What do we measure with luminol-, lucigenin- and penicillin-amplified chemiluminescence? 1. Investigations with hydrogen peroxide and sodium hypochlorite

Evidence is provided that the amplifiers luminol and lucigenin react with different reactive oxygen species (ROS), depending on the ROS-generating system used. H₂O₂ is used to produce calibration curves for luminol- and lucigenin-amplified chemiluminescence. With this chemiluminescence generator we characterized the specificity and sensitivity of luminol- and lucigenin-amplified chemiluminescence and also studied penicillin G, a known enhancer of luminol-amplified chemiluminescence. The combination of luminol and lucigenin in reciprocally changing concentrations is effective in an additive manner, but the weak amplifier penicillin increases luminol-amplified chemiluminescence distinctly more than in an additive manner in different combinations. Lucigenin-amplified chemiluminescence is increased by penicillin at about 1% of the optimum concentration of penicillin; increasing concentrations of penicillin are less and less effective. On the other hand, low lucigenin concentrations enhance penicillin-amplified chemiluminescence at optimum penicillin concentrations more than in an additive manner. Fe²⁺ does not alter luminol-, lucigenin- or penicillin-amplified chemiluminescence. Co²⁺ increases luminol-amplified chemiluminescence by a factor of 100. Lucigenin- and penicillin-amplified chemiluminescence are minimally enhanced by Co²⁺. Cu²⁺ enhances luminol-amplified chemiluminescence with increasing concentrations by a factor of 1000. Lucigenin-amplified chemiluminescence increases also by the factor of 1000, but the concentration–reaction curve is not as steep. NaOCl enhances H₂O₂/Fe²⁺-driven luminol-amplified chemiluminescence in a concentration-dependent manner by a factor of 10⁴ (in the highest concentration of 10 mmol/L) and lucigenin amplified chemiluminescence only by a factor of about 25. Catalase (CAT) abolishes luminol-, lucigenin- and penicillin-amplified chemiluminescence completely, whereas superoxide dismutase (SOD) has no effect on luminol- or penicillin-amplified chemiluminescence, but enhances lucigenin-amplified chemiluminescence five-fold increasingly with increasing SOD activity. © 1998 John Wiley & Sons, Ltd.     

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.     

Plant chemiluminescence

Light production by plants was confirmed by measuring chemiluminescence from root and stem tissue of peas (Pisum sativum), beans (Phaseolus vulgaris), and corn (Zea mays) in a modified scintillation spectrophotometer. Chemiluminescence was inhibited by treating pea roots with boiling ethanol or by placing them in a N₂ gas phase. Chemiluminescence was increased by an O₂ gas phase or by the addition of luminol. NaN₃ and NaCN blocked both in vitro and in vivo chemiluminescence.

It is postulated that the source of light is the hydrogen peroxide-peroxidase enzyme system. It is known that this system is responsible for chemiluminescence in leukocytes and it seems likely that a similar system occurs in plants.

     

Increased spontaneous chemiluminescence from liver homogenates and isolated hepatocytes upon inhibition of o2− and h2o2 utilization

The intracellular steady-state concentrations of hydrogen peroxide or Superoxide anion were increased by inhibiting either catalase, glutathione peroxidase, or Superoxide dismutase activities. Catalase was inhibited with aminotriazole while glutathione peroxidase activity was blocked by eliminating reduced glutathione after addition of either iodoacetamide diethylmaleate or phorone. The concentration of aminotriazole that stimulated chemiluminescence in 50% (60 mM) was very similar to the K_i for catalase activity (70 mM). Cyanide, an inhibitor of both catalase and Superoxide dismutase, stimulated chemiluminescence in 50% at a concentration (0.15 mM) which is much closer from the K_i for Superoxide dismutase (0.25 mM) than from the K_i for catalase (15 μM). The Superoxide dismutase inhibitor diethyldithiocarbamate also increased chemiluminescence six- to ten-fold. Depletion of reduced glutathione stimulated spontaneous chemiluminescence when its concentration decreased below 4.5 μmol · g liver⁻¹. The results shown herein suggest that the changes in the intracellular steady-state concentration occurring after inhibition of any antioxidant enzyme are responsible for the increased spontaneous chemilumi-nescence. Spontaneous chemiluminescence from intact cells may be used as a noninvasive method for monitoring intracellular free radical metabolism.     

