Historically a wide variety of organic fluorescent reagents have been used as tags or labels, such as fluorescein, rhodamine, and various cyanin dyes. Different from these organic reagents, certain lanthanide complexes, especially those of Eu3+ and Tb3+, are also recognized as efficient fluorescent labels, owing to their distinct properties specific to lanthanide complexes; they are excited in the UV region (310-340nm) and emit fluorescence at ca. 615nm (Eu3+) and ca. 545nm (Tb3+), with the long lifetimes of several hundred microseconds to more than 1 milliseconds. By taking advantage of these properties, time-resolved fluorometric measurement can remove background fluorescence from the sample matrix and often gives detectability better than one order of magnitude compared to those of conventional fluorometric assays.
The other reagent, ATBTA-Eu3+, has an amino group instead of dichlorotriazinyl in DTBTA-Eu3+, and is more stably stored, since it does not have the hydrolysable dichlorotriazinyl group. DTBTA-Eu3+ can be directly labeled to amino groups of biomolecules, whereas ATBTA-Eu3+ is used as a label after conversion to DTBTA-Eu3+ by reacting with trichlorotriazene. Scheme 1 summarizes these reactions and the labeling of DTBTA-Eu3+ to the primary amine groups of proteins and nucleic acids. Although ATBTA-Eu3+ is not so strongly fluorescent as DTBTA-Eu3+, the fluorescence becomes strong after reaction with trichlorotriazene. The fluorescence spectrum of ATBTA-Eu3+ is basically the same with that of DTBTA-Eu3+.
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