Luminol Concentration Research Articles

Experimental model for the study by chemiluminescence of the activation of isolated equine leucocytes

The activation of human polymorphonuclear leucocytes (the respiratory burst) can be studied by measuring their chemiluminescent response. This technique was adapted to equine leucocytes to investigate the effects of cell number, activator concentration, enhancers of chemiluminescence, pH, temperature and inhibitors. Leucocytes were isolated from citrated blood from healthy horses and chemiluminescence was measured with a Bio-Orbit luminometer sensitive to 900 nm light. The optimal cell density for the maximal chemiluminescent response ranged from 10 6 to 10 7 leucocytes 600 μl −1. Chemiluminescence increased as a function of temperature, and the concentrations of luminol, lucigenin and phorbol myristate acetate ( pma), and was pH related (optimal pH value = 8·0 for lucigenin and 8·5 for luminol). The inhibition of chemiluminescence by 5 × 10 −5M azide was 88 per cent for luminol and 37 per cent for lucigenin. Superoxide dismutase (100 iu) totally inhibited the chemiluminescence response. Approximately 30 per cent variability in chemiluminescence was observed under the same assay conditions, depending on the origin of the leucocytes. Based on these results, the conditions selected for the measurement of equine leucocyte chemiluminescence were: 10 6 to 10 7 leucocytes 600 μl −1, 1 × 10 −6M pma, 1 mM luminol or 0·4 mM lucigenin, physiological pH (7·4) and physiological temperature (37·8°C). These conditions were similar to those used for measuring the chemiluminescent response of human leucocytes.

The role of antioxidants in the prevention of t-butyl hydroperoxide-induced chemiluminescence.

An experimental system which assesses the antioxidant potential of ascorbic acid, glutathione, uric acid, and taurine was developed. The system comprised hemoglobin, luminol, t-butyl hydroperoxide, and different concentrations of antioxidants in TRIS-HCl buffer (pH 7.4). Control assays were performed by excluding antioxidants. Chemiluminescence was detected using a liquid scintillation counter in single photon mode. All antioxidants, when applied in the appropriate concentrations, decreased the maximum chemiluminescence values. The minimum concentrations which decreased the chemiluminescence values were defined for each of the antioxidants.

Electrochemiluminescence detection of glucose oxidase as a model for flow injection immunoassays

Glucose oxidase was covalently attached to aminosilanized indium tin oxide coated glass wafers and these were used as working electrodes in an electrochemiluminescence flow injection analyser as a model for enzyme immunoassays. Light from the chemiluminescent reaction between enzymatically produced hydrogen peroxide and electrochemically oxidized luminol was detected and the effect of pH and luminol concentration on this reaction was determined. Repetitive assays for glucose were carried out to investigate the surface chemistry. Results suggested that electrochemiluminescence enzyme immunoassays with glucose oxidase as the antibody label could be carried out.

TNF-alpha induces peroxynitrite-mediated depletion of lung endothelial glutathione via protein kinase C.

We tested the hypothesis that tumor necrosis factor-alpha (TNF) induces a peroxynitrite (ONOO-)-mediated depletion of glutathione via a protein kinase C (PKC)-dependent mechanism in pulmonary artery endothelial monolayers (PAEM). PAEM were incubated with TNF (1,000 U/ml) for 6 and 18 h. The PAEM were assayed for ONOO(-)-dependent changes in the concentration of luminol, free glutathione [Gfree; i.e., reduced glutathione and oxidized glutathione (GSSG)] and GSSG. TNF treatment decreased luminol and Gfree, and increased GSSG and GSSG/Gfree, compared with treatment with control media. The TNF-induced effects were prevented by co-incubation with the nitric oxide synthase inhibitors NG-monomethyl-L-arginine (1 mM), NG-nitro-L-arginine methyl ester (1 mM), or NG-nitro-L-arginine (1 mM). In addition, the TNF-induced effects were prevented by superoxide dismutase (10 U/ml), which removes O2-, and by urate (0.5 mM) and L-cysteine (3 mM), putative scavengers of ONOO-. The treatment of PAEM with the PKC activator phorbol 12-myristate 13-acetate (PMA, 1 microM) induced similar alterations in luminol and glutathione as TNF. TNF and PMA induced a protein of similar molecular weight (approximately 90 kDa) in the focal contact-rich fraction of PAEM lysate. TNF- and PMA-induced effects were prevented with the specific PKC inhibitor calphostin C (1 microM). The data indicate that TNF-induced PKC activation mediates ONOO- generation, which results in the oxidation and depletion of glutathione in PAEM.

