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maximum of the one-photon excitation spectrum (375 nm); extrapolation based on molar
absorption coefficients yields s
(2)
9 GM, which is sizeable, but still much
smaller than the cross-sections reported for mononuclear complexes with ligands opti-
mized for multiphoton absorption [79]. Despite this low performance, HeLa cells have
been incubated in the presence of 200 mM[Eu
2
(L11)
3
] and their image recorded on a
luminescence multiphoton confocal microscope upon excitation at 750 nm; internalized
helicates are quite visible, which opens interesting perspectives (Figure 6.9, right) [78].
All these experiments show that dinuclear lanthanoid helicates qualify as efficient and
versatile luminescent stains for imaging live cells by time-resolved luminescence micros-
copy, a technique which produces highly contrasted images in view of the elimination of
the fluorescence background by time discrimination. However, the staining is nonspecific,
the helicates being uptaken by several cell lines, malignant or not. A more targeted
approach is needed for the specific detection of cancerous cells, which is described in the
next section.
(760 nm)
¼
6.2.5.2 Specific Bioimaging of Breast Cancer Cells and Tissues
Attaining specificity in the detection of a given cell line by simple chemical probes is
difficult, even almost impossible. A much easier way is to make use of biochemical reac-
tants which target specific markers expressed by the cells and to attach a luminescent tag
onto these reactants, usually monoclonal antibodies. To this end, helicates have to be
modified for coupling with the biochemical molecules [75]. The coupling is achieved
either directly, that is the lanthanoid probe is linked to a monoclonal antibody mAb, or
indirectly with the chelate being covalently bound first to avidin (or biotin); the resulting
duplex is then fixed onto a biotinylated (or avidin derivatized) monoclonal antibody,
B-mAb, via the strong avidin-biotin interaction (log
K
10
15
) [80], as described in Fig-
ure 6.10. Alternatively, avidin may be substituted by streptavidin [81] or bovine serum
albumin (BSA) [82].
A relatively easy way to achieve bioconjugation is to graft a carboxylic acid group
onto the ligand, for example H
4
L20 (Scheme 6.4), which will then react with amine
groups of the biochemical molecule. This has been done, yielding [Ln(H
2
L20)
3
]-avidin
conjugates (LnL20-A). MALDI-TOF spectra and a quantitative UV-vis analysis point to
the binding being covalent, with an average of 3.2 helicates bound per avidin molecule.
The properties of the Eu
III
helicate and bioconjugate have been checked. The
addition of carboxylic acid groups leads to a decrease in quantum yield to 15
2% for
[Eu(H
2
L20)
3
] and 9.3
2% for [Eu
2
(L7)
3
].
But all other photophysical properties are maintained, in particular the Eu
0.9% for EuL20-A, as compared with 21
5
D
0
Þ
ð
lifetime
(2.45
0.01 ms, respectively) and the overall shape of the emission
spectra. Most important too, the biochemical activity of avidin is unchanged. Finally,
bioaffinity assays demonstrate that LnL20-A linked to a specific monoclonal antibody
can target the 5D10 antigen expressed by MCF-7 human breast cancer cells and that it
performs as well as a commercially available lanthanoid tag and much better than an
all-organic conjugate [83].
The method is particularly effective in the detection of cultured cells grown in micro-
channels, a way of achieving screening tests more rapidly (cells grow more rapidly in
fibronectin-coated microchannels than in a bulk culture medium) and in a more cost-
0.04 and 2.17
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