Chemiluminescence of lucigenin by electrogenerated superoxide ions in aqueous solutions

The chemiluminescence reaction of lucigenin (Luc²⁺?2NO₃^?, N,N′‐dimethyl‐9,9′‐biacridinium dinitrate) at gold electrodes in dioxygen‐saturated alkaline aqueous solutions (pH 10) was investigated in detail by the use of electrochemical emission spectroscopy. We noted that both O₂ and Luc²⁺ are reduced on a gold electrode in aqueous solution of pH 10 in almost the same potential region. From this fact, we expected chemiluminescence based on a radical–radical coupling reaction of superoxide ion (O₂·^?) and one‐electron reduced form of Luc²⁺ (Luc·⁺, a radical cation). Chemiluminescence was actually observed in the potential range where O₂ and Luc²⁺ were simultaneously reduced at the electrodes. The effects were examined upon addition of enzymes, i.e. superoxide dismutase (SOD) and catalase, into the solution and the substitution of heavy water (D₂O) for light water (H₂O) as a solvent on the chemiluminescence. In the presence of native and active SOD, chemiluminescence was completely absent. On the other hand, chemiluminescence was observed, unchanged in the presence of either denatured and inert SOD or catalase. In addition, the amount of chemiluminescence in D₂O solution was about three times greater than that in H₂O solution. These results, together with cyclic voltammetric results, suggest that O₂·^? participates directly in the chemiluminescence but H₂O₂ does not, and the chemiluminescence results from the coupling reaction between O₂·^? and Luc^·+ under the present experimental conditions. These chemically unstable species, O₂·^? and Luc·⁺, are produced during the simultaneous electroreduction of O₂ and Luc²⁺. The coupling reaction between those radical species would lead to the formation of a dioxetane‐type intermediate and, finally, to chemiluminescence. The chemiluminescence reaction mechanism is discussed. Copyright © 2002 John Wiley & Sons, Ltd.     

Light generation with Fenton's reagent its relationship to granulocyte chemiluminescence

A simple chemical system consisting of FeSO₄ and H₂O₂ (Fenton's reagent) was shown to emit light (chemiluminescence). The addition of tryptophan to the reaction markedly enhanced light production. Very little chemiluminescence was observed when H₂O₂ was omitted from the reaction and when ferric, instead of ferrous, ions were used. Hydroxyl radical (OH^.) and singlet oxygen (¹Δ_gO₂) quenchers suppressed chemiluminescence of the FeSO₄ + tryptophan + H₂O₂ system; and, deuterium oxide (²H₂O) enhanced chemiluminescence of both FeSO₄ reactions. These observations suggest that a radical chain reaction involving both OH^. and ¹Δ_gO₂ is responsible for the chemiluminescent reactions. Six iron-containing proteins, some of which are located within granulocytes, all emitted light in the presence of H₂O₂. Since iron and H₂O₂ are present in metabolically stimulated granulocytes, it is likely that chemiluminescent reactions similar to the ones demonstrated in this study account for part of the chemiluminescence of activated granulocytes.     

Thioctic Acid and Dihydrolipoic Acid are Novel Antioxidants Which Interact With Reactive Oxygen Species

Thioctic acid (TA) and its reduced form dihydrolipoic acid (DHLA) have recently gained somc recognition as useful biological antioxidants. In particular, the ability of DHLA to inhibit lipid peroxidation has been reported. In the present study, the effects of TA and DHLA on reactive oxygen species (ROS) generated in the aqueous phase have been investigated. Xanthine plus xanthine oxidase-generated superoxide radicals (O₂), detected by electron spin resonance spectroscopy (ESR) using DMPO as a spin trap. were eliminated by DHLA but not by TA. The sulhydryl content of DHLA, measured using Ellman's reagent decreased subsequent to the incubation with xanthine plus xanthine oxidase confirming the interaction between DHLA and O₂^-. An increase of hydrogen peroxide concentration accompanied the reaction between DHLA and O₂x, suggesting the reduction of O₂^- by DHLA. Competition of O₂^- with epinephrine allowed us to estimate a second order kinetic constant of the reaction between O₂^- and DHLA, which was found to be a 3.3 × 10⁵ M^-1 s^-1. On the other hand, the DMPO signal of hydroxyl radicals (HO ·) generated by Fenton's reagent were eliminated by both TA and DHLA. Inhibition of the Fenton reaction by TA was confirmed by a chemiluminescence measurement using luminol as a probe for HO ·. There was no electron transfer from Fe²⁺ to TA or from DHLA to Fe^{3 +} detected by measuring the Fe²⁺ -phenanthroline complex. DHLA did not potentiate the DMPO signal of HO · indicating no prooxidant activity of DHLA. These results suggest that both TA and DHLA possess antioxidant properties. In particular. DHLA is very effective as shown by its dual capability by eliminating both O₂^-; and HO ·.     

