Current research in life science targets the comprehensive genomic analysis of all living species from humans to micro organisms. Such extensive research is further extended to elucidation of all the proteins and their functions that are regulated by genes. Such comprehensive knowledge will finally contribute to understand all biological events at the molecular level, and elucidate the relations of genes, proteins with health and diseases. Highly sensitive fluorometric assay are used to detect proteins and nucleic acids by using various fluorescent labeling reagents, and the method is applied in the areas of medical diagnostics and treatments, new drug development, food safety testing, and agricultural and live-stock farming.

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 (310-340 nm) and emit fluorescence at ca. 615 nm (Eu3+) and ca. 454 nm (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 new lanthanide chelate reagent, TBTA-Eu3+, has a high stability constant, and therefore the problem of fluorescence intensity change in different buffers has been greatly reduced. DTBTA-Eu3+ has also several advantages such as the intensity stability in water for a long period, and the stability against photo-bleaching. The excitation and emission spectra are shown in Fig. 1.
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+.

Scheme 1. Conversion of ATBTA-Eu3+ to DTBTA-Eu3+ and the labeling reaction to amino groups of proteins and nucleic acids.

Fig. 1 Exitation and emission spectra of DTBTA-Eu3+ in 0.05M borate buffer at pH9.0 (1.5 x 10^-6M).
The excitation spectrum was right and the emission spectrum was left.

The DTBTA-Eu3+ labeled to proteins and nucleic acids is highly stable and the labeled biomolecules can be separated and purified with electrophoresis. Different from other lanthanide chelate reagents, DTBTA-Eu3+ does not lose its fluorescence intensity even after it is dried on a solid support array like DNA chips. It can be applied to immunoassay, hybridization assay, and immunohistochemistry, and is expanding its application area.

A2083 ATBTA-Eu3+
[=Sodium [4'-(4'-Amino-4-biphenylyl)-2,2':6',2''-terpyridine-6,6''-diylbis(methyliminodiacetato)]europate(III)]