Luminol-peroxide

Chemiluminescence and bioluminescence are also used in immunoassays to detect conventional enzyme labels (eg, alkaline phosphatase, P-galactosidase, glucose oxidase, glucose 6-phosphate dehydrogenase, horseradish peroxidase, microperoxidase, xanthine oxidase). The enhanced chemiluminescence assay for horseradish peroxidase (luminol-peroxide-4-iodophenol detection reagent) and various chemiluminescence adamantyl 1,2-dioxetane aryl phosphate substrates, eg, (11) and (15) for alkaline phosphatase labels are in routine use in immunoassay analyzers and in Western blotting kits (261—266). 

Burdo and Seitz reported in 1975 the mechanism of the formation of a cobalt peroxide complex as the important intermediate leading to luminescence in the cobalt catalysis of the luminol CL reaction [116]. Delumyea and Hartkopf reported metal catalysis of the luminol reaction in chromatographic solvent systems in 1976 [117], while Yurow and Sass [118] reported on the structure-CL correlation for various organic compounds in the luminol-peroxide reaction. 

The oxidative behaviour of glycolaldehyde towards hexacyanoferrate(III) in alkaline media has been investigated and a mechanism proposed, which involves an intermediate alkoxide ion. Reactions of tetranitromethane with the luminol and luminol-peroxide radical anions have been shown to contribute substantially to the tetranitromethane reduction in luminol oxidation with hexacyanoferrate(III) in aerated aqueous alkali solutions. The retarding effect of crown ethers on the oxidation of triethylamine by hexacyanoferrate(III) ion has been noted. The influence of ionic strength on the rate constant of oxidation of ascorbic acid by hexacyanofer-rate(III) in acidic media has been investigated. The oxidations of CH2=CHX (where X = CN, CONH2, and C02 ) by alkaline hexacyanoferrate(III) to diols have been studied. ... 

Determination of metal ions (Cr3+, Co2+, Cu2+) has been achieved using the metal-catalyzed luminol-peroxide system. The EOF delivery of the CL reagent (H202 pH 11.7) in the aqueous system can be achieved using a side channel (with luminol present in the separation buffer with a pH of 6.0) [168]. 

In order to understand the effect of these catalysts, it is important to observe that they do not act themselves as enhancers (no effect on light output is observed, when used alone). An explanation is proposed by considering in more detail the reaction steps from diazaquinone (L) to the luminol peroxide intermediate. This reaction involves nucleophilic attack on one of the diazaquinone carbonyls by hydrogen peroxide monoanion. A hydroperoxide species is formed, which can rearrange to the endoperoxide. Either compound is very unstable and collapses to 3-amino-phthalate in its excited state. Perhaps pyridine acylation catalysts could facilitate hydrogen peroxide attack by converting L into a more reactive intermediate. 

In Section 2.6, there was a brief discussion of the issues involved in efficient detection of chemiluminescence. Because it has been so thoroughly studied, the luminol/peroxide system has been employed as a chemiluminescent standard (with accurately determined quantum yield), either for instmments (L4) or for other chemiluminescent reactions (H7, Nl). A recent determination of its quantum yield by Lind and Merenyi (Lll) gives a value of 1.2%, which is identical to a much earlier measurement (L4). Interestingly, the quantum yield (although not the emission wavelength) was found to be the same in aqueous media and in DMSO when a photodiode detector was employed (close source) (MIO), but required a... 

In Section 3.1.3, reference was made to the fact that luminol/peroxide chemi-... 

An aqueous-alkaline solution containing hydrogen peroxide, but no molecular oxygen was used. The rate of the luminol peroxide formation was assayed at different pH values. 

Detecting the presence of small, even invisible, amounts of blood is routine. Physical characteristics of dried stains give minimal information, however, as dried blood can take on many hues. Many of the chemical tests for the presence of blood rely on the catalytic peroxidase activity of heme (56,57). Minute quantities of blood catalyze oxidation reactions between colorless materials, eg, phenolphthalein, luco malachite green, luminol, etc, to colored or luminescent ones. The oxidant is typically hydrogen peroxide or sodium perborate (see Automated instrumentation,hematology). 

Chemiluminescent Immunoassay. Chemiluminescence is the emission of visible light resulting from a chemical reaction. The majority of such reactions are oxidations, using oxygen or peroxides, and among the first chemicals studied for chemiluminescence were luminol (5-amino-2,3-dihydro-l,4-phthalazinedione [521-31-3]) and its derivatives (see Luminescent materials, chemiluminescence). Luminol or isoluminol can be directly linked to antibodies and used in a system with peroxidase to detect specific antigens. One of the first appHcations of this approach was for the detection of biotin (31). 

