The influence of dioxygen on luminol chemiluminescence

Assays of peroxy compounds are commonly performed after chromatographic separation of analysed mixtures. In high‐performance liquid chromatography (HPLC), solvent reservoirs are sparged by helium or inline vacuum‐degassed in order to control the compressibility of the solvents for efficient pumping. In this study, we investigated the influence of degassing the reaction solution on the light output of the hemin‐catalyzed luminol oxidation by various oxidants. We found that, when t‐butyl hydroperoxide, hydrogen peroxide, n‐butyl hydroperoxide, iodosobenzene and iodobenzene diacetate were used as oxidants, the luminol chemiluminescence was lowered by 50–70% compared with an equilibrated and degassed solution. The opposite effect was observed when dibenzoyl peroxide and 3‐chloroperoxybenzoic acid were used as oxidants, as the chemiluminescence increased by approximately 20–30%. The reduced chemiluminescence was explained based on the known role of dioxygen in luminol chemiluminescence. The enhancement of chemiluminescence was rationalized by suggesting an alternative mechanism of luminol oxidation valid for peroxyacids and diacyl peroxides in which the reaction of a peroxyacid anion with the diazaquinone led to light emission with a higher quantum yield than the usual path, which is suppressed by the removal of dioxygen from the reaction solution. Copyright © 2009 John Wiley & Sons, Ltd.     

The effect of pH on chemiluminescence of different probes exposed to superoxide and singlet oxygen generators

The compromised optima for high intensity chemiluminescence (CL), using superoxide generators, were all above pH 9.0 for the CL probes luminol and lucigenin. With luminol the optima were at pH 9.0 and 9.4 for the generators KO₂ and hypoxanthine/xanthine oxidase (HX/XO), respectively. Lucigenin, with the same generators, produced optima at pH 9.5 and 10.0, respectively. The probe methyl-Cypridina-luciferin analogue (MCLA) produced optima closer to neutral pH, which is preferred for physiological assessments. MCLA had optima at pH 6.0, 8.7 and 9.5 with KO₂ and with HX/XO optima at pH 4.8, 6.0, 7.0 and 8.7. When CL was assessed at physiological pH, MCLA observed superoxide radicals with a sensitivity of 100- and 330-fold more than luminol or luicigenin respectively. For singlet oxygen, the sensitivity of MCLA at this pH was 45- and 5465-fold more than for the said probes respectively. H₂O₂ did not elicit CL between pH 4 and 9.5 with any of the probes and did not influence the production of superoxide or singlet oxygen when co-assessed. Therefore CL could only be obtained when enzymes were used as converters. The optima for the enzyme-conversion system horseradish peroxidase (HRP)/H₂O₂, and luminol, were at pH 8.0 and 9.2. Lucigenin and HRP/H₂O₂ also had a biphasic CL profile with optima at pH 7.4 and 9.6. MCLA and HRP/H₂O₂ had five optima, with the major ones at pH 6.1 and beyond 10. The optima for the myeloperoxidase/H₂O system were at 8.6 and beyond 10.0 when luminol and 0.15 mol/L NaBr were used. © 1997 John Wiley & Sons, Ltd.     

Scavenger effect of flavonols on HOCl-induced luminol chemiluminescence.

Hypochlorous acid (HOCl), the main product of the myeloperoxidase system, is a strong oxidant and a potent chlorinating agent, which can damage host tissues. In the present work, the scavenger effect of three aglycone flavonols (myricetin, quercetin and kaempferol) and of the natural glycoside flavonol, rutin, was studied towards HOCl using luminol-dependent chemiluminescence (CL). At 1 micro mol/L fi nal concentration, rutin was the most powerful scavenger of HOCl with an inhibitory luminol oxidation of 91.4% +/- 3.2%. Quercetin, kaempferol and myricetin inhibited the luminol-dependent CL at the same concentration only by 75.9% +/- 3.4%, 57.7% +/- 5.3% and 43.3% +/- 3.5%, respectively. With increasing concentration of these flavonols, a dose-dependent inhibition of luminol CL was observed. In order to prove to what extent flavonols scavenge HOCl, their concentrations that gave 50% inhibition of luminescence (IC50) were compared to IC50 values of the sulphur-containing compounds N-acetyl cysteine (NAC) and taurine. The scavenging activities of compounds tested decrease in the order: rutin > NAC > quercetin > kaempferol > taurine. The present study revealed that rutin was the most effective scavenger agent.     

