Cryptates europium

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 derivative of the (bpy.bpy.bpy) cryptand, obtained by modifying one of the chains, Lbpy, forms a di-protonated cryptate with EuCb in water at acidic pH, [EuCl3(H2Lbpy)]2+ in which the metal ion is coordinated to the four bipyridyl and two bridgehead nitrogen atoms, and to the three chlorine ions (Fig. 4.25). The polyamine chain is not involved in the metal ion coordination, due to the binding of the two acidic protons within this triamine subunit. In solution, when chlorides are replaced by perchlorate ions, two water molecules coordinate onto the Eu(III) ion at low pH and one at neutral pH, a pH at which de-protonation of the amine chain occurs, allowing it to coordinate to the metal ion. As a result, the intensity of the luminescence emitted by Eu(III) is pH dependent since water molecules deactivate the metal ion in a non-radiative way. Henceforth, this system can be used as a pH sensor. Several other europium cryptates have been developed as luminescent labels for microscopy. 

Europium cryptates are excited in the UV wavelength range either by a xenon flash lamp or by a nitrogen laser. Their fluorescence occurs in a wavelength range between 550 and 710 nm with typical narrow emission lines. Since the electronic transitions of the europium ion are forbidden by quantum mechanical rules, the cryptate fluorescence lifetime is exceptionally long, in the range of 100-1000 J,s. 

Europium cryptates can be associated with various acceptors, originally with a cross-linked allophycocyanin called XL665 or with small fluorescent near infrared dyes that have been selected so that their photophysical properties fit those of the europium cryptate for an efficient FRET process ... 

Figure 4 Spectral selectivity of the HTRF signal. Characteristics of the emission spectra both of the europium cryptate (purple line) and the acceptor (red line) are responsible for the spectral selectivity between the acceptor fluorescence emission at 665 nm and the donor fluorescence emission at 620 nm...

Figure 5 Temporal selectivity of the HTRF signal. At the acceptor emission wavelength (665 nm), the FRET signal from the sensitized acceptor (C) and the small signal from the europium cryptate (B) can be easily distinguished from the free acceptor signal and from the background caused by chemical compounds or biological media (A)...

Figure 6. A typical europium cryptate featuring the antenna effect [19].

S-Streptavidin, APC-allophycocyanin, Eu-Europium cryptate bound to an antiphosphotyrosineantibody, FRET-Fluoiescence Resonance Ehs Transfer. 

The foundation for HTRF lies in the Nobel prize chemistry of Professor Jean Marie Lehn (Strasbourg University) [1]. This chemistry was further developed by Professor Lehn and Dr. Gerard Mathis (CIS bio international) into a europium cryptate (EuK) based assay technology (Figure 1). This innovative structure has five distinct and critically important functions ... 

Numerous bioassays format have been designed using derivatives of the mono-functionalized europium cryptate such as 47 (commercially available from CISbio, see http //www.htrf.com). Since the last reviews [127] some new applications Eu " C [BP.BP.BP] cryptate were developed through the HTRF technology, as caspase assay [128], telomerase assay [129], leukotriene b4 assay [130], inositol-1-phosphate assay [131], minisequencing [132], and MutS-DNA interaction [133]. Recently, the HTRF technology has been used in the study of protein-protein interactions and the authors demonstrated that measured values compare favorably with those calculated from independent experiments [134]. 

Europium chelates are most often used donors in the commercialized TR-FRET product lines. Europium cryptates are applied in the diagnostic system of Brahms, Kryptor [24], and in the HTS system of Cis-Bio International (HTRF ). Various... 

The very slow outer-sphere reactions of Eu and Eu " have long been considered good candidates for nonadiabaticity, in view of the poor overlap of / orbitals. New data from europium cryptate complexes do not support this view. From cross reactions, self-exchange rates of two cryptate couples [Eu(2.2.1)] and [Eu(2.2.2)] t are calculated to be much higher than for EUaq", whereas for nonadiabatic reactions the rate is expected to fall with increasing metal-metal distance. Studies of cobalt ammines, including the encapsulated [Co(sep)], [Co(sar)], and [Co(azacapten)] , have led to a similar conclusion. " ... 

Alpha-Bazin B., H. Bazin, L. Boissy, G. Mathis. Europium cryptate-tethered ribonucleotide for the labeling of RNA and its detection by time-resolved amplification of cryptate emission. Anal. Biochem., 286, 17-25 (2000). 

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