Determination of viable mammalian cells by luminol chemiluminescence using microperoxidase

Chemiluminescent assay for menadione-catalyzed H₂O₂ production by mammalian cells was modified by luminol chemiluminescence with microperoxidase instead of peroxyoxalate chemiluminescence with carcinogenic fluorescent materials. Luminol can be used as a common chemiluminescent reagent for the determination of viable mammalian cells and bacteria.     

Chemiluminescence method based on the KIO4−K2CO3−Mn2+ reaction for rapid and sensitive determination of forchlorfenuron in dried fruit

Forchlorfenuron is a low-toxic phenylurea plant growth regulator. Excessive intake of forchlorfenuron can lead to metabolic disorders of the matrix and be harmful to human health. The chemiluminescence intensity of the KIO₄–K₂CO₃–Mn²⁺ reaction decreased in the presence of forchlorfenuron. Based on this result, a rapid and sensitive chemiluminescence method was established to determine forchlorfenuron by combining it with a batch injection static device. The injection speed, injection volume and reagent concentration of the forchlorfenuron–KIO₄–K₂CO₃–Mn²⁺ chemiluminescence reaction were optimized. Under these optimized conditions, the linear range of the method was 1.0–200.0 μg/L, and the limit of detection was 0.29 μg/L (S/N = 3). The chemiluminescence method for the determination of forchlorfenuron could be completed in 10 s. The method was applied to detect the residual forchlorfenuron in dried fruit samples, and the results are consistent with high-performance liquid chromatography-mass spectrometry. This method has the advantages of high sensitivity, rapid response, less reagent consumption, and convenient operation. It will provide a new perspective for chemiluminescence for the rapid and sensitive determination of forchlorfenuron in various complex samples.     

Oscillating chemiluminescence with thiosemicarbazide in a batch reactor

Oscillating chemical reactions are complex systems involving a large number of chemical species. In oscillating chemical reactions, some species, usually reaction intermediates, exhibit fluctuations in their concentration. In this report, a novel slowly‐damped oscillating chemiluminescence produced by the addition of thiosemicarbazide (TSC) to the oscillating system H₂O₂–KSCN–CuSO₄–NaOH was investigated. Narrow and slightly asymmetric light pulses of 1.5 s half‐width are emitted at 440 nm, with an oscillation period of 22–363 s, an induction period of 9–397 s and an emitted light time of 700–1500 s, depending on reagent concentrations. In this study the dependence of the induction period and the oscillation period on the reagent concentrations was investigated and both parameters were plotted with respect to reagent concentrations. Copper concentration showed a significant effect on the oscillation period. A possible mechanism for the oscillating chemiluminescence reaction is discussed. Copyright © 2008 John Wiley & Sons, Ltd.     