Chemiluminescence in Wounded Root Tissue : EVIDENCE FOR PEROXIDASE INVOLVEMENT

Root and stem segments from soybean (Merrill cv. `Bragg') showed an enhanced chemiluminescence upon mechanical injury. Roots emitted more light than did stems. Light emission was diminished by CN⁻ and N₃⁻ but was not affected by rotenone and antimycin A. Catalase quenched chemiluminescence in wounded root segments as did ascorbic acid and hydroquinone. Superoxide dismutase addition resulted in a small diminution in light emission, but mannitol, an OH· scavenger, was without effect. The addition of H₂O₂ to wounded root segments markedly elevated chemiluminescence in the presence of air as well as under N₂. It is concluded that peroxidases, found abundantly in roots, predominantly contribute to light emission in wounded plant tissue.     

Highly luminescent S,N co‐doped carbon quantum dots‐sensitized chemiluminescence on luminol–H2O2 system for the determination of ranitidine

S,N co‐doped carbon quantum dots (N,S‐CQDs) with super high quantum yield (79%) were prepared by the hydrothermal method and characterized by transmission electron microscopy, photoluminescence, UV–Vis spectroscopy and Fourier transformed infrared spectroscopy. N,S‐CQDs can enhance the chemiluminescence intensity of a luminol–H₂O₂ system. The possible mechanism of the luminol–H₂O₂–(N,S‐CQDs) was illustrated by using chemiluminescence, photoluminescence and ultraviolet analysis. Ranitidine can quench the chemiluminescence intensity of a luminol–H₂O₂–N,S‐CQDs system. So, a novel flow‐injection chemiluminescence method was designed to determine ranitidine within a linear range of 0.5–50 μg ml^?1 and a detection limit of 0.12 μg ml^?1. The method shows promising application prospects.     

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.     

The role of H2O2 formation in the insulin-like and insulin antagonistic effects on fat cells of omega-aminoalkyl glycosides.

A class of ω-aminoalkyl glycosides previously found to antagonize insulin's action on glucose oxidation in fat cells and to stimulate glucose oxidation in insulin's absence is now shown to mimic insulin also on the conversion of glucose to free fatty acids and to glycerol and glycerides. These glycosides also act like insulin by inhibiting hormone- and cholera toxin-stimulated lipolysis. Various lines of evidence demonstrate that most, if not all, of the insulin-like activity of these glycosides results from H₂O₂ formed from an amine oxidase-catalyzed oxidation of the aminoalkyl moiety of these compounds. A contaminant in the bovine plasma albumin (BPA) preparations used in the bioassays was found to represent a major source of the amine oxidase activity. Membrane (ghost) preparations were also found to possess amine oxidase activity capable of forming H₂O₂ from the glycosides in amounts sufficient to express insulin-like activity. Preliminary experiments with intact adipocytes suggest that this activity is located on the cell surface. The BPA-associated activity corresponds to the known Cu²⁺-containing “plasma-type” amine oxidase (EC 1.4.3.6) on the basis of its substrate specificity and susceptibility to selective inhibitors. The plasma membrane activity appears to correspond to neither the plasma-type nor to the flavin-containing mitochondrial-type (EC 1.4.3.4) and remains to be identified. The observed potent antilipolytic effects of both H₂O₂ and the aminoalkyl glycosides points out that any mechanism used to explain the insulin-like action of H₂O₂ must account for this ability to inhibit lipolysis as well as to stimulate glucose utilization. That catalase inhibits the insulin-like action of the glycosides and H₂O₂, but not that of insulin indicates that insulin's action is not mediated by cell surface-produced H₂O₂. Also, since the insulin antagonistic activity of these glycosides was not inhibited by catalase, H₂O₂ formation is not responsible for this antagonism. The latter finding, added to present and previous evidence on the carbohydrate structural requirements involved in H₂O₂ production and in the insulin-like biological and binding properties of the aminoalkyl glycosides, is consistent with a role(s) for their carbohydrate moieties in both the insulin antagonistic and agonistic activities of these compounds.     