The influence of age and sex on phagocyte chemiluminescence

The process of ageing is associated with increased susceptibility to infection. Phagocytes form the primary defence mechanism against infecting microorganisms, but the influence of ageing on phagocyte function remains controversial. In this study we have applied a microtitre plate phagocyte chemiluminescence (CL) assay suitable for clinical use to compare phagocyte oxidative metabolism in younger healthy subjects (age 20–60 years) and healthy older (60–70 years) subjects. Polymorphonuclear leukocytes (PMNL) and monocytes were stimulated using phorbol myristate acetate (PMA), serum opsonized zymosan (SOZ), and non-opsonized zymosan (ZYM) in the presence of both lucigenin and luminol. Monocytes showed a higher luminolenhanced CL response to PMA in males compared with females in the younger age group. No PMNL differences were observed between the sexes. Although no difference were found in relation to age when cells were stimulated with PMA and SOZ, significantly lower background (unstimulated) CL was obtained from PMNL with luminol. PMNL luminol-enhanced CL responses were also lower in response to ZYM. The findings suggest a reduced response of PMNL from older subjects to minimal stimulation. This could be related to abnormalities in the triggering of the respiratory burst or myeloperoxidase release due to ageing. The influence of age and sex should be taken into account in clinical studies of phagocyte CL.     

Free fatty acid determination by peroxidase catalysed luminol chemiluminescence

A sensitive, specific, and partly automatic method for the analysis of free fatty acids is described. The assay involves activation of free fatty acids by acyl-CoA synthetase (EC 6.2.1.3) followed by oxidation of the thioesters by acyl-CoA oxidase. The H₂O₂ formed is determined in a reaction catalysed by horseradish peroxidase (EC 1.11.1.7) using luminol as electron donor. The assay has a linear range of 0.05 to 5 nmol of different free fatty acids (C₁₀-C₁₈) in the original sample. The efficiency of the method toward capric, lauric, myristic, palmitic, palmitoleic, stearic, oleic, and linoleic acid measured as recovery of light emission compared to that of H₂O₂ standards, was over 90%. AffiGel 501 was used to covalently bind the free thiol group in CoASH eliminating interference of this substance in the peroxidase-luminol reaction.     

Electrogenerated chemiluminescence reaction of lucigenin with isatin at a platinum electrode

The electrogenerated chemiluminescence (ECL) reaction of lucigenin with isatin was investigated at a platinum electrode in a neutral aqueous solution. The ECL intensity of lucigenin at ?0.65 V was greatly enhanced by isatin, and the ECL intensity was about 50 times higher than that of lucigenin without isatin. The enhanced ECL was believed to be produced by the chemiluminescence reaction between reduced lucigenin and superoxide anion that was generated by the reaction of electrochemically reduced isatin with dissolved oxygen. The conditions for the determination of isatin were optimized. Under the optimized condition, the enhanced ECL intensity vs. isatin concentration was linear in the range 4.8 × 10^?7?1.9 × 10^?5 g/mL; with a detection limit of 3.3 × 10^?8 g/mL, and the relative standard derivation 1.0 × 10^?6 g/mL isatin was 3.8%. Copyright © 2004 John Wiley & Sons, Ltd.     

Enhanced chemiluminescence of lucigenin with epinephrine in cationic surfactant micelles containing periodate

Epinephrine (EP) species involved in the lucigenin chemiluminescence (CL) were identified in alkaline solution by comparing the time course of the CL response and the formation of EP oxidation products. EP quinone and adrenolutine (AL) were found to be responsible for the lucigenin-CL reaction. The mechanism of the lucigenin-CL enhancement was investigated using cationic micellar hexadecyltrimethylammonium hydroxide (CTAOH), periodate, and a mixture of micellar CTAOH and periodate. The CL enhancement in the presence of micellar CTAOH and periodate could be explained in terms of increases in the oxidation rate of EP to EP quinone and the intramolecular oxidation rate of adrenochrome to AL.     

Mechanism of alcohol‐enhanced lucigenin chemiluminescence in alkaline solution

The chemiluminescence (CL) of lucigenin (Luc²⁺) can be enhanced by different alcohols in alkaline solution. The effect of different fatty alcohols on the CL of lucigenin was related to the carbon chain length and the number of hydroxyl groups. Glycerol provides the greatest enhancement. UV/Vis absorption spectra and fluorescence spectra showed that N‐methylacridone (NMA) was produced in the CL reaction in the presence of different alcohols. The peak of the CL spectrum was located at 470 nm in all cases, indicating that the luminophore was always the excited‐state NMA. The quenching of lucigenin CL by superoxide dismutase (SOD) and the electron spin resonance (ESR) results with the spin trap of 5,5‐dimethyl‐1‐pyrroline N‐oxide (DMPO) demonstrated that superoxide anions (O₂^?–) were generated from dissolved oxygen in the CL reaction and that glycerol and dihydroxyacetone (DHA) can promote O₂^?? production by the reduction of dissolved oxygen in alkaline solution. It was assumed that the enhancement provided by different alcohols was related to the solvent effect and reducing capacity. Glycerol and DHA can also reduce Luc²⁺ into lucigenin cation radicals (Luc^?+), which react with O₂^?? to produce CL, and glycerol can slowly transform into DHA, which is oxidized quickly in alkaline solution. Copyright © 2015 John Wiley & Sons, Ltd.     