Internal enzyme fiber-optic biosensors for hydrogen peroxide and glucose

The response characteristics and ideal operating conditions are described for a novel fiber-optic internal enzyme biosensor for hydrogen peroxide. The sensing mechanism involves hydrogen peroxide entering an enzyme containing internal solution by crossing a gas-permeable membrane. Once in the internal solution, the enzyme horseradish peroxidase (HRP) catalyzes the chemiluminescent reaction between hydrogen peroxide and luminol. The resulting light intensity is monitored and related to the analyte concentration. Sensor response properties strongly depend on the composition of the internal enzyme solution. Specifically, the magnitude, rate, and stability of the sensor response depend on the pH, buffer salts, and the concentrations of luminol and horseradish peroxidase. Under optimal conditions, the steady-state signal is linearly related to the square of the hydrogen peroxide concentration according to the kinetics of the HRP catalyzed reaction. The resulting hydrogen peroxide biosensor is used as the internal sensing element of a glucose biosensor by immobilizing glucose oxidase on the outer surface of the gas-permeable membrane. This glucose biosensor possesses a linear dynamic range from 2 to 18 mM with response times shorter than 60 s. Accurate measurements of glucose in blood are demonstrated.

In vitro phagocytosis assay of nano- and microparticles by chemiluminescence. I. Effect of analytical parameters, particle size and particle concentration

Abstract Chemiluminescence (CL) was used to study the uptake of differently sized model drug carriers (polystyrene latex particles) by human granulocytes. The polystyrene particles were incubated with the granulocytes on microtitre plates and the CL monitored for a period of 3 hours. The sensitivity of the chemiluminescence (CL) assay could be increased by optimization of the luminol concentration, cell number and particle number (mass) per well. The intensity (uptake)/time profiles were characterized by the intensity I max (uptake velocity) and the area under the curve (AUC, total uptake of particles). The reproducibility within one cell isolation was good. To compare CL measurements from different isolations internal standards were included to compensate for biological variations in the granulocyte function. The total uptake of particles increased with increasing particle size and at low to medium particle concentrations. It levelled or even decreased at high particle concentrations reducing the sensitivity of the assay. The AUC correlated with the total mass of phagocytosed particles and could therefore be used to compare the uptake of nano- and microparticles differing in size.

Interaction of luminol with the oscillating system hydrogen peroxide-potassium thiocyanate-copper sulfate-sodium hydroxide

Two types of oscillating chemiluminescence are observed when luminol is added to the oscillating system H 2 O 2 -KSCN-CuSO 4 -NaOH. Narrow bursts of light, with intensity proportional to the concentration of luminol, occur at the same time as the sharp increase in the concentration of a copper peroxide complex that is a key species in the oscillation of the luminol-free system. A second set of broad, low-intensity oscillations is apparent at luminol concentrations below 10 −4 M. These are ascribed to the slower reaction of luminol with superoxide. A skeleton mechanism for the action of luminol is proposed, but testing it will require refinement of the mechanism of the H 2 O 2 -KSCN-CuSO 4 -NaOH oscillator

Chemiluminescence detection of haem-containing microsomal proteins

In the purification and characterization of cytochrome P-450 isoenzymes from rat liver microsomes, a luminol-mediated chemiluminescence in microsomes after addition of peroxides was discovered. The pH dependence of the kinetics of the chemiluminescence reaction was investigated and the reactions conditions were optimized with respect to the peroxide and luminol concentrations. The detection limits appear to be a factor of 10 3 lower than those obtained with other techniques. The speed of detection was also improved substantially. The nature of the strong chemiluminescence was further investigated by (partial) purification of the cytochrome P-450 isoenzymes. It appeared that the chemiluminescence activity was closed related to the presence of cytochrome P-450.

