Chemiluminescence precursors

An indirect chemiluminescence immunoassay is an assay, with another component than the primary chemiluminescent emitter coupled to the antigen or antibody. This can be a cofactor or a catalyst or even a molecule capable of converting a non-chemiluminescent precursor to a chemiluminescent or potentially chemiluminescent species. Most indirect assays are enzyme mediated. 

Chemiluminescent compounds and their precursors in P. stipticus. Although P. stipticus is negative in the luciferin-luciferase reaction, crude extracts of this fungus are chemiluminescent, like the luciferin obtained from Ompbalia flavida by Kuwabara and Wassink (1966). The chemiluminescence is elicited by the addition of H2O2 and Fe2+ under a mild condition of pH 5-8, and the luminescence is strongly... 

Fig. 9.13 Absorption spectrum of one of the luciferin precursors of Mycena cit-ricolor in methanol (dash-dot line, A.max 369 nm). The absorption and fluorescence emission spectra of the decylamine-activation product of the same precursor in neutral aqueous solution (solid lines abs. Amax 372 nm and fl. Xmax 460 nm), and in ethanol (broken lines abs. Amax 375 nm and fl. Amax 522 nm). The chemiluminescence spectrum of the same activation product (dotted line, A.max 580 nm). The dotted line (7max 320 nm) is the absorption spectrum of M. citricolor natural luciferin reported by Kuwabara and Wassink (1966).

Treatment of the precursors with decylamine resulted in a high level of chemiluminescence activity. Taking the activity obtained with decylamine as 100%, the activities obtained with other amines were methylamine, 5% hexylamine, 23% octylamine, 39% and dode-cylamine, 55%. For comparison, the Panellus precursors, PS-A and PS-B, are best activated with methylamine, and a synthetic model compound, K-l, is best activated with hexylamine. 

We have also investigated other oxalate esters as a potential means to improve the efficiency. The most commonly used oxalates are the 2,4,6-trichlorophenyl (TCPO) and 2,4-dinitrophenyl (DNPO) oxalates. Both have severe drawbacks namely, their low solubility in aqueous and mixed aqueous solvents and quenching of the acceptor fluorescence. To achieve better solubility and avoid the quenching features of the esters and their phenolic products, we turned to difluorophenyl oxalate (DFPO) derivatives 5 and 6 (Figure 14). Both the 2,4- and the 2,6-difluoro esters were readily synthesized and were shown to be active precursors to DPA chemiluminescence. In fact, the overall efficiency of the 2,6-difluorophenyl oxalate 5 is higher than for TCPO in the chemical excitation of DPA under the conditions outlined earlier. Several other symmetrical and unsymmet-rical esters were also synthesized, but all were less efficient than either TCPO or 2,6-DFPO (Figure 14). 

Figure 14 Some examples of endpoint determination in titrations using chemiluminescent indicators. (A) Acid-base titration the endpoint is detected by the emission of light (B) complexometric titration the endpoint is detected by disappearance of light. M, metal acting as a catalyst X, excited state from the CL precursor acting as indicator.

An interesting phenomenon which may be related to this precursor state is the chemiluminescence which has been reported in the disproportionation of alkoxy radicals.16 In the case of CH30 radicals the reaction is ... 

A slightly different application is where species produced electrochem-ically lead to photon emission in the visible spectrum, via the formation of organic radicals by homogeneous reaction from electrochemically generated precursors. The electrode controls the quantity of precursor, enabling quantitative parameters of the homogeneous reaction to be elucidated. This is known as electrogenerated chemiluminescence or electrochemiluminescence (ECL). 

In sum, then, a good deal of experimental evidence has been gathered which supports, although indirectly, the intermediacy of a 1,4-biradical in the chemiluminescent reaction of simple dioxetanes. Yet there is no direct evidence that such biradicals exist with finite lifetimes. An attempted independent generation of a 1,4-biradical by decomposition of a dinitrite proved inconclusive (Suzuki, 1979). The influence of quenchers, radical scavengers, and external heavy atoms on the chemiluminescent reaction of trimethyldioxe-tane (Simo and Stauff, 1975) and adamantylideneadamantane-l,2-dioxetane [8] (Neidl and Stauff, 1978) was studied. While the authors interpret their results in terms of a relatively long-lived precursor to the excited-state product, namely the 1,4-biradical, the results are open to alternative explanations (Horn etal., 1978-79). 

The most recent threshold determination for the radical process (I) utilizes the.Meinel band chemiluminescence of the O(- P) + HCO - OH + CO reaction (20). The basic photochemical questions have been and remain the determination of the true precursors for the two product channels and the energy (or excitation wavelength)-dependent quantum yields for each channel. 

Trapping experiments would constitute the most unequivocal proof for the intervention of diradical intermediates in the decomposition of 1,2-dioxetanes. Although such experiments have not been reported to date, the interesting observation that tri-Z-butylphenol extinguished the trimethyldioxetane chemiluminescence more efficiently than piperylene, was construed as evidence that the phenol scavenged a relatively long-lived precursor, presumably a diradical to the electronically excited product. ... 

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