to efficiently catalyse luminol oxidation by hydrogen peroxide, forming a long-term chemiluminescence (CL) signal. Like other anionic plant peroxidases, SPP is able to catalyse this enzymatic reaction efficiently in the absence of any enhancer. Maximum intensity produced in SPP-catalysed oxidation of luminol was detected at pH 7.8-7.9 to be lower than that characteristic of other peroxidases (8.4-8.6). Varying the concentrations of luminol, hydrogen peroxide and Tris buffer in the reaction medium, we determined favourable conditions for SPP catalysis (100 mmol/L Tris-HCl buffer, pH 7.8, containing 5 mmol/L hydrogen peroxide and 8 mmol/L luminol). The SPP detection limit in luminol oxidation was 1.0 x 10(-14) mol/L. High sensitivity in combination with the long-term CL signal and high stability is indicative of good promise for the application of SPP in CL enzyme immunoassay.     

DNA/HSA interaction and nuclease activity of an iron(III) amphiphilic sulfonated corrole

The DNA binding of amphiphilic iron(III) 2,17‐bis(sulfonato)‐5,10,15‐tris(pentafluorophenyl)corrole complex (Fe–SC) was studied using spectroscopic methods and viscosity measurements. Its nuclease‐like activity was examined by using pBR322 DNA as a target. The interaction of Fe–SC with human serum albumin (HSA) in vitro was also examined using multispectroscopic techniques. Experimental results revealed that Fe–SC binds to ct‐DNA via an outside binding mode with a binding constant of 1.25 × 10⁴ M^–1. This iron corrole also displays good activity during oxidative DNA cleavage by hydrogen peroxide or tert‐butyl hydroperoxide oxidants, and high‐valent (oxo)iron(V,VI) corrole intermediates may play an important role in DNA cleavage. Fe–SC exhibits much stronger binding affinity to site II than site I of HSA, indicating a selective binding tendency to HSA site II. The HSA conformational change induced by Fe–SC was confirmed by UV/Vis and CD spectroscopy. Copyright © 2015 John Wiley & Sons, Ltd.     

Luminol-enhanced chemiluminescence of rabbit polymorphonuclear leukocytes: the nature of oxidants that directly induce luminol oxidation

The present work deals with the reaction pathways, including the formation of hydroxyl radicals and chloroamines, which lead to luminol chemiluminescence caused by hypochlorite generation in a suspension of stimulated rabbit polymorphnonuclear leukocyte. Luminol-enhanced (0.02 mM) chemiluminescence of leukocytes stimulated by phorbol 12-myristate 13-acetate does not change in the presence of dimethyl sulfoxide at moderate concentrations (0.02-2.6 mM) at which it must show the specific ability to scavenge hydroxyl radicals. It suggests that no generation of hydroxyl radical with the participation of hypochlorite and superoxide anion takes place after the stimulation of polymorphnonuclear leukocytes. A high dimethyl sulfoxide concentrations (260 mM) a significant fall in chemiluminescence intensity, due to direct interaction of the scavenger with hypochlorite, is observed. Chemiluminescence intensity rose if luminol was added to a leukocyte suspension preliminary stimulated for 10 min. The effect results from the accumulation of hydrogen peroxide but not chloroamines. Exogenic amino acids and taurin at high concentrations (3-15 mM) weaken the chemiluminescence. The data obtained suggest that chemiluminescence in the system studied results predominantly from the direct initial reaction of hypochlorite with luminol. The chemiluminescence intensity is enhanced by hydrogen peroxide via the oxidation of luminol oxidation products.