Effect of sulphite on the oxidative metabolism of human neutrophils: Studies with lucigenin- and luminol-dependent chemiluminescence

To assess the effect of sulphite on the oxidative metabolism of human neutrophils, chemiluminescence (CL) measurements were performed using lucigenin and luminol as chemiluminigenic probes. Lucigenin-dependent CL was used for measuring superoxide anion (O) production, and luminol-dependent CL was used for determination of myeloperoxidase (MPO)-connected processes. With sulphite concentrations of 0.01 to 1 mmol/L, resting neutrophils showed an up to sixfold increase of lucigenin-dependent CL, but only a 1.9-fold increase of luminol-dependent CL. Subsequent stimulation of sulphite-treated neutrophils with phorbol myristate acetate (PMA) (soluble stimulant) or zymosan (particulate stimulant) resulted in an additional significant increase of lucigenin-dependent CL compared to stimulated control cells, whereas luminol-dependent CL increased slightly by 0.01 mmol/L sulphite and decreased then continuously. Sulphite concentrations above 1 mmol/L decreased both lucigenin- and luminol-dependent CL of resting and PMA- or zymosan-stimulated neutrophils. Lucigenin-dependent CL of sulphite-treated and subsequently stimulated neutrophils was strongly inhibited by extracellularly added superoxide dismutase, whereas luminol-dependent CL was markedly reduced by the MPO inhibitor azide. The intracellular activity of MPO in neutrophils stimulated with PMA in the presence of sulphite (2 mmol/L) was reduced by 55%. Sulphite (0.1 mmol/L) also inhibited strongly the activity of MPO in a cell-free system. These results indicate that micromolar concentrations of sulphite exert a stimulating effect on the O production of neutrophils extracellularly, but have an inhibitory effect on MPO-catalysed reactions intracellularly.     

Time-resolved chemiluminescence study of the TiO2 photocatalytic reaction and its induced active oxygen species.

The time-resolved chemiluminescence (CL) method has been applied to study the TiO(2) photocatalytic reaction on a micros-ms timescale. The experimental set-up for time-resolved CL was improved for confirmation of the unique luminol CL induced by the photocatalytic reaction. The third harmonic light (355 nm) from an Nd:YAG laser was used for the light source of the TiO(2) photocatalytic reaction. Luminol CL induced by this reaction was detected by a photomultiplier tube (PMT) and a preamplifier was used for amplifying the CL signal. Experimental conditions affecting the photocatalytically induced CL were discussed in detail. The involvement of active oxygen species such as .OH, O(2) (.-) and H(2)O(2) in the CL was examined by adding their scavengers. It is concluded that .OH was greatly involved in the CL on a micros-ms timescale, especially in time periods <100 micros after illumination of the pulse laser. On the other hand, CL generated by O(2) (.-) began to increase after 100 micros and became dominant after 2.5 ms. A small part of the CL might be generated by H(2)O(2) on the whole micros-ms timescale. A CL reaction mechanism related with .OH and dissolved oxygen was proposed to explain the photocatalytically induced luminol CL on a micros-ms timescale, especially in periods <100 micros.     

Mathematical model for the luminol chemiluminescence reaction catalyzed by peroxidase

A kinetic model that accurately describes intensity vs. time reaction profiles for the chemiluminescence reaction between luminol and hydrogen peroxide, as catalyzed by horseradish perioxdase, is derived and evaluated. A set of three differential equations is derived and solved to provide intensity time information for the first 200 seconds of the reaction. The model accurately predicts intensity-time profiles when literature values are used for all but one of the reaction rate constants. Furthermore, the model predicts a nonlinear curve for plots of light intensity versus the initial hydrogen peroxide concentration. Experimental data confirm that such plots are nonlinear. Finally, a linear double-reciprocal plot is predicted by the model and the experimental data verify this relationship. (c) 1993 Wiley & Sons, Inc.     

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.     

