Detection lanthanide ions

This PCR -> hybridization -> DELFIA type of assay can be performed in a multi-color format. Samiotaki et al. (1997) reported this type of assay using seven different hybridization probes, which are three singly-labeled probes (labeled with Eu, Tb, and Sm), three doubly-labeled probes (labeled with Eu and Tb, Eu and Sm, and Tb and Sm), and one triply-labeled probe (labeled with Eu, Tb, and Sm) (see table 5 and scheme 15 for the chelate ligand). After PCR amplification, the PCR product was allowed to hybridize with one of the seven hybridization probes, washed, detected lanthanide ions by DELFIA , and washed. This cycle was repeated for the remaining six hybridization probes. This procedure enabled detection and identification of seven types of human papilloma virus in one single assay. 

Another antibody-based immunosorbent assay that can be used to determine HAT activity uses a secondary anti-IgG antibody, which is directed against the primary antibody and is labeled with the lanthanide Europium (Eu). After another washing step that removes all nonbound secondary antibody, one last incubation step is performed, which releases the lanthanide ion from the antibody, so that the final detection of time-resolved fluorescence (340/615 nm) caused by the released metal ion correlates with the acetylation level of the oligepeptide histone substrate, which is correlated with enzymatic activity. So far. 

Figure 19-16 shows how Eu3+ can be incorporated into an immunoassay. A chelating group that binds lanthanide ions is attached to antibody 2 in Figure 19-13. While bound to the antibody, Eu3+ has weak luminescence. After all steps in Figure 19-13 have been completed, the pH of the solution is lowered in the presence of a soluble chelator that extracts Eu3+ into solution. Strong luminescence from the soluble metal ion is then easily detected by a time-resolved measurement.

The detection of aromatic carboxylates via the formation of ternary complexes using lanthanide ion complexes of functionalised diaza-crown ethers 30 and 31 has been demonstrated [134]. Like the previous examples, these complexes contained vacant coordination sites but the use of carboxylic acid arms resulted in overall cationic 2+ or 1+ complexes. Furthermore, the formation of luminescent ternary complexes was possible with both Tb(III) and Eu(III). A number of antennae were tested including picolinate, phthalate benzoate and dibenzoylmethide. The formations of these ternary complexes were studied by both luminescence and mass spectroscopy. In the case of Eu-30 and Tb-30, the 1 1 ternary complexes were identified. When the Tb(III) and Eu(III) complexes of 30 were titrated with picolinic acid, luminescent enhancements of 250- and 170-fold, respectively, were recorded. The higher values obtained for Tb(III) was explained because there was a better match between the triplet energy of the antenna and a charge transfer deactivation pathway compared to the Eu(III) complex. 

As far as NMR studies are concerned, 2D-EXSY spectra show that the complexes exhibit fast intramolecular rotation of the ligand with respect to the lanthanide ions on the NMR time scale, which corresponds to the rotations of the cyclohexane backbones about the threefold axis in the homotrimetallic complexes A3 (1.16—311 )2(OI I2)r> l3+ (Chapon et al., 2002). Only two NMR signals are detected for protons HI andH2, and plots of 5 ara/( z) vs Cj/(Sz)j (eq. (74)) and Sf /Cj vs (Sf,/Cj (eq. (75)) for [tf3(L16-3H)2(OH2)6]3+ (R = Pr-Yb except Pm and Gd) display linear correlations which have been assigned to isostructurality along the complete lanthanide series (Chapon et al., 2001 fig. 63),... 

Determination of lanthanide ions with a fairly low detection limit of 10-5-10-6 wt% is achieved with Ln111 /i-diketonates. Relying on this fact new, rapid, selective, and highly sensitive analytical methods essentially based on the luminescence properties of these complexes have been developed. After a preliminary work on Smm and Eum chelates (Topilova et al.,... 

The L1 complexes of the middle lanthanides Gd(III), Eu(III), and Tb(III) decompose less rapidly at pH 7.4, 37 °C than do the L1 complexes of La(III) or Lu(III) (14). The fit of the lanthanide ion into the macrocycle may be important here. Certainly, the macrocycle fit will vary for La3+ (116 pM) compared to Lu3+ (97.7 pM) (41). A recent study using luminescence measurements suggests a greater lability of the Eu(L1)3+ complex than previously reported (28). Detection of the Eu(DPTA)-complex produced upon addition of diethylenetriaminepentaacetic acid (DTPA) to Eu(L1)3+ indicates that the complex decomposes approximately 12% in 48 h at 37 °C, pH 7.4. It is noteworthy that solutions of Eu(L1)3+ contain two different species (28). One of them, possibly a hydroxy-bridged dimer, is present in greater amounts at high concentrations of Eu(L1)3+. 

NMR nuclei will enter into an exchange process in which the ligand will displace one or two molecules of water from an aquo lanthanide ion depending upon whether the ligand is a monodentate or bidentate. Thus a variety of mixed complexes are possible, Ln(L) (H20)m. Hence NMR detects the averaged effects which varies from one lanthanide to another. 

In principle, paramagnetic ions also may be used to induce hyperfine shifts in nucleic acids to aid detection of binding sites. Ions with high relative magnetic anisotropy and short unpaired electron relaxation times (i.e., Co Fe and trivalent lanthanide ions except for Gd ) are candidates for such studies. Indeed, Tb and Eu ions have been used as fluorescent probes of nucleic acid structures it is expected that NMR studies also would be informative. " ... 

Ancillary ligands can dramatically improve detection strategies based on sensitized Ln luminescence by stabilizing the lanthanide to pH variations. Such enhancements in stability and reproducibility allow the use of lanthanide sensors in situ and also potentially in vivo, where free lanthanide ions might precipitate and/or have toxic effects. 

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