Chemiluminescence analysis of oil oxidizing bacteria <Emphasis Type="Italic">Actinetobacter calcoaceticus</Emphasis> extracts: Effects of the extracts on pSoxS-lux biosensor

A comparative H₂O₂-luminol- and Fe(II)-induced chemiluminescence analysis of extracts of two strains of marine oil oxidizing bacteria Actinetobacter calcoaceticus cultivated either in the presence or absence of oil was carried out. Effects of these extracts on E. coli MG1655 biosensor (pSoxS-lux) were studed. Activation of H₂O₂-induced chemiluminescence in the presence of oil was observed. This suggests activation of free radical lipid peroxidation. Aqueous extracts of microorganisms cultivated in the presence of oil were shown to activate reactive oxygen species production (ROS) in Fe(II)-induced chemiluminescence reaction mixture. Acetone-ethanol extracts induced antioxidative systems of both strains. Chemiluminescence analysis in a biological system carried utilizing E. coli MG1655 (pSoxS-lux) revealed that aqueous extracts of the strains cultivated in the absence of oil contained potential antioxidants.     

Interaction of trout hemoglobin with H2O2: a chemiluminescence study

Erythrocytes from trout Salmo irideus are characterized by four different hemoglobin components (HbI, HbII, HbIII and HbIV), HbI and HbIV being predominant. In this study we describe the interaction between trout hemoglobin (HbI and HbIV) and H₂O₂ using a chemiluminescence assay. Our data show that the reaction of hemoglobins with H₂O₂ produces a time-limited and significant increase of chemiluminescence signal. The half-life of the decay of this chemiluminescence signal was characteristic for each type of hemoglobin used. These results indicate the formation of excited molecules related to the interaction between trout hemoglobin and H₂O₂. © 1997 John Wiley & Sons, Ltd.     

Enhanced chemiluminescent sandwich enzyme immunoassay for hen egg lysozyme

A sensitive chemiluminescent sandwich-type enzyme immunoassay for hen egg lysozyme was developed. The assay was performed on polystyrene microtitre plates using immobilized specific polyclonal rabbit antibody against lysozyme, a peroxidase conjugate and the H₂O₂/luminol-enhanced chemiluminescence detection reagent. The chemiluminescent signal was detected using either a microplate luminometer, or photographic film in a camera luminometer. The detection limit for lysozyme was 0.3 ng/mL, and this was three times lower than that obtained using a colorimetric method with H₂O₂ and o-phenylendiamine as substrates. Recovery of the assay was 97–112% and the relative standard deviation ranged from 3.6% to 10.3%. The immunoassay overcame interference from the food sample matrix when lysozyme, used as a bacteriostatic agent, was measured.     

Determination of amine oxidases in tissues by peroxidation-induced chemiluminescence of phthalazines

A method is proposed for the determination of the activity of amine oxidases in purified samples and tissue homogenates. The method is based on the chemiluminescence of luminol and other cyclic hydrazides elicited by the horseradish peroxidase-catalyzed peroxidation using H₂O₂ produced in the amine oxidase reaction. Several aspects of the chemiluminescence method for determining enzymatic activity in crude tissue extracts are discussed.     

Quantitative generation of singlet (1Δg) oxygen from acidified aqueous peroxynitrite produced by the reaction of nitric oxide and superoxide anion

Simple acidification of aqueous alkaline peroxynitrite quantitatively generates singlet (¹Δ_g) molecular oxygen, detected and quantitated spectroscopically (1270 nm). This observation provides a chemical basis for physiological cytotoxicity of ONOO^? generated in the diffusion - controlled reaction of cellular NO^? and O. The experiments consist of (i) chemical generation of ONOO^? from NO^? gas and KO₂ powder in alkaline aqueous solution; (ii) absorption spectral identification of ONOO^? in the near-UV with maximum at 302 nm; (iii) spectroscopic identification of ¹O₂ by its emission band at 1200–1340 nm with maximum at 1275 nm; and (iv) quantitation of ¹O₂ generated in ONOO^?/H⁺ reaction by comparison of the chemiluminescence intensity at 1270 nm with that from H₂O₂/OCl^? reaction that generates ¹O₂ with unit efficiency at alkaline pH. ¹O₂ was generated with unit efficiency with respect to ONOO^? concentration by the ONOO^?/H⁺ reaction.     

Reactivities of diphenylfuran (a singlet oxygen trap) with singlet oxygen and hydroxyl radical in aqueous systems.