Sensitive determination of bromhexine hydrochloride based on its quenching effect on luminol/H2O2 electrochemiluminescence system

In this paper, the electrochemiluminescence (ECL) behavior of luminol/H₂O₂ system in the presence of bromhexine hydrochloride (BrH) was investigated. It was found that the ECL intensity of luminol/H₂O₂ system on a platinum electrode could be intensely quenched by BrH owing to the scavenging superoxide radical ability of BrH, and therefore the sensitive determination of BrH was possible. Under optimal conditions, the quenched ECL intensity was linear to the concentration of BrH in a wide range of 0.08 to 500 μM, with a detection limit of 0.02 μM (signal‐to‐noise ratio (S/N) = 3). This ECL method possessed the merits of rapid, simple and sensitive, and was successfully applied to the BrH quantification in pharmaceutical preparations with satisfactory recoveries of 91.0 ± 4.0 to 106.5 ± 3.4%. The possible route of the quenched ECL of luminol/H₂O₂ in the presence of BrH was also discussed.     

Effect of different inhibitors on the intracellularly and extracellularly generated chemiluminescence induced by formylmethionyl-leucyl-phenylalanine in polymorphonuclear leukocytes. Cellular response in the presence of mannitol,benzoate, taurine,indomethacin and NDGA

When polymorphonuclear leukocytes (PMNL) interact with the soluble stimulus formylmethionyl-leucyl-phenylalanine (FMLP), the cells increase their production of oxidative metabolites. This increased production can be measured as lumino-amplified light emission or chemiluminescence (CL). In the present report, experimental systems which allow a quantitation of extracellularly and intracellularly generated metabolites have been used, and the effect of mannitol, benzoate, taurine, indomethacin and nordihydroguaiaretic acid has been investigated. The presence of the hypochlorous acid scavenger taurine had no effect on the intracellular response, whereas the extracellular response was reduced with around 50%. The hydroxyl radical scavenger mannitol had only minor effects on the response, whereas benzoate, another hydroxyl radical scavenger, reduced the extracellular response with around 50% and the intracellular response with more than 90%. Indomethacin, an inhibitor of arachidonic acid metabolism, did not influence the response, whereas NDGA, also an inhibitor of the arachidonic acid metabolism, totally abolished both the extracellular and the intracellular response. The use of scavengers/inhibitors as a means of determining the mechanisms of light emission, and the origin of chemiluminescence produced by neutrophils stimulated by FMLP is discussed.     

New luminol chemiluminescence reaction using diperiodatoargentate as oxidate for the determination of amikacin sulfate

A new chemiluminescence (CL) reaction between luminol and diperiodatoargentate {K₂ [Ag (H₂IO₆) (OH) ₂]} was observed in alkaline medium. The CL intensity could be greatly enhanced by amikacin sulfate. Therefore a new CL method for the determination of amikacin sulfate was built by combining with flow injection technology. A possible mechanism of the CL reaction was proposed via the investigation of the CL kinetic characteristics, the CL spectrum and the UV absorption spectra of some related substance. The concentration range of linear response was 5.1 × 10^?8 to 5.1 × 10^?6 mol L^?1 with a detection limit of 1.9 × 10^?8 mol L^?1 (3σ). The proposed method had good reproducibility with a relative standard deviation of 2.8% (n = 7) for 5.1 × 10^?7 mol L^?1 of amikacin sulfate. It was successfully applied to determine amikacin sulfate in serum. Copyright © 2009 John Wiley & Sons, Ltd.     

Interaction of metals with peroxidase--mediated luminol-enhanced, chemiluminescence (PLmCL).

The peroxidase-mediated luminol-enhanced chemiluminescence (PLmCL) method has been used to study the in vitro effect of contaminants such as heavy metals on the reactive oxygen species production by immunocytes. We were interested to know whether metals could directly affect peroxidase-mediated luminescence, taking horseradish peroxidase (HRP) as a model enzyme, since this could contribute to the inhibition of immunocyte LmCL. Copper inhibited PLmCL in a dose-dependent manner, while cadmium, iron, silver and lead only partly decreased the signal in the concentration range tested. In contrast, zinc enhanced the signal at high concentrations. Eventually, chromium, mercury and aluminium did not affect PLmCL. It is suggested that these effects reflect the ability of the metals to interact with the active site of the peroxidase. These results demonstrate that such interactions have to be considered when interpreting the effects of metals on immunocytes using the LmCL method.     

