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.     

Influence of the storage time and the method of stimulation on whole blood chemiluminescence

The ultra‐weak light, chemiluminescence (CL), of stimulated leukocytes is a well‐known phenomenon. Parameters of this CL are modified by many factors including laboratory procedures. The order of stimulation and enhancement (two possibilities) and two concentrations of luminol create four types of procedure, which were accomplished in five sample storage ‘time points’. We received the strongest signals of CL using higher concentrations of luminol (and DMSO), but only when stimulation (FMLP) was used before enhancement (luminol); luminol used before FMLP strongly inhibited CL. For lower luminol concentration (and DMSO), the order of stimulation and enhancement was of no importance. There were comparable but weaker signals of CL in this case. We received stronger signals with storage time for all procedures. It may be dependent on the priming of phagocytes by releasing cell factors. Stimulation (FMLP) before enhancement (luminol) eliminates the inhibitory effect of DMSO on CL. Copyright © 2002 John Wiley & Sons, Ltd.     

Laccase–luminol chemiluminescence system: an investigation of substrate inhibition

Chemiluminescence (CL) reactions are widely used for the detection and quantification of many types of analytes. Laccase has previously been proposed in CL reactions; however, its light emission behaviour has not been characterized. This study was conducted to characterize the laccase–luminol system, determine its kinetic parameters, and analyze the effects of protein and OHˉ concentration on the CL signal. Laccase from Coriolopsis gallica was combined with different concentrations of luminol (125 nM to 4 mM), and the enzyme kinetics were evaluated using diverse kinetic models. The laccase–luminol system was able to produce CL without an intermediate molecule, but it exhibited substrate-inhibition behaviour. A two-site random model was used and suggested that when the first luminol molecule was bound to the active site, laccase affinity for the second luminol molecule was increased. This inhibition effect could be avoided using a low luminol concentration. At 5 μM luminol concentration, 1 mg/ml (0.13 U) laccase is needed to achieve nearly 90% of the maximum CL signal, suggesting that the available luminol could not bind to all active sites. Furthermore, the concentration of NaOH negatively affected the CL signal. The laccase–luminol system represents an alternative to existing CL systems, with potential uses in molecular detection and quantification.     

Enhanced chemiluminescence of the luminol–AgNO3 system by Ag nanoparticles

The oxidation reaction of luminol with AgNO₃ can produce chemiluminescence (CL) in the presence of silver nanoparticles (NPs) in alkaline solution. Based on the studies of UV‐vis absorption spectra, photoluminescence (PL) spectra and CL spectra, a CL enhancement mechanism is proposed. The CL emission spectrum of the luminol–AgNO₃–Ag NPs system indicated that the luminophore was still 3‐aminophthalate. On injection of silver nanoparticles into the mixture of luminol and AgNO₃, they catalysed the reduction of AgNO₃ by luminol. The product luminol radicals reacted with the dissolved oxygen, to produce a strong CL emission. As a result, the CL intensity was substantially increased. Moreover, the influences of 18 amino acids, e.g. cystine, tyrosine and asparagine, and 25 organic compounds, including gallic acid, tannic acid and hydroquinone, on the luminol–AgNO₃–Ag NPs CL system were studied by a flow‐injection procedure, which led to an effective method for detecting these compounds. Copyright © 2011 John Wiley & Sons, Ltd.     

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

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

Comparison of chemiluminescence from luminol solution and luminol–TiO2 suspension after illumination of a 355 nm pulse laser

Chemiluminescence (CL) from luminol solution and luminol–TiO₂ suspension after illumination of a 355 nm pulse laser is compared. Both the CL systems showed the CL spectra with maximum wavelength of 430 nm, suggesting that the emission was from the excite state of 3‐aminophthalate ion. The TiO₂ photocatalytically induced luminol CL could be separately detected either when the pulse laser power was smaller than 0.15 mJ/pulse or a slit was placed beyond ?2–2 mm in the vertical direction of the laser beam. The TiO₂ photocatalytically induced luminol CL intensity was linear to the laser power, while that of the 355 nm pulse laser‐induced was nonlinear. A log–log plot between the 355 nm pulse laser‐induced luminol CL intensity and laser power showed a near‐linear regression fit with a slope of 2.11, suggesting that a two‐photon absorption process of luminol was present in the 355 nm pulse laser‐induced luminol CL. Adsorbed oxygen on the surface of TiO₂ seemed to greatly contribute to the photocatalytically induced CL. Copyright © 2010 John Wiley & Sons, Ltd.     

Flow injection analysis of riboflavin with chemiluminescence detection using a N-halo compounds-luminol system.