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.     

A highly sensitive detection of chloramphenicol based on chemiluminescence immunoassays with the cheap functionalized Fe3O4@SiO2 magnetic nanoparticles

A strategy has been applied to chloramphenicol (CAP) detection with chemiluminescence immunoassays (CLIA) based on cheap functionalized Fe₃O₄@SiO₂ magnetic nanoparticles (Fe–MNPs). The strategy that bovine serum albumin (BSA) was immobilized on cheap functionalized Fe–MNPs and that the CAP molecules were then immobilized on BSA, avoided the long process of dialysis for preparation of the BSA‐CAP conjugates. The samples were detected for both methods that utilized two different kinds of functionalized Fe–MNPs (amine‐functionalized Fe₃O₄@SiO₂ and carboxylic acid‐functionalized Fe₃O₄@SiO₂). The sensitivities and limits of detection (LODs) of the two methods were obtained and compared based on inhibition curves. The 50% inhibition concentrations (IC₅₀) values of the two methods were about 0.024 ng ml^?1 and 0.046 ng ml^?1 respectively and LODs were approximately 0.0002 ng ml^?1 and 0.001 ng ml^?1 respectively. These methods were much more sensitive than that of any traditional enzyme‐linked immunosorbent assay (ELISA) previously reported. Therefore, such chemiluminescence methods could be easily adapted for small molecule detection in a variety of foods using Fe–MNPs.     

Spontaneous Chemiluminescence of Soybean Embryonic Axes during Imbibition

Isolated soybean (Glycine max L. var Hood) embryonic axes have a spontaneous chemiluminescence (about 150 counts per minute per embryo) that increases showing two phases, upon water imbibition. The first photoemission burst was measured between 0 and 7 hours of imbibition with a maximum of about 350 counts per minute per embryo after 2 hours. The second photoemission phase, between 7 and 30 hours, increased from about 220 to 520 counts per minute per embryo. Both chemiluminescence phases were inhibited by infused butylated hydroxyanisole while only the second phase was inhibited by infused salicylhydroxamic acid. On the basis of the sensitivity of the lipoxygenase reaction to both inhibitors (about 90%), the first burst is tentatively assigned to oxy-radicals mobilized upon water uptake by the embryonic axes, and the second phase is tentatively identified as due to lipoxygenase activity. The in vivo lipoxygenase activity of the embryonic axes was estimated by both the fraction of total oxygen uptake that was inhibited by butylated hydroxyanisole and by the fraction of photoemission that was inhibited by butylated hydroxyanisole and by salicylhydroxamic acid. Both approaches indicated marked increases (5-fold and 12-fold, respectively) of lipoxygenase activity between 2 and 30 hours of imbibition. The measured chemiluminescence per O₂ uptake ratio (the experimental quantum yield) for the lipoxygenase reaction (3.3 × 10⁻¹⁴ counts per O₂ molecule) was used to estimate the O₂ uptake due to lipoxygenase activity from the photoemission of the embryonic axes after 30 hours of imbibition. The value (0.54 microliters per minute per axis) was close to the butylated hydroxyanisole-sensitive O₂ uptake (1.2 microliters O₂ per minute per axis) of the same embryonic axes. Chemiluminescence may afford a noninvasive assay for lipoxygenase activity in intact plant tissues.     

Investigation on 1O2 generation ability of di‐sulfonic phthalocyanine zinc isomers using an HPLC–CL system

An HPLC system combining a chemiluminescence detector was applied to estimate the singlet oxygen (¹O₂) generation ability of di‐sulfonic phthalocyanine zinc (ZnPcS₂) isomers. As photosensitizers, ZnPcS₂ produces ¹O₂ in air‐saturated solutions under photoirradiation. The latter reacts with methyl Cypridina luciferin analogue (MCLA) to initiate chemiluminescence. This photoinduced chemiluminescence (PCL) of MCLA provides an easy method for evaluating the isomers' ¹O₂ generation ability during a simultaneous HPLC separation procedure. The cis‐isomers and trans‐isomers of ZnPcS₂ show different ¹O₂ generation abilities, which are in accordance with differences in their absorption spectra. Copyright © 2013 John Wiley & Sons, Ltd.     