The assaying of haemoglobin using luminol chemiluminescence and its application to the dating of human skeletal remains

The luminol chemiluminescence reaction has, for some time, been used as a tool for the detection of haemoglobin at crime scenes. More recently, the luminol test has been suggested as a possible tool for estimating the post‐mortem interval (PMI) of skeletal remains. The preliminary results from the following study indicate that the chemiluminescent luminol test is a relatively easy and economical method for distinguishing between remains of medico‐legal (≤100 years) and historical (>100 years) interest. The femur was the preferred bone for PMI measurements using the luminol test, due to its robustness and relative resistance to diagenesis. Initial results suggest that bone that was historical in nature, produced a demonstrably weaker reaction than that of medico‐legal interest. These results suggest that the luminol test is a promising technique, albeit with some limitations, for the assessment of skeletal material that may be potentially of medico‐legal interest. Copyright © 2009 John Wiley & Sons, Ltd.     

The photodynamic effect: the comparison of chemiexcitation by luminol and phthalhydrazide

The presence of light, oxygen and photosensitizer (organic dye) is required for the photodynamic effect. Light and photosensitizer are harmless by themselves, but when combined with oxygen, reactive oxygen species (ROS) can be produced. This photodynamic effect is used in photodynamic therapy (PDT); the production of ROS as lethal cytotoxic agents can inactivate tumor cells. However, during PDT, there are many difficulties, so it is not possible to excite the photosensitizer using a laser, a source of light at the wavelengths specific to the photosensitizer (in visible region of the spectrum). Chemiluminescence is the light emission as a result of a chemical reaction. It is possible to use a chemiluminescent mixture to excite the photosensitizer even if the light emission does not conform to the absorption maximum of the photosensitizer. Luciferin and luminol have been used as chemiluminescent compounds (energizers) for the excitation of the photosensitizers. The aim of this work was to compare the chemiexcitation of some selected photosensitizers (e.g. fluorescein, eosin, methylene blue, hypericin and phthalocyanines) by chemiluminescent mixtures containing luminol (high chemiluminescent quantum yield) or phthalhydrazide (low chemiluminescent quantum yield) on some Gram‐positive (Enterococcus faecalis, Staphylococcus aureus) and Gram‐negative (Pseudomonas aeruginosa, E. coli) bacteria and some cell lines (NIH3T3 and MCF7). The efficiency of the chemiexcitation was dependent on the kind of the photosensitizer and on the type of the bacterial strain or cell line and was independent of the energizers. Copyright © 2010 John Wiley & Sons, Ltd.     

Chemiluminescence of lucigenin is dependent on experimental conditions.

The aim of the study was to test the effect of experimental conditions such as light radiation and temperature on chemiluminescence (CL) of 10(-2)-10(-5) mol/L lucigenin dissolved in various types of solvents. Irradiation by UV light (280, 297, 313 or 400 nm) induced a significant increase in CL of lucigenin dissolved in borate buffer. This effect was the most obvious for 10(-2)-10(-3) mol/L lucigenin. All wavelengths used had a similar effect. UV irradiation did not induce changes in the CL activity of lucigenin dissolved in dH2O or in dimethyl sulphoxide (DMSO). Different results for various solvents were not dependent on pH. The CL activity of 10(-2) mol/L and 10(-3) mol/L lucigenin dissolved in borate buffer increased depending on the solution temperature (25 degrees C, 30 degrees C, 37 degrees C or 40 degrees C) already at the beginning of the analysis, with a further increase during 16 h incubation period. It can be summarized that temperatures higher than 25 degrees C and intensive light irradiation are among those factors which significantly affect the result of analysis when lucigenin is used as a luminophor.     

The forensic use of luminol chemiluminescence to detect traces of blood inside motor vehicles.

The luminol test for blood was carried out on a set of interior fittings and surfaces inside three different makes of modern motor car. The surfaces and fittings provided little interference with the test for blood, although there was some detectable chemiluminescence when the test was applied to blood-free material from a seatbelt, a boot-lining and a gear-knob. The case with which haemoglobin samples could be washed off interior car surfaces was also examined for seat fabrics, carpets, roof-linings and various other plastic interior surfaces. A standard wash with water alone was not very effective and removed only ca. 50% of the haemoglobin. A standard wash with soapy water or with a proprietary multipurpose car cleaner removed ca. 90% of the haemoglobin from the tested surface. The effect of high car interior temperatures on haemoglobin samples that were subsequently used in the luminol test was also examined. It was shown that the sensitivity of the luminol test was not decreased but was increased by the prior heating of a haemoglobin sample. This effect was attributed to the thermal conversion of haemoglobin to the more brighter catalyst for chemiluminescence, methaemoglobin. The enthalpy of this conversion in the solid state was found to be 14.1 kJ/mol.