A chemiluminescent method for the determination of riboflavin is described. The method is based on the chemiluminescence (CL) generated during the oxidation of luminol by N-bromosuccinimide (NBS) and N-chlorosuccinimide (NCS) in alkaline medium. It was found that riboflavin could greatly enhance this CL intensity when present in the luminol solution. Based on this observation, a new flow-injection CL method for the determination of riboflavin is proposed in this paper. The detection limits were 7.5 ng/mL and 3.5 ng/mL of riboflavin for the NBS- and NCS-luminol CL systems, respectively. The relative CL intensity was linear, with riboflavin concentration in the range 19-600 ng/mL and 600-2000 ng/mL for the NBS-luminol CL system, and 12-200 ng/mL and 200-2000 ng/mL for the NCS-luminol CL system. The results obtained for the assay of pharmaceutical preparations compared well with those obtained by the official method and demonstrated good accuracy and precision.     

Myeloperoxidase-Based Chemiluminescence of Polymorphonuclear Leukocytes and Monocytes

Luminol and lucigenin chemiluminescence (CL) responses produced by separated human blood polymorphonuclear leukocytes (pmn) and monocytes (mono) have been studied following stimulation with the surface-receptor agonist fMLP (a synthetic chemotactic peptide) and the protein kinase C activator phorbol myristate acetate (PMA). Pmn produced two- to threefold the luminol CL and superoxide anion (O₂⁻) levels of mono; lucigenin CL was similar for both cell-types. The myeloperoxidase (MPO) inhibitor salicylhydroxamic acid (SHA) abrogated luminol but not lucigenin CL in both cell types, but did not further inhibit the already grossly subnormal luminol CL responses seen with MPO-deficient cells which produced normal lucigenin CL. SHA also profoundly inhibited the luminol CL response in a cell-free MPO–H₂O₂ system. Mono lucigenin CL does not appear to specifically measure O₂⁻ production. These data show that luminol CL provides a useful measure of pmn and also mono MPO activity. However, analysis of the effects of various reactive oxygen species (ROS) scavengers, assessed on phagocyte and cell-free CL systems (both MPO–H₂O₂ and superoxide generating) suggest that the luminol CL signal is not entirely dependent on MPO activity.     

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.     

Characterization of peroxidases in luminol chemiluminescence coupled with copper-catalysed oxidation of cysteamine

Hydrogen peroxide formed during the course of the copper(II)-catalysed oxidation of cysteamine with oxygen was continuously determined by a peroxidase (POD)-catalysed luminol chemiluminescence (CL) method. Horseradish peroxidase (HRP), lactoperoxidase (LPO) and Arthromyces ramosus peroxidase (ARP) were used as a CL catalyst. The respective PODs gave specific CL intensity-time profiles. HRP caused a CL delay, and ARP gave a time-response curve which followed the production rate of H₂O₂. LPO gave only a weak CL flash which decayed promptly. These differences of CL response curves could be explained in terms of the different reactivities of PODs for superoxide anion and the different formation rate of luminol radicals in the peroxidation of luminol catalysed by POD.     

Effects of inhibitors on chicken polymorphonuclear leukocyte oxygenation activity measured by use of selective chemiluminigenic substrates

Chicken heterophil polymorphonuclear leukocytes (CPMNLs) have NADPH oxidase activity, but lack myeloperoxidase (MPO). Stimulation of CPMNLs by phorbol 12-myristate 13-acetate or chicken opsonified zymosan results in luminol-dependent chemiluminescence (CL) activity, which is small relative to that of human peroxidase-positive neutrophils (HPMNLs), as well as lucigenin-dependent CL, comparable to HPMNL responses. Inhibitors were used to investigate and characterize the CL activity of CPMNLs. Inhibition constants were calculated, using Dixon inhibition analysis, or were reported as the concentration producing 50% inhibition of the magnitude of CL responses. Azide and cyanide are effective inhibitors of luminol CL in HPMNLs, although these peroxidase inhibitors do not inhibit either luminol or lucigenin CL of CPMNLs. Since these agents also inhibit eosinophil peroxidase, lack of inhibition of CPMNL CL indicates that the small percentages of peroxidase-positive eosinophils in CPMNL preparations are not responsible for the luminol CL observed. Iodoacetate and fluoride, pre-oxidase and pre-peroxidase inhibitors of glycolytic metabolism, effectively inhibit lucigenin and luminol CL activities in CPMNLs. Superoxide dismutase competitively inhibits lucigenin and luminol CL in CPMNLs, but catalase is an ineffective inhibitor. Although luminol is efficiently dioxygenated by a MPO-dependent mechanism in HPMNL, use of peroxidase-deficient CPMNLs indicates that this substrate does not exclusively measure peroxidase activity.     