Chemiluminescence of rainbow trout (Salmo gairdneri) phagocytes and factors affecting their response.

Factors affecting the chemiluminescent (CL) response of rainbow trout (Salmo gairdneri) phagocytes were investigated. The cells were stimulated by the addition of Vibrio anguillarum or zymosan to the cultures in the presence of luminol as an amplifier. The CL was dose-dependent on the concentrations of luminol and zymosan, and the number of phagocytes and V. anguillarum. Living V. anguillarum elicited rapid and strong CL of phagocytes regardless of opsonization of the bacteria with fresh, normal rainbow trout serum. Opsonized formalin-killed bacteria induced a slow, low CL response, and no CL was elicited by non-opsonized, killed bacteria. Phagocytes isolated from the peritoneal cavity, head kidney, spleen and peripheral blood showed similar CL patterns.

Chemiluminescence Accident Dosimetry with Drugs and Other Solid Materials

Abstract It has been shown that it is possible to measure radiation doses using the chemiluminescence (CL or lyoluminescence) of different solids. The purpose of this study was to find out whether chemiluminescence from drugs, as well as from other solid materials collected in the neighbourhood of a radiation accident, could be used to estimate the radiation dose to the people involved in the radiation accident. CL glow curves of 18 gamma ray irradiated powdered drugs were investigated. For seven selected pharmaceuticals, optimisation of the CL yield according to sample mass, pH of solution and concentration of luminol was performed. We found that with many powdered drugs, doses as low as about 1 Gy can be measured. The CL of 10 irradiated solid materials (hair, finger-nail clippings, chalk, cellulose, etc.) was investigated using luminol solutions. The results showed that it would be very difficult to observe a radiation induced chemiluminescence with doses of about 1 Gy, when using solid materials of minimum solubility in the luminol sensitiser system.

Chemiluminescence Accident Dosimetry with Drugs and Other Solid Materials

It has been shown that it is possible to measure radiation doses using the chemiluminescence (CL or lyoluminescence) of different solids. The purpose of this study was to find out whether chemiluminescence from drugs, as well as from other solid materials collected in the neighbourhood of a radiation accident, could be used to estimate the radiation dose to the people involved in the radiation accident. CL glow curves of 18 gamma ray irradiated powdered drugs were investigated. For seven selected pharmaceuticals, optimisation of the CL yield according to sample mass, pH of solution and concentration of luminol was performed. We found that with many powdered drugs, doses as low as about 1 Gy can be measured. The CL of 10 irradiated solid materials (hair, finger-nail clippings, chalk, cellulose, etc.) was investigated using luminol solutions. The results showed that it would be very difficult to observe a radiation induced chemiluminescence with doses of about 1 Gy, when using solid materials of minimum solubility in the luminol sensitiser system.

Variables in xanthine oxidase-initiated luminol chemiluminescence: Implications for chemiluminescence measurements in biological systems

We tested the effects of generally used chemiluminescence inhibitors on an example of luminol chemiluminescence elicited by xanthine oxidase/hypoxanthine system, and attempted to assess their capabilities in discovering the reaction pathways leading to chemiluminescence. Luminol itself is a xanthine oxidase inhibitor and its concentration affects the reaction mechanism. Maximal chemiluminescence response was observed at luminol concentration inhibiting urate production. Chemiluminescence was totally inhibited by superoxide dismutase, the inhibition by catalase depended on luminol concentration. Ferricytochrome c, a detector of superoxide, either stimulated or inhibited chemiluminescence in a concentration-dependent manner. Chemiluminescence was highly stimulated by peroxidases. A pronounced inhibition of chemiluminescence was caused by chelators; 1 m m desferal and 0.01 m m diethyldithiocarbamate. It is suggested that measurement of luminol chemiluminescence is not a suitable method for discrimination among individual reactive oxygen species and their quantitative determination in biological systems.

Aqualuminescence of alkaline luminol in the presence of fluorescein

The light yield from both luminol and fluorescein is studied at a fixed concentration of luminol and by varying the concentration of fluorescein in 3 × 10 −2 M basic solution by direct chemical reaction as well as by aqualuminescence technique. The emission spectra of chemiluminescence and aqualuminescence of the alkaline luminol-fluorescein mixture are recorded on a Fuoss spectrograph. A 4-fold increase in the aqualuminescence intensity of luminol has been observed in the presence of fluorescein as compared to that of pure luminol. The results are explained on the basis of reactions of colour centres with the activators.