An Improved Chemiluminescence Method for Hydrogen Peroxide Determination in Plant Tissues

As a consequence of the increasing importance of hydrogen peroxide in plant metabolism, more efficient methods are required for accurate determinations of its concentration in plant tissue and organs. Here we present a highly sensitive chemiluminescence (CL) method based on the Co (II) catalysed oxidation of luminol by H₂O₂. The replacement of ferricyanide, the traditional catalyst of luminol luminescence by Co (II), enhanced the sensitivity of the reaction towards H₂O₂ in three orders of magnitude. Thus, plant extracts can be diluted to such a level that quenching effects of phenols and ascorbic acid (ASA), which are normally present at high concentrations in plant tissues is avoided, and therefore, pre-treatments with PVP and ascorbate oxidase to remove these quenchers from plant-extracts become unnecessary. To exemplified the high performance of the method, measurements of H₂O₂ were carried out in PVP treated and non-treated extracts of grapevine leaf, a plant tissue that contain high levels of phenols and ASA. Moreover, increases in H₂O₂ levels were detected in disc-leaf treated with aminotriazole, a specific Cat inhibitor, showing the importance of Cat as a H₂O₂ scavenging enzyme in leaves of grapevine.     

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.     

Microflow injection potassium bioassay based on G‐quadruplex DNAzyme‐enhanced chemiluminescence

By taking advantage of microflow injection chemiluminescence analysis, we developed a distinctive microfluidic bioassay method based on G‐Quadruplex DNAzyme‐enhanced chemiluminescence for the determination of K⁺ in human serum. AGRO100, the G‐rich oligonucleotide with high hemin binding affinity was primarily selected as a K⁺ recognition element. In the presence of K⁺, AGRO100 folded into G‐quadruplex and bound hemin to form DNAzyme, which catalyzed the oxidation of luminol by H₂O₂ to produce chemiluminescence. The intensity of chemiluminescence increased with the K⁺ concentration. In the study, the DNAzyme showed both long‐term stability and high catalytic activity; other common cations at their physiological concentration did not cause notable interference. With only 6.7 × 10^?13 mol of AGRO100 consumption per sample, a linear response of K⁺ ranged from 1 to 300 µmol/L, the concentration detection limit 0.69 µmol/L (S/N = 3) and the absolute detection limit 1.38 × 10^?12 mol were obtained. The precision of 10 replicate measurements of 60 µmol/L K⁺ was found to be 1.72% (relative standard deviation). The accuracy of the method was demonstrated by analyzing real human serum samples. Copyright © 2014 John Wiley & Sons, Ltd.     

M3 muscarinic acetylcholine receptor antagonists: SAR and optimization of bi-aryl amines

Exploration of multiple regions of a bi-aryl amine template led to the identification of highly potent M₃ muscarinic acetylcholine receptor antagonists such as 14 (pA₂ = 11.0) possessing good sub-type selectivity for M₃ over M₂. The structure–activity relationships (SAR) and optimization of the bi-aryl amine series are described.     

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.     

Fe3O4 and bimetal–organic framework Zn/Mg composite peroxide-like catalyze luminol chemiluminescence for specific measurement of atropine in Datura plant

Atropine (AT) is an anticholinergic drug. AT is abundantly in Datura plant seeds. Fe₃O₄@Zn/Mg MOF (Fe₃O₄@MOF) composite was synthesized. The compound had a high peroxidase-like activity in a chemiluminescence (CL) reaction. Addition of AT quenched CL. The linear range and limit of detection were 5–600 μg L⁻¹ and 2 × 10⁻² μg L⁻¹. This method is fast, reversible, and selective, without biodegradability effects, high accuracy, and precision for measuring AT in the Datura plant.     

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.     

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.