It has been studied whether 2,5-diphenylfuran is a specific singlet oxygen trap in aqueous systems. With certain ¹O₂ generating systems (Rose Bengal photooxygenation and NaOClH₂O₂ systems) and·OH generating systems (Fenton's reagent and acetaldehyde-xanthine oxidase system), diphenylfuran was chiefly converted in all cases to cis-dibenzoyl-ethylene, but not to trans-dibenzoylethylene. Low but detectable conversion of diphenylfuran to a hydroperoxide, probably a distinct ¹O₂-derived reaction in aqueous media, was found only in the Rose Bengal photooxygenation system.     

Fast and sensitive chemiluminescence determination of H2O2 concentration in stimulated human neutrophils

A fast and sensitive chemiluminescence assay for the determination of H₂O₂ in stimulated neutrophils without the use of enzymes was developed. The method is based on the oxidation of luminol by hypochlorous acid. The chemiluminescence of this reaction is highly dependent on the concentration of hydrogen peroxide. Changes in H₂O₂ concentration in PMA-stimulated neutrophils were followed by injection of NaOCI to cell suspension at different times after cell stimulation. The short integration time of 2 s permits calculation of actual concentrations of H₂O₂ without influence of H₂O₂ decomposition by cellular enzymes or newly produced H₂O₂ due to dismutation of superoxide anion radicals. Concentrations of H₂O₂ were diminished by catalase and enhanced by sodium azide owing to inhibition of cellular catalase and myeloperoxidase. Changes in H₂O₂ concentration upon stimulation could be observed at 3000 cell/mL.     

1,10‐Phenanthroline–H2O2–KSCN–CuSO4–NaOH oscillating chemiluminescence system

In this paper, oscillating chemiluminescence (CL), 1,10‐phenanthroline H₂O₂–KSCN–CuSO₄–NaOH system, was studied in a batch reactor. The system described is a novel, slowly damped oscillating CL system, generated by coupling the well‐known Epstein–Orban, H₂O₂–KSCN–CuSO₄–NaOH chemical oscillator reaction with the CL reaction involving the oxidation of 1,10‐phenanthroline by hydrogen peroxide, catalyzed by copper(II) in alkaline medium. In this system, the CL reaction acts as a detector or indicator system of the far‐from‐equilibrium dynamic system. Narrow and slightly asymmetric light pulses of 1.2 s half‐width are emitted at 440 nm with an emitted light time of 200–1000 s, induction period of 3.5–357 s and oscillation period of 28–304 s depending on the reagent concentrations. In this report the effect of the concentration variation of components involved in the oscillating CL system on the induction period, the oscillation period and amplitude was investigated and the parameters were plotted with respect to reagent concentrations. Copper concentration showed a significant effect on the oscillation period. The possible mechanism for the oscillating CL reaction was also discussed. Copyright © 2008 John Wiley & Sons, Ltd.     

Killing of Bacillus Spores by Aqueous Dissolved Oxygen, Ascorbic Acid, and Copper Ions

An approach to decontamination of biological endospores is discussed. Specifically, the performance of an aqueous modified Fenton reagent is examined. A modified Fenton reagent formulation of cupric chloride, ascorbic acid, and sodium chloride is shown to be an effective sporicide under aerobic conditions. The traditional Fenton reaction involves the conversion of hydrogen peroxide to hydroxyl radical by aqueous ionic catalysts such as the transition metal ions. Our modified Fenton reaction involves the conversion of aqueous dissolved oxygen to hydrogen peroxide by an ionic catalyst (Cu²⁺) and then subsequent conversion to hydroxyl radicals. Results are given for the modified Fenton reagent deactivating spores of Bacillus globigii. A biocidal mechanism is proposed that is consistent with our experimental results and independently derived information found in the literature. This mechanism requires diffusion of relatively benign species into the interior of the spore, where dissolved O₂ is then converted through a series of reactions which ultimately produce hydroxyl radicals that perform the killing action.     