Chemiluminescence study of active oxygen species produced by TiO2 photocatalytic reaction.

Two chemiluminescence approaches have been used for study of active oxygen species produced by the TiO2 photocatalytic reaction. One is based on flow injection analysis (FIA)-luminol chemiluminescence (CL); another is a time-resolved CL method. In the FIA-CL experiment, an UV-illuminated TiO2 suspension and water were passed into a mixing cell by two separate flow lines. Luminol solution was injected into the water flow line at different times. The injected luminol reacted with active oxygen species generated by the TiO2 photocatalytic reaction in a mixing coil and produced CL. It was found that the maximum CL was detected at the first injection of luminol. CL intensity decreased with time of injection. When the luminol was injected after 5 min, the CL intensity was almost unchanged. Addition of scavengers of active oxygen species indicated that the CL produced early in the 5 min was caused by O2- and H2O2, while CL after 5 min was only from H2O2. In the time-resolved CL, the third harmonic wavelength of Nd:YAG laser (355 nm) was used as a UV light source, and CL was detected by a PMT and recorded in a millisecond time scale using a digital oscilloscope. It was found that CL induced by the photocatalytic reaction increased with concentration of the TiO2 suspension. Scavengers of active oxygen species of *OH, O2- and H2O2 were added to study the involvement of the active oxygen species.     

Chemiluminescence flow biosensor for glucose using Mg-Al carbonate layered double hydroxides as catalysts and buffer solutions

In this work, serving as supports in immobilizing luminol reagent, catalysts of luminol chemiluminescence (CL), and buffer solutions for the CL reaction, Mg-Al-CO(3) layered double hydroxides (LDHs) were found to trigger luminol CL in weak acid solutions (pH 5.8). The silica sol-gel with glucose oxidase and horseradish peroxidase was immobilized in the first half of the inside surface of a clear quartz tube, and luminol-hybrid Mg-Al-CO(3) LDHs were packed in the second half. Therefore, a novel CL flow-through biosensor for glucose was constructed in weak acid solutions. The CL intensity was linear with glucose concentration in the range of 0.005-1.0mM, and the detection limit for glucose (S/N=3) was 0.1μM. The proposed biosensor exhibited excellent stability, high reproducibility and high selectivity for the determination of glucose and has been successfully applied to determine glucose in human plasma samples with satisfactory results. The success of this work has broken the bottleneck of the pH incompatibility between luminol CL and enzyme activity.     

Luminol‐, isoluminol‐ and lucigenin‐enhanced chemiluminescence of rat blood phagocytes stimulated with different activators

Luminol‐, isoluminol‐ or lucigenin‐enhanced chemiluminescence (CL) was used to measure the production of reactive oxygen species by rat blood leukocytes. Opsonized zymosan (OZ), phorbol‐12‐myristate‐13‐acetate (PMA), calcium ionophore A23187 (Ca‐I) or N‐formyl‐Met‐Leu‐Phe (fMLP) were used as activators. The CL signal of isolated blood leukocytes decreased in rank order of luminol > isoluminol > lucigenin. The kinetic pro?les of luminol‐ and isoluminol‐enhanced CL were similar upon stimulation by each activator tested. The remarkably higher luminol and isoluminol CL responses were obtained after OZ stimulation when compared with other activators. However, when lucigenin was used, the PMA‐ and OZ‐stimulated CL were comparable. The presence of plasma increased OZ‐activated CL because of the enhanced phagocytosis of OZ. This was demonstrated by determining the phagocytosis of the ?uorescent OZ using a ?ow cytometer. In contrast, the presence of plasma decreased PMA‐activated CL, due to the antioxidant properties of plasma as determined by the CL method. As far as whole blood is concerned, only OZ activated luminol‐enhanced CL was reliable. Blood volumes over 5 µL decreased CL activity due to the scavenging ability of erythrocytes. The results suggest that 0.5 µL whole blood is suf?cient for routine luminol‐enhanced CL analysis of whole blood oxidative burst in rats. Copyright © 2004 John Wiley & Sons, Ltd.     

