Europium, resonance energy transfer

When the proteins are in close proximity the Europium-cryptate emission can be absorbed by the acceptor (such as allophycocyanin [APC], or XL) which emits at a higher wavelength. When the two proteins are far apart, no fluorescence resonance energy transfer (FRET) occurs. 

A homogeneous and sensitive HTRF binding assay was developed to allow prosecution of an HTS campaign for novel small molecule Hsp90 inhibitors. The HTRF assay was based on a non-radio-active resonance energy transfer between a donor label (europium chelate) and an acceptor label (allophycocyanin [APC]) brought into close proximity by a specific binding interaction. 

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. 

Harma H, Dahne L, Pihlasalo S et al (2008) Sensitive quantitative protein concentration method using luminescent resonance energy transfer on a layer-by-layer europium(III) chelate particle sensor. Anal Chem 80 9781-9786... 

Two simple examples will be given in order to illustrate the instrumentation and the primary data treatment. The discussion is focused on the technical problems and the interpretation of the results is left to a minimum. The first example deals with the resonance energy transfer (RET) from europium to an organic fluorophore. The distance between the donor and acceptor is kept constant by an oligonucleotide. RET is a very common technique in the case of lanthanides, and therefore the discussion is somewhat lengthy and detailed. The second example deals with a sample of upconversion phosphor containing yttrium, ytterbium and erbium. 

Heyduk, E. and Heyduk, T. Thiol-reactive, luminescent europium chelates Luminescence probes for resonance energy transfer distance measurements in biomolecules. Anal. Biochem. 248 216-227, 1997. 

Figure 38 shows the result of this analysis. It is quite clear that a very good correlation exists between the europium-emission intensities and the number of resonances found. From this result Axe and Weller concluded that resonant-energy exchange is the likely mechanism by which the energy is transferred. 

Similar examples for energy transfer from ligand localized levels to highly localized 4f levels are represented by the rare-earth chelates. Voloshin and Savutskii (1976) studied europium benzoylacetonate imder pressures up to 6 GPa. Exciting the triplet level they could observe the luminescence from the Eu " " ion. It was possible to describe the observed initial increase in the quantum yield of the Eu " " luminescence up to 2.5 GPa and the following decrease by the exchange resonance theory (Dexter, 1953). A more detailed study on different Tris chelates of Sm +, Eu +, Gd " ", and Tb " with -diketonates was performed by Hayes and Drickamer (1982), where the most dramatic effects of pressure on energy transfer phenomena were found for the Eu + chelates. 

The authors observe that other rare earths also quench terbium in the same solution. The quenching effects appear to correlate well with a resonance-exchange mechanism. This strongly points to the fact that the terbium-to-europium-transfer process is probably the same. Perhaps the most important aspect of this work is that it vividly shows that energy may migrate from one rare-earth ion to another without the necessity of a crystal or glass lattice. ... 

---