Chemiluminescence luminol

Luminol chemiluminescence has also been recommended for measuring bacteria populations (304,305). The luminol—hydrogen peroxide reaction is catalyzed by the iron porphyrins contained in bacteria, and the light intensity is proportional to the bacterial concentration. The method is rapid, especially compared to the two-day period required by the microbiological plate-count method, and it correlates weU with the latter when used to determine bacteria... 

Assayed by the luminol chemiluminescence method (Puget and Michelson, 1974). One unit corresponds to 0.35 unit of the cytochrome c method (McCord and Fridvich, 1969). 

Lee, J., and Seliger, H. H. (1972). Quantum yields of the luminol chemiluminescence reaction in aqueous and aprotic solvents. Photochem. Pho-tobiol. 15 227-237. 

The overall reaction scheme of the luminol chemiluminescence in an aqueous medium is shown in Figure 1. The luminol oxidation leads to the formation of an aminophthalate ion in an excited state, which then emits light on return to the ground state. The quantum yield of the reaction is low ( 0.01) compared with bioluminescence reactions and the emission spectrum shows a maximum1 at 425 nm. 

Figure 1. Overall luminol chemiluminescence reaction in aqueous medium.

Bioluminescence and chemiluminescence are very powerful analytical tools, since in addition to the direct measurement of ATP, NAD(P)H or hydrogen peroxide, any compound or enzyme involved in a reaction that generates or consumes these metabolites can be theoretically assayed by one of the appropriate light-emitting reactions. Some of these possibilities have been exploited for the development of optical fibre sensors, mainly with bacterial bioluminescence and with luminol chemiluminescence. 

Observations Table 2 shows the levels of free radicals determined by luminol chemiluminescence, H202 levels, and membrane lipid peroxidation determined by conjugated dienes. Total free radicals significantly increased by 1.4 fold, hydrogen peroxide significantly decreased 1.3 fold, and lipid peroxidation increased significantly 1.5 fold. 

It has been established 106> that 3-aminophthalate is the emitting species in luminol chemiluminescence and that in all known cases of other cyclic hydrazides the corresponding dicarboxylate is the emitter (e.g. 97,107)). 

The maximum of luminol chemiluminescence emission is at 425 nm in aqueous and at 480 nm in DMSO-containing solvent. It was suggested that different anions of 3-aminophthalate were responsible for this phenomenon, namely 51 (in water) and 52 (in aprotic solvents). 

D. S. Bersis and J. Nikokavouras 108> came to the conclusion, on the basis of their chromatographic investigations of luminol preparations, that these two emission maxima of luminol chemiluminescence were actually due to impurities in luminol, not to different states of 3-aminophthalate. Recent investigations of 3-aminophthalate in solution 109)... 

Gorsuch and Hercules 109> stated that certain discrepancies between the fluorescence spectrum of 3-amino-phthalate dianion and the chemiluminescence spectrum of luminol are partly due to reabsorption of the shorter-wavelength chemiluminescence light by the luminol monoanion. These authors confirmed the results of E. H. White and M. M. Bursey 114> concerning the very essential solvent effect on luminol chemiluminescence the relative intensity of the latter in anhydrous DMSO/t-BuOK/ oxygen was found to be about 30,000 times that in DMSO/28 mole % water/potassium hydroxide/oxygen. 

The emission maxima of luminol chemiluminescence and of 3-amino-phthalate dianion in some solvents are listed in Table 3 ... 

Stopped-flow experiments of luminol chemiluminescence in the system luminol/pure DMSO/tert.butylate/oxygen 109> with independent variations of the concentrations of reactants confirmed the results obtained previously by E. H. White and coworkers 117> as to pseudo-first-order dependence of the chemiluminescence intensity upon each of the reactants. Moreover, the shapes of the decay curves obtained... 

The luminol chemiluminescence reactions can be divided into two classes depending on the oxidation conditions 115> ... 

Table 5. Luminol chemiluminescence quantum yields in different solvents with some oxidative systems (after J. Lee and H. H. Seliger 115>)...

The photosensitized results are from I.B.C. Matheson and J. Lee 118h It is seen that the quantum yields in photosensitized oxidation depend on the concentrations of luminol and base, and on temperature. At higher temperature (50°) and low luminol concentrations, the quantum yields reached those of hemin-catalyzed hydrogen peroxide oxidation of luminol in aqueous-alkaline solution. Primary products of the photosensitized oxidation are singlet oxygen (1Ag02) or a photoperoxide derived from methylene blue, but neither of these is directly responsible for the luminol chemiluminescence. 

Finally, we mention another experimental result which points to a radical mechanism in luminol chemiluminescence. 

As early as in his first paper on luminol chemiluminescence H. O. Albrecht 128> postulated that diazaquinone 63 ( dehydroluminol ) is in-... 