Divalent copper, cobalt, nickel, and vanadyl ions promote chemiluminescence from the luminol—hydrogen peroxide reaction, which can be used to determine these metals to concentrations of 1—10 ppb (272,273). The light intensity is generally linear with metal concentration of 10 to 10 M range (272). Manganese(II) can also be determined when an amine is added to increase its reduction potential by stabili2ing Mn (ITT) (272). Since all of these ions are active, ion exchange must be used for deterrnination of a particular metal in mixtures (274). 

Chromium (ITT) can be analy2ed to a lower limit of 5 x 10 ° M by luminol—hydrogen peroxide without separating from other metals. Ethylenediaminetetraacetic acid (EDTA) is added to deactivate most interferences. Chromium (ITT) itself is deactivated slowly by complexation with EDTA measurement of the sample after Cr(III) deactivation is complete provides a blank which can be subtracted to eliminate interference from such ions as iron(II), inon(III), and cobalt(II), which are not sufficiently deactivated by EDTA (275). 

Iron(II) can be analy2ed by a luminol—air reaction in the absence of hydrogen peroxide (276). Iron in the aqueous sample is reduced to iron(II) by sulfite other metals which might interfere are also reduced to valence states that are inactive in the absence of hydrogen peroxide. The detection limit is 10 ° M. 

Hydrogen Peroxide Analysis. Luminol has been used for hydrogen peroxide analysis at concentrations as low as 10 M using the cobalt(III) triethanolamine complex (280) or ferricyanide (281) as promoter. With the latter, chemiluminescence is linear with peroxide concentration from... 

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... 

A method of detecting herbicides is proposed the photosynthetic herbicides act by binding to Photosystem II (PS II), a multiunit chlorophyll-protein complex which plays a vital role in photosynthesis. The inhibition of PS II causes a reduced photoinduced production of hydrogen peroxide, which can be measured by a chemiluminescence reaction with luminol and the enzyme horseradish peroxidase (HRP). The sensing device proposed combines the production and detection of hydrogen peroxide in a single flow assay by combining all the individual steps in a compact, portable device that utilises micro-fluidic components. 

Rost, M., Karge, E., and Klinger, W. (1998). What do we measure with luminol-, lucigenin- and penicillin-amplified chemiluminescence 1. Investigations with hydrogen peroxide and sodium hypochlorite. J. Biolumin. Chemilumin. 13 355-363. 

Among the different synthetic compounds used for hydrogen peroxide determination, only luminol and oxalate esters have found widespread use and were really evaluated for H2O2 detection. 

Another way to produce light from luminol is electrogenerated chemiluminescence6, 1. Luminol is oxidized using a positively biased electrode and in the presence of hydrogen peroxide the light emission occurs. 

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. 

Cormier M.J.,Prichard P.M., An investigation of the mechanism of the luminescent peroxidation of luminol by stopped flow techniques, J. Biol. Chem. 1968 243 4706-4714. 

Freeman T.M., Seitz W.R., Chemiluminescence fiber optic probe for hydrogen peroxide based on the luminol reaction, Anal. Chem. 1978 50(9) 1242-1246. 

Abdel-Latif M.S., Guilbault G.G., Peroxide optrode based on micellar-mediated chemiluminescence reaction of luminol, zlna/. Chim. Acta 1989 221 11-17. 

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. 

Special review articles published since 1968 on these topics are one by E. H. White and D. F. Roswell 2> on hydrazide chemiluminescence M. M. Rauhut 3) on the chemiluminescence of concerted peroxide-decomposition reactions and D. M. Hercules 4 5> on chemiluminescence from electron-transfer reactions. The rapid development in these special fields justifies a further attempt to depict the current status. Results of bioluminescence research will not be included in this article except for a few special cases, e.g. enzyme-catalyzed chemiluminescence of luminol, and firefly bioluminescence 6>. 

DPA) in dimethylphthalate at about 70°, yields a relatively strong blue Umax =435 nm) chemiluminescence the quantum yield is about 7% that of luminol 64>. The emission spectrum matches that of DPA fluorescence so that the available excitation energy is more than 70 kcal/mole. Energy transfer was observed on other fluorescers, e.g. rubrene and fluorescein. The mechansim of the phthaloyl peroxide/fluorescer chemiluminescence reaction very probably involves radicals. Luminol also chemiluminesces when heated with phthaloyl peroxide but only in the presence of base, which suggests another mechanism. The products of phthaloyl peroxide thermolysis are carbon dioxide, benzoic acid, phthalic anhydride, o-phenyl benzoic acid and some other compounds 65>66>. It is not yet known which of them is the key intermediate which transfers its excitation energy to the fluorescer. 

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