Abstract
Acridinium chemiluminescent labels have been extensively used in diagnostic immunoassays. When acridinium is triggered with basic hydrogen peroxide, a highly strained dioxetanone system is produced which, with the release of carbon dioxide, converts to an acridone in an electronically excited state. The acridone returns to the ground state releasing a single photon around 440 nm. Here, we describe the acridinium and acridone constructs linked to two structural isomers, 5- or 6-carboxy-fluorescein. Triggering the acridinium, or exciting the acridone, results in a nearly 100% energy transfer to the fluorescein moiety. The acridinium (chemiluminescence) and acridone (fluorescence) emission spectra are identical and characteristic to each isomer. The absorption maximum of the acridone-6-fluorescein is shifted from 495 nm to 502 nm, and its emission is shifted from 520 nm to 523 nm, as compared to the acridone-5-fluorescein. However, the maxima of the excitation spectra of the isomers are much closer than the absorption peaks, 494 nm and 496 nm, respectively. Moreover, the emission intensity of the acridone-6-fluorescein is 50% weaker than the emission of the acridone-5-fluorescein. Such discrepancies are caused by the presence of a dimmer fluorescent species, which has been confirmed by measuring fluorescence lifetimes. In fact, the lifetime of the isomer-6 contains two components: 2.9 ns (62%) and 0.8 ns (38%), while the decay of the isomer-5 is mono-exponential with the lifetime of 4 ns typical to fluorescein. The spectral differences between the isomers can be explained by the intramolecular orientation of the acridone and fluorescein moieties. In both, 5- and 6-carboxy-fluorescein-acridinium constructs, as well as in their acridone analogs, the acridone emission is completely quenched. This indicates an effective energy transfer pathway, perhaps by the Dexter excited state electron transfer mechanism.
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