Luminol oxidation chemiluminescence

In the first proposal of a mechanism for chemiluminescent luminol oxidation, Albrecht postulates a bicyclic endoperoxide as the high-energy intermediate. The endoperoxide is presumably formed by nucleophilic attack of hydrogen peroxide monoanion on one of the diazaquinone 27 carbonylic groups to form 28, followed, after deprotonation to 29, by ring closure to 30 (Scheme 21) . 

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). 

The emission yield from the horseradish peroxidase (HRP)-catalyzed luminol oxidations can be kicreased as much as a thousandfold upon addition of substituted phenols, eg, -iodophenol, -phenylphenol, or 6-hydroxybenzothiazole (119). Enhanced chemiluminescence, as this phenomenon is termed, has been the basis for several very sensitive immunometric assays that surpass the sensitivity of radioassay (120) techniques and has also been developed for detection of nucleic acid probes ia dot-slot. Southern, and Northern blot formats (121). 

Titration Indicators. Concentrations of arsenic(III) as low as 2 x 10 M can be measured (272) by titration with iodine, using the chemiluminescent iodine oxidation of luminol to indicate the end point. Oxidation reactions have been titrated using siloxene the appearance of chemiluminescence indicates excess oxidant. Examples include titration of thallium (277) and lead (278) with dichromate and analysis of iron(II) by titration with cerium(IV) (279). 

CHEMILUMINESCENT ANALYSIS BASED ON THE LUMINOL OXIDATION REACTION IN SPECIFIC REGIMES... 

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. 

Whereas substitution of the hydrazide ring in luminol leads to complete loss of chemiluminescence capacity 121>, N-methylated linear hydrazides such as 57 a and 57 b were found to exhibit chemiluminescence on oxidation with oxygen in DMSO 120>. 

In this part of the chapter we will give a more detailed description of some highly efficient organic chemiluminescence systems, which occur with the involvement of peroxide intermediates. We have chosen to begin the subject with the well-known and widely applied luminol oxidation and will show that, even though this reaction has been exhaustively studied, several critical points in its mechanism remain unclear and are still the subject of... 

The chemiluminescence emission resulting from the oxidation of luminol (5-amino-2,3-dihydro-l,4-phthalazinedione) has been extensively studied since its discovery by Albrecht in 1928. Although luminol oxidation is one of the most commonly applied chemiluminescent reactions, to date no definitive mechanism is known . Efficient chemiluminescence emission is only observed when luminol (25) is oxidized under alkaline conditions. Depending on the medium, co-oxidants are required in addition to molecular oxygen for the observation of light emission, but under any condition, 3-aminophthalate (3-AP) and molecular nitrogen are the main reaction products (equation 10). 

Scheme 28) Quantum yields of lucigenin oxidation by hydrogen peroxide in alkaline media are comparable with the values obtained in luminol oxidation (1.24 x 10 E mol ) ° . However, the use of other peroxides, such as tcrt-butyl hydroperoxide, results in a decrease of chemiluminescence quantum yields of two orders of magnitude, confuming the hypothesis that a 1,2-dioxetane is the HEI, since its formation would be impossible with alkyl peroxides . 

J. K. Robinson, M. J. Bollinger, and J. W. Birks, Luminol/H202 Chemiluminescence Detector for the Analysis of NO in Exhaled Breath, Anal. Chem. 1999, 71, 5131. Many substances can be analyzed by coupling their chemistry to luminol oxidation. See, for example, O. V. Zui and J. W. Birks, Trace Analysis of Phosphorus in Water by Sorption Preconcentration and Luminol Chemiluminescence, Anal. Chem. 2000, 72, 1699. 

Chemiluminescence of oxidized luminol has been the basis of several lumino-metric methods of estimation of TAC (Table 1). The mostcommon is to measure the induction time of the reaction. Often the chemiluminescence is first induced by an oxidant and then attenuated by addition of a sample, and the time to recover the initial fluorescence is measured. The enhanced chemiluminescent assay introduced a decade ago is based on the oxidation of luminol by perborate or by hydrogen peroxide in a reaction catalyzed by horseradish peroxidase. Enhancement (and stabilization) of luminescence is achieved by addition of p-iodophenol. The original procedure used a commercial reagent kit (ECL Anti-oxidant Detection Pack... 

