emission (545 nm) under neutral pH conditions after oxidation
by ROS, was successfully designed and synthesized. The
chemiluminescence intensity of KBI was much higher than
the intensities of known commercially available Cypridina
luciferin analogs such as MCLA and FCLA, and at a longer
wavelength than the chemiluminescence of MCLA. The direct
bonding of the imidazopyrazine moiety to the BODIPY
fluorophore evoked high-performance chemiluminescence.
The selectivity measurements of KBI towards various ROS
revealed that KBI has high specificity for O2À. Finally, the
highly sensitive detection of ROS generated from HL-60
cells was demonstrated with KBI. Consequently, a new type
of functional chemiluminescent probe for bioanalytical
application has been developed. In the future, the detection
of ROS generated from particular organelles (e.g. mitochondria)
will be attempted by modifying KBI with organelle specific
targeting moieties.
Fig.
3
The signal to background ratio for chemiluminescence
generated from stimulated cells and non-stimulated cells (reference)
using KBI and MCLA.
are described in the ESIw). In order to demonstrate the
applicability of ÀKBI for bioanalytical applications, the
detection of O2 generated from living cells (the human
leukemia cell line: HL-60) was investigated.
We kindly acknowledge Mr Hidehiro Kubota, Mr Atsushi
Asakawa, and Mr Tsutomu Irie from ATTO corporation for
granting us access to the chemiluminescence measurement
devices.
HL-60 cells (106 cells) were loaded with KBI (HBSS buffer,
pH 7.4 containing 1% DMSO: 1 mM, 200 ml) or MCLA
(HBSS buffer, pH 7.4: 1 mM, 200 ml) by incubation at
37 1C/CO2 5% for 40 min. Then, the suspensions containing
probe-loaded cells were transferred to a black 96-well microplate,
phorbol 12-myristate 13-acetate (PMA; HBSS buffer, pH 7.4
0.1% DMSO: 1 mM, 20 ml) was added as a stimulating agent to
each well, and the chemiluminescence was detected on an
AB-2350 PHELIOS (ATTO corporation). ROS are known
to be generated when HL-60 cells are stimulated with PMA.17
The chemiluminescence at each well was collected every seven
seconds for KBI, and every ten seconds for MCLA, respectively.
The averaged chemiluminescence intensities are represented as
bars in Fig. 3. The signal to background ratio, that is the
chemiluminescence intensity observed for stimulated cells
(KBI-CL and MCLA-CL, respectively) compared to the
intensity observed for non-stimulated cells (KBI-reference
and MCLA-reference, respectively), is higher for KBI than
for MCLA. This indicates that KBI responds to ROS generated
from HL-60 cells, and that the sensitivity of KBI is higher
than that of MCLA. Furthermore, the absolute integrated
chemiluminescence of KBI is much larger than that of MCLA
(see Fig. S-3 of the ESIw). Significantly, the signal to noise
ratio of the emission spectra of KBI is obviously high, whereas
that of MCLA is too low to be distinguished as a significant
signal from the background (see Fig. S-3 of the ESIw).
Thus, KBI enables the sensitive detection of ROS generated
from stimulated cells, which is difficult to achieve by using
commercially available luciferin-based chemiluminescent
probes such as MCLA. Furthermore, it can be assumed with
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À
high probability that O2 was generated from HL-60 cells,
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À
response towards O2
.
In conclusion, a novel luciferin-based long-wavelength
chemiluminescent probe (KBI), which shows yellow-green
ꢁc
This journal is The Royal Society of Chemistry 2009
Chem. Commun., 2009, 3047–3049 | 3049