T. Nagano et al.
tion). In contrast, the analogous phosphate derivative of
[Tb-1] was not very stable in neutral aqueous buffer, pre-
sumably due to the intramolecular catalytic effect of the car-
boxylate group (data not shown). Kinetic parameters were
also determined (Figure S8 in the Supporting Information),
and the turnover number (kcat) of [Tb-5] seemed comparable
to that of 4-nitrophenyl phosphate (PNPP), a standard chro-
mogenic substrate of ALP.[24] Taken together, these results
indicate that [Tb-5] is a good substrate of ALP, being hydro-
lyzed to [Tb-2]. Upon excitation at l=330 nm, the Tb3+ lu-
minescence increased approximately fivefold within 10 min
(Figure 3C), whereas there was a threefold decrease of lu-
minescence intensity in response to excitation at l=280 nm.
The time-dependent change of time-resolved excitation
spectra showed a clear isosbestic point at l=315 nm (Fig-
ure 3D). When the ratio between excitation wavelengths lEx
=330 nm and lEx =280 nm was plotted as a function of time,
a dramatic increase was observed compared with single-
wavelength measurement (Figure S9 in the Supporting In-
formation). Finally, the applicability of the system to high-
throughput screening was demonstrated by monitoring the
change of the ratio (lEx >310 nm/lEx =280 nm) on addition
of ALP in a 96-well plate (Figure 3E, ethylenediaminetetra-
acetic acid (EDTA) was used as a model inhibitor). As
shown in Figure S10 in the Supporting Information, this
method provided higher sensitivity than can be obtained
with the standard colorimetric detection of ALP activity by
using PNPP.
In conclusion, we have developed a strategy to create ra-
tiometric luminescent probes based on lanthanide com-
plexes. The idea of exploiting the absorption shift accompa-
nying the target reaction is quite simple, but as far as we
know, its applicability to Tb3+ or Eu3+ complexes has never
been demonstrated. As the scaffold of the probes, we fo-
cused on SA–DTPA–Tb3+, which can respond to reactions
involving both the hydroxyl and carboxyl groups. Compari-
son of four model complexes, [Tb-1]–[Tb-4], showed that
both lmax and FLum are influenced by derivatization at the
two groups. We utilized these results to develop [Tb-5] as a
probe for ratiometric measurement of ALP activity. Of
course, SA-based sensors are unlikely to be suitable for all
target molecules of biological interest, and their excitation
wavelengths are relatively short for biological applications.
However, we believe that this strategy is complementary to
other currently available methods. Our approach should at
least be easily adaptable to other hydrolases. Notably, there
are a few luminescent probes in the literature that exhibit
absorption changes in response to certain ions (although
they were not studied as ratiometric probes),[25] and this fact
suggests that our strategy is potentially generalizable. Fur-
thermore, other scaffolds suitable for probes might be
found, for example, by screening aromatic compounds. The
availability of ratiometric sensing should further enhance
the established advantages of lanthanide complexes, consid-
ering that dual-excitation-wavelength fluorescent probes are
already in widespread use due to their utility and robust-
ness.[26]
Acknowledgements
We thank Yasuteru Urano and Hirotatsu Kojima for helpful advice. This
work was supported in part by the Ministry of Education, Culture,
Sports, Science and Technology of Japan (Grant Nos. 22000006 to T.N.
and T.T., and 23651231 to T.T.). T.T. was also supported by the Cosmetol-
ogy Research Foundation and Mochida Memorial Foundation for Medi-
cal and Pharmaceutical Research, Japan.
Keywords: hydrolases
·
lanthanides
·
luminescence
·
ratiometric detection · sensors
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