Since the emission spectra of enhanced and unenhanced luminol oxidations are similar, it seems clear that emission is from the excited-state aminophthalate derived from luminol itself and not from the enhancer. The enhancement is also specific for peroxidase i.e., it does not occur under conditions in which the heme would dissociate from the enzyme. Furthermore, luminol chemiluminescence triggered by heme-containing compounds, such as hemoglobin or cytochrome c, is actually reduced by enhancers such as p-iodophenol (T7). This latter phenomenon explains the potential usefulness of enhanced chemiluminescence, i.e., an amplified signal combined with a reduced background and reduced interference from the reagents as well as from endogenous heme compounds. 

Since the reports on enhanced chemiluminescence in the mid-1980s (e.g., TIO, T12-T14, T16, T17), numerous assays have been described in the literature. Table 5 represents a summary of the types of analyte that have more recently proved amenable to detection by enhanced luminol oxidation via horseradish peroxidase labels. This table is meant to be illustrative rather than comprehensive. 

Armbruster, D. A., Jirinzu, D. C., and Williams, J. V., Enhanced luminescence immunoassays for free thyroxine (FT4). Clin. Chem. (Winston-Salem, N.C.) 34, 1153-1154 (abstr.) (1988). Amhold, J., Mueller, S., Arnold, K., and Grimm, E., Chemiluminescence intensities and spectra of luminol oxidation by sodium hypochlorite in the presenee of hydrogen peroxide. J. Biolumin. Chemilumin. 6, 189-192 (1991). 

By a similar mechanism, 5-amino-2,3-dihydrophthalazine-l,4-dione 3 (luminol, see p 434) displays an intensely blue chemiluminescence on oxidation with hydrogen peroxide in the presence of complex iron salts, e.g. haemin. 

Electrochemistry can be coupled with other physical methods such as fluorescence spectroscopy. An XO-based electrochemiluminescent biosensor for hypoxanthine has been reported. The enzyme was immobilized in a carbon paste electrode with bovine serum albumin cross-linked with glutar-aldehyde. The working principle of the biosensor is illustrated in Scheme 5.6. As already shown (eqn (5.3a)), H2O2 is produced by the catalytic reaction between hypoxanthine and XO immobilized on the electrode surface. In an alkaline or neutral solution, luminol is electrochemically oxidized to a compound that reacts spontaneously with H2O2 to generate chemiluminescent luminol and the ensuing luminescence was used to quantify the amount of hypoxanthine present. 

Our final example is one of the classic cases of chemiluminescence, the oxidation of luminol [aminophthalhydrazide (116)] by alkaline hydrogen peroxide in the presence of a ferrous salt (i.e., alkaline Fenton s reagent). 

The parent of the series of which luminol is the best known member, phthalic acid hydrazide itself, provides few useful clues to the understanding of luminol chemiluminescence. It is not chemiluminescent on oxidation by Fe -hemin in alkaline aqueous solution. In aprotic solvents however a yellow (526 nm) emission is seen. As will be seen later, most cyclic hydrazides produce the excited state of the corresponding dicarboxylate. In this case the emission comes from the monoanion of the starting phthalhydrazide, by back-transfer of energy from the product. One can only assume that the primary excited product is the dicarboxylic acid dianion, but there is not direct evidence for this [7]. 

The would-be acceptor, 1,4-dimethylnaphthalene, is not fluorescent in the visible range of the spectrum. However, (24) again chemiluminesces on oxidation in the aqueous system, with an efficiency of 1% of that of luminol (ca. 1 x lO"" ). 

The anthracene derivative (44), naphtho-[2,3-g]-phthalazine-l,4-dione (NPD) is fairly stable, and it chemiluminesces on oxidation with basic hydrogen peroxide with an emission maximum of 430 nm, similar to the luminol emission maximum [43, 56, 57]. 

That the chemiluminescence of the monoacyl hydrazides follows mechanisms other than that of the luminol type hydrazides, is evident from the fact that methyl substitution in the hy dr azide group does not lead, as with luminol to the loss of chemiluminescence. The hydrazides (69) and (70), for example, chemiluminesce on oxidation in the DMSO system ... 

Chemiluminescent labels, in which the luminescence is generated by a chemical oxidation step, and bioluminescent labels, where the energy for light emission is produced by an enzyme-substrate reaction, are additional labeling types (39,42). Luminol [521 -31 -3] CgHyN202, and acridine [260-94-6] C H N, derivatives are often used as chemiluminescent labels. 

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