STUDIES ON NITRIC OXIDE FREE RADICALS GENERATED FROM POLYMORPHONUCLEAR LEUKOCYTES (PMN) STIMULATED BY PHORBOL MYRISTATE ACETATE (PMA)

The interaction of NO and O^?₂free radicals generated from PMA (phorbol myristate acetate)-stimulated PMN (polymorphonuclear leukocytes) was studied by a nitroxide spin trap, DMPO (5,5-dimethyl-1-pyrroline-1-oxide). It was found that addition of L-arginine to the system would significantly decrease the trapped O^?₂by DMPO and addition of N^G-monomethyl-arginine (NGMA) would significantly increase the trapped O^?₂by DMPO. It was proved that the formation of ONOO^?by the reaction of NO and O^?₂was the main reason for the decrease of trapped O^?₂in the experiment with xanthine/xanthine oxidase and irradiation of riboflavin systems. The yield of NO during this process was calculated. The generation dynamic of NO was studied by a luminol-dependent chemiluminescence technique and it was found that after stimulation of PMN by PMA, there would be an immediate, significant chemi-luminescence, which came mainly from the active oxygen free radicals generated by PMN. If L-arginine was added to this system, the chemiluminescence would increase about 100-fold, but NGMA inhibited the increase of the chemiluminescence. Ten minutes after addition of L-arginine, this increase did not change, the chemiluminescence peak decreased gradually, but the half life increased. The ESR and chemiluminescence properties of NO and ONOO^?synthesized were also studied in model systems.     

Determination of absolute chemiluminescence quantum yields for reactions of bis-(pentachlorophenyl) oxalate,hydrogen peroxide and fluorescent compounds

Absolute chemiluminescence quantum yields (?_CL) for reactions of bis-(pentachlorophenyl) oxalate (PCPO), hydrogen peroxide (H₂O₂) and 9:10 diphenyl anthracene (DPA) have been determined. A fully corrected chemiluminescence monitoring spectrometer was calibrated for spectral sensitivity using the chemiluminescence of the bis-(pentachlorophenyl) oxalate system as a liquid light source, the total photon output of which had previously been determined by chemical actinometry. At high (PCPO)/(H₂O₂) ratios ?_CL was found to be independent of PCPO and H₂O₂ concentrations.     

The role of superoxide anion in peroxidase-catalyzed chemiluminescence of luminol

The chemiluminescence associated with peroxidation of luminol in buffered aqueous solution is a complex process involving several intermediates. It can be inhibited by removal of oxygen from the incubation medium. Superoxide radical is both an intermediate in this reaction and an essential component in light-producing steps. The importance of O₂^? in propagating this reaction was shown by the inhibition of luminescence by superoxide dismutase. A mechanism was proposed which is consistent with the data. It appears likely that the diverse biological effects of peroxidases are largely due to the reactivities of these intermediates and products.     

Pyrophosphate as substrate for alkaline phosphatase activity: A convenient flow‐injection chemiluminescence assay

A sensitive and convenient flow‐injection chemiluminescence (FI‐CL) turn‐on assay for alkaline phosphatase (ALP) activity without any label and synthesis is developed. Cu²⁺ can catalyze the luminol–H₂O₂ CL reaction. Pyrophosphate (PPi) can chelate Cu²⁺ and therefore the Cu²⁺‐mediated luminol‐H₂O₂ CL reaction is inhibited. The addition of ALP can catalyze the hydrolysis of PPi into phosphate ions, Cu²⁺ is released and the chemiluminescence recovers. A detection limit of 1 mU/mL ALP is obtained.     

Chemiluminescent determination of esterases in monocytes

Esterase from monocytes promotes the hydrolysis of 2-methyl-1-propenylbenzoate (MPB) yielding 2-methyl-1-propenol, which is oxidized by horseradish peroxidase/H₂O₂ producing triplet acetone. The chemiluminescence of this reaction can be enhanced by the addition of 9,10-dibromoanthracene-2-sulphonate. The non-specific esterase present in monocytes is responsible for MPB hydrolysis, since (a) the chemiluminescence of the reaction was inhibited by fluoride, and (b) cells that do not contain a significant amount of non-specific esterases, e.g. lymphocytes and neutrophils, did not trigger light emission. The analytical application of this reaction is considered. © 1998 John Wiley & Sons, Ltd.