Development of a highly sensitive and selective microplate chemiluminescence enzyme immunoassay for the determination of free thyroxine in human serum

A microplate chemiluminescence enzyme immunoassay (CLEIA) with high sensitivity, selectivity and reproducibility was developed for the determination of free thyroxine (FT4) in human serum. A competitive assay has been utilized with horseradish peroxidase (HRP) labeled thyroxine analog in the chemiluminescence (CL) detection. The CL signal produced by the emission of photons from luminol was directly proportional to the amount of analyte. The linear range was 0.45-7.5 ng dL(-1 )and the detection limit was 0.09 ng dL(-1). Experimental conditions, such as temperature, pH, incubation time, titration level and other relevant variables upon the CL signal have been examined and optimized. A coefficient of variance of less than 16% was obtained for intra- and inter-assay precision. The present method has been successfully applied to the analysis of FT4 in human serum. The positive and negative coincidence ratios are satisfactory. Good correlations were obtained between the results by the proposed method and radioimmunoassay (RIA), as well as a Bayer ACS-180SE detection system.     

Differences in the reactivity of phthalic hydrazide and luminol with hydroxyl radicals

The reactivity of 5-amino-2,3-dihydro-phthalazine-1,4-dione (luminol) and phthalic hydrazide with hydroxyl radicals was studied. HO^·-radicals were generated by the Fenton reaction as well as by water radiolysis. Both luminol and phthalic hydrazide react with hydroxyl radicals under intense chemiluminescence (CL) emission. However, exclusively the CL arising from phthalic hydrazide oxidation can be quenched by competition (e.g. by the addition of carbohydrates), whereas luminol CL is enhanced.

The reactivities of both compounds with HO^·-radicals were further studied by time-resolved spectroscopy (pulse radiolysis), competition methods, NMR spectroscopy and mass spectrometry. Whereas only slight differences were detectable by pulse radiolysis, the analysis of competition kinetics in the presence of p-nitroso-dimethylaniline (NDMA) gave a two-fold-enhanced reactivity for luminol (4.8 × 10⁹l mol^-1 s^-1) in comparison to phthalic hydrazide (2.0 × 10⁹l mol^-1s^-1).

NMR and mass spectrometric analyses revealed significant differences in the reactivity of HO^·-radicals: whereas in luminol solutions hydroxylation of the aromatic ring system predominated, hydroxylated products were not detectable upon irradiation of phthalic hydrazide. A hypothetical mechanism is proposed which may explain the observed differences.     

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.     

Nitric oxide synthase inhibitors decrease human polymorphonuclear leukocyte luminol-dependent chemiluminescence

Nitric oxide synthase (NOS) inhibitors have been reported to modulate luminol-dependent chemiluminescence (CL) in rat macrophages, whereas the potent oxidant peroxynitrite (ONOO^-) was shown to react with luminol to yield CL in a cellfree system. We evaluated the role of the -arginine/NOS pathway in luminol CL by phorbol ester-activated human polymorpho-nuclear (PMN) leukocytes using the NOS inhibitors N^G-monomethyl- -arginine ( -NMMA) and N-iminoethyl- -omithine ( -NIO). Nitric oxide (·NO) release was determined by oxidation of oxymyoglobin. In addition, the effect of NOS inhibitors on superoxide anion O₂^-) production was measured. Luminol CL was notably diminished by -NMMA in a dose-dependent manner. Superoxide dismutase (SOD) also decreased luminol CL and -NMMA potentiated light emission decrease produced by SOD. Nitric oxide and O₂^·- production was significantly decreased by -NMMA; moreover, luminol-dependent CL but not O₂^·- production was attenuated by -NIO. These data suggest that products of catalytic activity of both ·NO synthase and NADPH oxidase are required to elicit maximal luminol CL in this system. These studies demonstrate that the NOS synthase pathway is involved in luminol CL by human PMN, and they suggest that ONOO would be an unrecognized mediator in this phenomenon.     

A stopped‐flow study of the dual chemiluminescence for the luminol–KIO4–Mn2+ system in strong alkaline solutions

A novel phenomenon of dual chemiluminescence (CL) was observed for the KIO₄–luminol–Mn²⁺ system in strong alkaline solutions using the stopped‐flow technique. Scavenging study of the reactive oxygen species (ROS) suggested that the two CL peaks originated from different CL pathways precipated by distinct ROS (O₂^? and ^?OH for the first peak, mainly ¹O₂ for the second peak). Generation of these ROS at different time intervals from the reactions involving IO₄^?, O₂, and Mn²⁺ and their subsequent reactions with luminol induced the intense CL emission. The relative intensity of the two CL peaks can be tuned over a wide range by varying the concentrations of Mn^2?, luminol and KIO₄. Because of the involvement of different ROS in each pathway, the two CL peaks could respond quite differently to various substances. Moreover, variation of the intensity ratio of the two CL peaks altered the relative proportions of the corresponding ROS, thereby changing their responses to a given substance. The dual CL emission acts like a pair of tunable probes and it is believed that this CL system has great potential in analytical applications. Copyright © 2010 John Wiley & Sons, Ltd.