Chelates for Time-resolved Fluorescence

Different lanthanide metals also produce different emission spectrums and different intensities of luminescence at their emission maximums. Therefore, the relative sensitivity of time-resolved fluorescence also is dependent on the particular lanthanide element complexed in the chelate. The most popular metals along with the order of brightness for lanthanide chelate fluorescence are europium(III) terbium(III) samarium(III) dysprosium(III). For instance, Huhtinen et al. (2005) found that lanthanide chelate nanoparticles used in the detection of human prostate antigen produced relative signals for detection using europium, terbium, samarium, and dysprosium of approximately 1.0 0.67 0.16 0.01, respectively. The emission 

Organic fluorescent dyes with the appropriate spectral properties also can be paired with lanthanide chelates in FRET systems. For instance, many rhodamine dyes and the cyanine dye Cy5 have ideal excitation wavelengths for receiving energy from a nearby europium chelate. The LeadSeeker assay system from GE Healthcare incorporates various Cy5-labeled antibodies for developing specific analyte assays. In addition, if using a terbium chelate as the donor, then a Cy3 fluorescent dye can be used in assays as the acceptor. 

Terpyridine-bis (methylenamine) tetraacetic acid (TMT) chelate of europium 

In a fume hood, dissolve the TMT chelator in a 1 1 mixture of DMF DMSO at a concentration of lOmg/ml. 

Dissolve a protein to be labeled in 0.1 M sodium bicarbonate, pH 9.0, at a concentration of l-10mg/ml. 

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In the second step, the purified, transaminated DNA is brought to a higher pH (8.5-10) in order to decrease the protonation of the reactive amino group. Viscidi et al. (1986) labeled the N -substituted cytosine residue with biotin using the NHS-biotin ester, whereas, e.g., Hurskainen et al. (1991) labeled the same intermediate with a europium chelate (for time-resolved fluorescence). The Cq/qj increases about 1 log but this is compensated by the high probe concentration. This method yields probes with a similar detectability (10 molecules) as enzymatically labeled biotin probes, but the reagents are inexpensive and easily available. 

ConnaUy R., D. Jin, J. Piper. BHHST An improved lanthanide chelate for time-resolved fluorescence applications, SPIE Proceedings, 5704, 93-104 (2005). 

Morton RC, Diamandis EP (1990) Streptavidin-based macromolecular complex labeled with a Europium chelator suitable for time-resolved fluorescence immunoassay applications. Anal Chem 62 1841-1845... 

This class of ligands mostly consists of derivatives of pyridine, 2.2 -bipyridinc. 2,2, 2"-ter-pyridine, and 1,10-phenanthroline. Scheme 6 shows the structures of fourEu3+ chelates with aromatic amine derived ligands, that can be covalently bound to proteins, for time-resolved fluorometry, among which 4,7-bis(chlorosulfophenyl)-l,10-phenanthroline-2,9-dicarboxylic acid (BCPDA)-Eu3+ and trisbipyridine cryptate (TBP)-Eu3+ are widely used for europium fluorescence labels in TR-FIA. 

Another example of improved sensitivity due to modulation of lanthanide photophysics by ancillary ligands can be found in the europium and terbiiun chelates used in time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassays (100,101). Due to their line-type emissions and long decay times, the lanthanide chelate is used as a donor, with some visible-absorbing dye such as Alexa 647 or a rhodamine derivative as the acceptor. Without the helper ligand, the lanthanides would be unprotected from solvent and have much shorter decay times, making them unsuitable for such an assay. 

Phimphivong, S. and Scott Saavedra, S. (1998) Terbium chelate membrane label for time-resolved, total internal reflection fluorescence microscopy of substrate-adherent cells. Bioconjugate Chemistry, 9, 350-357. 

Heterogeneous fluorescent immunoassays for T4 based on lanthanide rare earth ions and time-resolved fluorescence were also developed. The use of europium chelates as fluorescent probes is particularly attractive because of their extraordinarily long Stokes shifts and long fluorescence decay times. Thus the sharp emission peak of europium (613 nm) can be easily separated fr om scattering caused by excitation light (340 nm) or by interfering substances in... 

Fig. 7.5. Lanthanide chelates, such as Eu, have exceptionally long fluorescent decay times (up to over 1 ms), whereas proteins and other macromolecules may have decay times of 10 to 10 s. At every 1 ms interval, an excitation pulse is sent through the sample in time-resolved fluorescence. After a delay of 400 p,s, fluorescence is measured for 400 (xs (two cycles shown).

This time-resolved fluorescence technique allows a measure of the time dependence of fluorescence intensity after a short excitation pulse. It consists of obtaining a spectrum measured within a narrow time window during the decay of the fluorescence of interest. The usefulness of this technique is now well proven for biochemical assays and immunoassays. Lanthanide chelates have luminescence decay times over 600 ps, which allows time-gated fluorecence detection, with a complete rejection of other fluorecence signals. For these quantitative applications, the primary source is generally a quartz lamp associated with a splitter. 

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