| Autor: |
Tikhomirova AA; Applied Research and Technology, Abbott Diagnostics Division, Abbott Laboratories, Abbott Park, Illinois 60064, United States of America., Swift KM; Applied Research and Technology, Abbott Diagnostics Division, Abbott Laboratories, Abbott Park, Illinois 60064, United States of America., Haack RA; Applied Research and Technology, Abbott Diagnostics Division, Abbott Laboratories, Abbott Park, Illinois 60064, United States of America., Macdonald PJ; Applied Research and Technology, Abbott Diagnostics Division, Abbott Laboratories, Abbott Park, Illinois 60064, United States of America., Hershberger SJ; Applied Research and Technology, Abbott Diagnostics Division, Abbott Laboratories, Abbott Park, Illinois 60064, United States of America., Tetin SY; Applied Research and Technology, Abbott Diagnostics Division, Abbott Laboratories, Abbott Park, Illinois 60064, United States of America. |
| Abstrakt: |
Acridinium 9-carboxylic acid derivatives have been extensively used as chemiluminescent labels in diagnostic assays. Triggering acridinium with basic hydrogen peroxide produces a highly strained dioxetanone intermediate, which converts into an acridone in an electronically excited state and emits light at 420-440 nm. Here, we introduce a novel acridinium-fluorescein construct emitting at 530 nm, in which fluorescein is covalently attached to the acridinium N-10 nitrogen via a propyl sulfonamide linker. To characterize the spectral properties of the acridinium-fluorescein chemiluminophores, we synthesized the analogous acridone-fluorescein constructs. Both acridinium and acridone were linked to either 5- or 6-carboxyfluorescein and independently synthesized as individual structural isomers. Using fluorescent acridone-fluorophore tandems, we investigated and optimized the diluent composition to prevent dye aggregation. As monomolecular species, the acridone isomers demonstrated similar absorption, excitation, and emission spectra, as well as the expected fluorescence lifetimes and molecular brightness. Chemical triggering of acridinium-fluorescein tandems, as well as direct excitation of their acridone-fluorescein analogs, resulted in a nearly complete energy transfer from acridone to fluorescein. Acridone-based dyes can be studied with steady-state spectroscopy. Thus, they will serve as useful tools for structure and solvent optimizations, as well as for studying chemiluminescent energy transfer mechanisms in related acridinium-fluorophore tandems. Direct investigations of the light-emitting molecules generated in the acridinium chemiluminescent reaction empower further development of chemiluminescent labels with red-shifted emission. As illustrated by the two-color HIV model immunoassay, such labels can find immediate applications for multicolor detection in clinical diagnostic assays. |