Simultaneous detection of native and luminol-dependent luminescence of stimulated human polymorphonuclear leukocytes

A method for investigating the cellular response of polymorphonuclear leukocytes to various stimuli was introduced using simultaneously native (luminol-independent) and luminol dependent luminescence as an indicator for myeloperoxidase (MPO)-H2O2-halide and O2- mediated reactions. In experimental systems containing low concentrations of luminol the total light emission was separated into contributions of native and luminol-dependent luminescence by making use of the different spectral behaviour of the two kinds of luminescence. Consequently the MPO-H2O2-halide system could be distinguished from the O2- dependent system by interpreting the recorded temporal traces of the emitted light.

A sensitive lipoxygenase assay based on chemiluminescence

The presence of luminol during lipoxygenase I-catalyzed oxygenation of linoleic acid caused a complex pattern of emission of chemiluminescent light. During the aerobic phase of the lipoxygenase reaction, the rate of formation of light was dependent on the enzyme, linoleic acid, and luminol concentrations. The correlation was linear between the rate of chemiluminescent light formation and lipoxygenase I concentration in the range 2–22 mU/ml of enzyme in the presence of saturation concentrations of linoleic acid (0.4 m m) and luminol (0.04 m m). Luminol and derivatives of isoluminol were all applicable as chemiluminescent probes without an inhibitory effect on the oxygenation rate itself. Free radicals that dissociated from the enzyme-substrate complex in the presence of oxygen in a constant ratio to the oxygenation rate are suggested to be responsible for luminol oxidation.

Luminol-induced neutrophil chemiluminescence

Our studies suggest that luminol directly enhances the chemiluminesce of human neutrophils. We show that a significant peak in chemiluminescence production in a particle-free system occurs between and 5 and 15 min following exposure of cells to micromolar concentrations of luminol. The response is directly related to dose over a wide range of luminol concentrations and can be inhibited by superoxide dismutase (90%), catalase (100%) and sodium azide (40%). Evidence is presented which suggests that the effect of luminol eliciting a peak in neutrophil chemiluminescence is mediated within intact cells rather than at the cell membrane. Luminol may produce a peak in chemiluminesce by stimulating very low levels of hexose monophosphate shunt activity and superoxide generation or it may simply amplify light production generated by the production of excited oxygen radicals resulting from surface interactions.

Lyoluminescence of sensitized glucose

Lyoluminescence studies have acquired considerable significance in recent years due to its potential as a cheap, convenient and versatile method for radiation dosimetry. It has advantages over other conventional techniques due to tissue equivalence of organic lyoluminescent materials and small size in which this type of dosimeter can be used. It is particularly attractive for therapeutic dosimetry. The paper reports the performance of glucose crystallised with known concentrations of sensitizer luminol. It also gives the relative performance of various lyoluminescence materials of similar type being studied currently.

Concentration effects in the chemiluminescence of luminol

A 10 −2 M basic solution of luminol undergoes an oxidation reaction accompanied by a chemiluminescence emission with λ max = 480 nm. By comparing the chemiluminescence spectra with the relevant fluorescence spectra at luminol concentrations of 10 −2 and 10 −3 M it can be concluded that the emitting species is the 3-aminophthalate ion. This observation is in agreement with the accepted mechanism for chemiluminescence.

The dye-sensitized photo-oxidation chemiluminescence of luminol.

Abstract— The chemiluminescence reaction of luminol has been investigated using conditions of methylene blue photosensitized oxidation. The quantum yields of chemiluminescence obtained were dependent upon temperature and the concentrations of luminol and base; and under the optimum conditions of high temperature and low luminol concentration, the value of the quantum yield approached that for the chemical reaction where the oxidant is hydrogen peroxide and catalyst. An analysis of the results suggests that it is not the primary species produced on photosensitization which is responsible for the chemiluminescent reaction, but a species produced by reaction of the primary species with base.