Europium, fluorescent decay

To test the above ideas, Weitz etal.(i2) performed experiments on the fluorescence decay from a thin layer of europium(III) thenoyltrifluoracetonate (ETA) deposited on a glass slide covered with Ag particles approximately 200 A in diameter. The fluorescence decay rate was found to increase by three orders of magnitude in comparison with that of ETA in solid form. In addition to the large increase in decay rate, there was also evidence for an increase in overall fluorescence quantum efficiency. It is not possible from Eq. (8.11) to say anything about the manner in which is partitioned between radiative and nonradiative processes, y should be written in terms of a radiative part yr and a nonradiative part ynr y = yr + y r. Since the radiative rate for dipole emission is given by... 

Rieke and Allison (97) also examined the fluorescent-decay time of europium dibenzoylmethide (EuD3). They found a marked difference between it and unchelated europium compounds. The material was prepared by the method described by Crosby et al. (142). The data were collected by illuminating the samples with a stroboscopic light source of 20-/zsec decay time. The results for EuD3 as compared with their data on EuC13-4H20 are ... 

Metlay (145) studied the fluorescent decay of both EuD3 and EuD4. These compounds were prepared by the method of Crosby et al. (142). As Metlay points out, Whan and Crosby (146) in a later paper describe their preparation in more detail. They indicate that a period of heating in vacuum is necessary to convert the chelate from one containing four molecules of dibenzoylmethane per atom of europium to one containing but three. 

The fluorescent decay data of europium tris-thenoyltrifluoroacetonate (EuTTA)3 as given in Table XI suggests that both mechanisms are operative. This may be inferred from the fact that not only is the lifetime longest in tri-iV-butyl phosphate (TBP), but the quantum efficiency of energy transfer to the emitting level is also greater. The quantum efficiency is found to increase by a factor greater than the lifetime. 

To a very large extent, most of the recent data on fluorescent decay times of the other trivalent ions (those beside terbium, neodymium, and europium) stems in some way from laser experiments. In this section some representative data on these are considered. 

The use of europium chelates, with their unusually long fluorescence decay times, as labels for proteins and antibodies has provided techniques that are referred to as time-resolved fluoroimmunoassays (TRFIA). Fluorophores as labels for biomolecules will be the topic of Sect. 3. Nevertheless, TRFIAs always have to compete with ELISA (enzyme-linked immunosorbent assays) techniques, which are characterized by their great versatility and sensitivity through an enzyme-driven signal amplification. Numerous studies have been published over the past two decades which compare both analytical methods, e.g., with respect to the detection of influenza viruses or HIV-1 specific IgA antibodies [117,118]. Lanthanide luminescence detection is another new development, and Tb(III) complexes have been applied, for instance, as indicators for peroxidase-catalyzed dimerization products in ELISAs [119]. 

Cha et al. (1999) used a variant of FRET called LRET for lanthanide-based fluorescence energy transfer. In this technique (Selvin, 1996) the donor is terbium or europium which, in fact, is luminescent. There are several advantages of this technique over regular FRET. It has been found that terbium emits isotropically, which means that the uncertainty due to the dipole orientation is decreased to a maximum error of 10%. This error can be decreased even further if the anisotropy of the acceptor is also known. The second advantage is that the fluorescence decay has a time constant of about 1.5 ms, making it easily measurable with conventional recording techniques. The third advantage is that the emission of terbium is peaked and one can find fluorophores that emit in between peaks. This means that the fluorescence of the acceptor can be measured with little or no contamination from the donor. In addition, as the acceptor has a fast decay, any measurement of the acceptor fluorescence with decays comparable to the donor will exclude any possible direct... 

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... 

In time-resolved fluorescence (TRF) (Maundrell et al., 1985), europium chelates are excited at 340 nm to emit two types of fluorescence, a shortlived background fluorescence (< 0.1 ms) and a fluorescence due to emitted photons of Eu " lasting up to over 1 ms. This difference in fluorescence decay rate can be exploited by measuring fluorescence only after background fluorescence has completely decayed to obtain a very high signal to noise ratio (detectability down to 10 5 M) as shown in Fig. 7.5. Originally, anti-hapten antibody was labeled with Eu " but in more recent procedures Eu " is directly attached to the nucleic acid (Sections 7.3.2.1 and 7.8.1). 

Figure 18-27. Fluorescence decay profile of an europium chelate as used in a time-resolved fluorescence immunoassay. Background fluorescence disappears after a few nanoseconds, whereas the chelate decays in the millisecond time range. Reproduced by permission, courtesy of LKB Produkter, Bromma, Sweden.

De Schryver et al. [44] applied this model to analyse the pyrene fluorescence quenching by metal ions in SDS micelles. The situation described by the inequality (40) was observed for nickel, copper and lead ions. The values were determined from the slope of the linear plot of S2/SS vs. [M] [see Eq. (43)]. For europium and chromium ions, both interfacial exchange processes in micelle-micelle and micelle-bulk solution are very slow as compared with pyrene fluorescence decay. Here, the kinetics fits well to Case 2 discussed above. For silver and thallium ions, the rates of the fluorescence... 

The fluorescence lifetime can be measured by time-resolved methods after excitation of the fluorophore with a light pulse of brief duration. The lifetime is then measured as the elapsed time for the fluorescence emission intensity to decay to 1/e of the initial intensity. Commonly used fluorophores have lifetimes of a few nanoseconds, whereas the longer-lived chelates of europium(III) and terbium(III) have lifetimes of about 10-1000 /tsec (Table 14.1). Chapter 10 (this volume) describes the advantages of phase-modulation fluorometers for sensing applications, as a method to measure the fluorescence lifetime. Phase-modulation immunoassays have been reported (see Section 14.5.4.3.), and they are in fact based on lifetime changes. 

Nardi and Yatsiv (141) studied the temperature dependence and the decay times of europium emissions in europium dibenzoylmethide. In this compound the ultraviolet radiation absorbed by the organic component is transferred to the rare-earth ion and fluorescence is emitted from two levels, namely, the SD0 and the 5DI. The compound was prepared by treating a solution of EuC13 in ethanol with a solution of dibenzoylmethane in ethanol. The compound was precipitated by the addition of piperidine. 

Figure 40. Fluorescence rise and decay in the piperidine adduct of a four-ligand europium dibenzoylmethide chelate [from Ref. (148)].

Metal complexes like lanthanide chelates (mainly europium or terbium), ruthenium phenanthrolines or bipyridyls, and platinum porphyrins can be used as fluorescent labels for biomolecules. Their long decay times are perfectly suited for a detection by time-resolved imaging, and the labeled target molecules can be used for the determination of intracellular recognition processes or for the screening of DNA and protein arrays. Ratiometric lifetime-based imaging methods in combination with sophisticated data acquisition and evaluation tools can substantially contribute to the development... 

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. 

There are two pathways available to the ligand triplet state energy. It can decay to the ground state (kr >.s) or it can be transferred to the europium ion. Grosby and co-workers showed that the fluorescence efficiency of europium chelates, or in other words kr- o, increases as the... 

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