Seitz, Suydam, and Hercules 186> recently developed on the basis of luminol chemiluminescence a method for chromium-III ion determination which has a detection limit of about 0.025 ppb. The method is specific for free chromium-III ions as chromium-VI compounds have no catalytic effect and other metal ions can be converted to a non-catalytic form by complexing with EDTA, since the chromium-III complex of EDTA, which is in any case not catalytically active, is formed kinetically slowly 186>. To detect extremely small light emissions, and hence very small metal concentrations, a flow system was used which allows the reactants to be mixed directly in front of a multiplier. (For a detailed description of the apparatus, see 186>). 

Penicillin Enhancement of luminol chemiluminescent reaction, batch method 100 ng/mol 100 ng/mL-100 pg/mL 71, 72... 

When neutrophils are stimulated with fMet-Leu-Phe, they generate a bi-phasic luminol chemiluminescence response. The first phase of chemilumi-... 

The molecular species responsible for luminol and lucigenin chemiluminescence are not fully defined. Clearly, luminol chemiluminescence requires the combined activities of both the NADPH oxidase and myeloperoxidase inhibitors of either enzyme (e.g. diphenylene iodonium for the oxidase and salicylhydroxamic acid for myeloperoxidase) completely prevent luminol... 

Merenyi, G., Lind, J., Eriksen, T. E. (1990). Luminol chemiluminescence Chemistry, excitation, emitter. J. Biolum. Chemilum. 5, 53-6. 

These approaches have been used to show conclusively that the initial, low formation of DAG that occurs during activation with soluble agonists comes from PLC activity, and that the later, more sustained generation of DAG comes from PLD activity. Such experiments have also shown that primary alcohols can inhibit the activity of the NADPH oxidase under some conditions. When neutrophils are pretreated with cytochalasin B, primary alcohols are potent inhibitors of 02" secretion, and the kinetics of phosphatidic acid formation are rapid, peaking within about 20 s and coinciding with oxidase activation. However, in the absence of cytochalasin B, primary alcohols have little effect on the initiation of O2" secretion, but decrease the duration of oxidase activity they also inhibit the later phase of luminol chemiluminescence, which is largely intracellular, and the kinetics of phosphatidic acid formation closely parallel the kinetics of this intracellular oxidase activity (Fig. 6.20). Thus, in cytochalasin-treated cells, PLD is activated rapidly, and this activation is required for 02" secretion in the absence of cytochalasin, PLD is activated more slowly and its function is not for the activation of the oxidase, but rather for sustained (and intracellular) activity. 

Figure 6.20. Role of phospholipase D in NADPH oxidase activation. In (a) neimophils were preincubated with [3H]-alkyl-lyso-PAF (5 /iCi/ml) for 60 nun at 37 C. The cells were then washed twice with RPMI 1640 medium and finally resuspended at 2 x 10 cells/ ml in the presence ( ) and absence ( ) of 100 mM ethanol. The cells were then stimulated with 1 pM fMet-Leu-Phe and, at time intervals,aliquots were removed for analysis ofphos-phatidic acid ( ) and phosphatidylethanol ( ) by thin layer chromatography (TLC). In (b), neutrophils were incubated in the presence and absence of 10 mM butanol, and luminol chemiluminescence (10 jUM, final concentration of luminol) was measured after stimulation by 1 jUM fMet-Leu-Phe. Source Experiment of Gordon Lowe and Fiona Watson.

Figure 7.12. Role of protein biosynthesis in the ability of neutrophils to generate reactive oxidants. Neutrophils were incubated in the presence (O) and absence ( ) of 30 pg/ml cycloheximide at 37 °C. At time intervals, portions were removed and luminol chemiluminescence measured after stimulation with 1 pM fMet-Leu-Phe. Values presented are a percentage of the control value measured at time zero.

Figure 8.4. Activation of neutrophils by soluble immune complexes isolated from the synovial fluid of a patient with rheumatoid arthritis. Neutrophils were isolated from control blood and incubated in the absence (O) or presence ( ) of 50 U/ml GM-CSF for 60 min at 37 °C. Thereafter, luminol chemiluminescence was measured following the addition of soluble immune complexes isolated from the synovial fluid of a patient with rheumatoid arthritis.

Kawasaki, S., Yamashoji, S., Asakawa, A., Isshiki, K., and Kawamoto, S. (2004). Menadione-catalyzed luminol chemiluminescence assay for the rapid detecHon of viable bacteria in foods under aerobic conditions. /. Food Prot. 67, 2767-2771. 

Miyazawa T, Fujimoto K, Suzuki T, Yasuda K. Determination of phospholipid hydroperoxides using luminol chemiluminescence high-performance liquid chromatography. Oxygen Radicals in Biological Systems, Pt. C 233, 324-332, 1994. 

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