A lanthanide-complex-based ratiometric luminescent probe specific for peroxynitrite

Article abstract of DOI:10.1002/chem.201000528

A lanthanide-complex-based ratiometric luminescence probe specific for peroxynitrite (ONOO^-), 4'-(2, 4-dimethoxyphenyl)-2,2':6',2 - terpyridine6,6 -diyl]bis(methylenenitrilo)tetrakis(acetate)-Eu ³⁺/Tb³⁺ ([Eu³⁺/Tb³⁺ (DTTA)]), has been designed and synthesized. Both [EU³⁺(DTTA)] and [Tb ³⁺(DTTA)] are highly water soluble with large stability constants at ≈10²⁰, and strongly luminescent with luminescence quantum yields of 10.0 and 9.9%, respectively, and long lumines-cence lifetimes of 1.38 and 0.26 ms, respectively. It was found that the luminescence of [Tb ³⁺(DTTA)] could be quenched by ONOO^- rapidly and specifically in aqueous buffers, while that of [Eu³⁺(DTTA)] did not respond to the addition of ONOO^-. Thus, by simply mixing [Eu ³⁺(DTTA)] and [Tb³⁺(DTTA)] in an aqueous buffer, a ratiometric luminescence probe specific for time-gated luminescence detection of ONOO^- was obtained. The performance of [Tb³⁺(DTTA)] and [Eu³⁺/ Tb³⁺(DTTA)] as the probes for luminescence imaging detection of ONOO^- in living cells was investigated. The results demonstrated the efficacy and advantages of the new ratiometric luminescence probe for highly sensitive luminescence bioimaging application.

Full text of DOI:10.1002/chem.201000528

DOI: 10.1002/chem.201000528
A Lanthanide-Complex-Based Ratiometric Luminescent Probe Specific for
Peroxynitrite
Cuihong Song,[a] Zhiqiang Ye,[b] Guilan Wang,[b] Jingli Yuan,*[a, b] and Yafeng Guan*[a]
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FULL PAPER
Abstract: A lanthanide-complex-based
ratiometric luminescence probe specific
for peroxynitrite (ONOOꢀ), 4’-(2,4-di-
methoxyphenyl)-2,2’:6’,2’’-terpyridine-
6,6’’-diyl]bis(methylenenitrilo)tetrakis-
cence lifetimes of 1.38 and 0.26 ms, re-
spectively. It was found that the lumi-
[Tb3+
ACTHNGUTER(NNUG DTTA)] in an aqueous buffer, a
ratiometric luminescence probe specific
for time-gated luminescence detection
of ONOOꢀ was obtained. The perfor-
nescence of [Tb3+
ACHTNUTRGNEUNG(DTTA)] could be
quenched by ONOOꢀ rapidly and spe-
cifically in aqueous buffers, while that
mance of [Tb3+
(DTTA)] and [Eu3+
ACHTUNGTERNNUNG /
A
([Eu3+/Tb3+
of [Eu3+
A
Tb3+
ACTHNUTRGNE(NUG DTTA)] as the probes for lumi-
A
nescence imaging detection of ONOOꢀ
in living cells was investigated. The re-
sults demonstrated the efficacy and ad-
vantages of the new ratiometric lumi-
nescence probe for highly sensitive lu-
minescence bioimaging application.
E
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
with large stability constants at ꢁ1020,
and strongly luminescent with lumines-
cence quantum yields of 10.0 and
9.9%, respectively, and long lumines-
Keywords: analytical methods · cell
imaging · lanthanides · lumines-
cence · peroxynitrite
Introduction
(DHR-123), dihydrodichlorofluorescein (DCFH), and so
forth, the specificity of these probes is low and the exact
mechanism of the oxidation of these probes by ONOOꢀ is
still a question of debate. Thus, there is an imperative need
to develop new ONOOꢀ-specific fluorescent probes. In this
regard, several such probes have been synthesized recently
by using mechanism-based molecular design, involving
either aromatic nitration (NiSPYs)[6] or ketone oxidation re-
actions (HKGreen).[7] These probes are based on a photoin-
duced electron transfer (PET) mechanism and show dramat-
ic fluorescence enhancement upon reaction with ONOOꢀ.
They suffer, however, from a few limitations: poor water
solubility and photostability, small Stokes shift, and inferior
intracellular retentions. Furthermore, all the reported
ONOOꢀ fluorescent probes only show changes in fluores-
cent intensity, which could be influenced by many factors,
for example, excitation intensity, dye concentration, sample
environment, and so forth. Thus, a ratiometric fluorescent
probe that can eliminate most or all ambiguities by self-cali-
bration of two emission bands[8] for ONOOꢀ is highly desir-
able.
It has long been appreciated that lanthanide complexes
afford considerable scope for the development of novel
chemical entities that can be used as luminescence probes,
as components of optoelectronic devices, or as key sensor
materials.[9] Luminescent lanthanide complexes have long lu-
minescence lifetimes, large Stokes shifts, and sharp emission
profiles; these properties enable them to be used for micro-
second time-gated (or time-resolved) luminescence meas-
urements to eliminate fast decaying autofluorescence from
biological specimens, scattering lights, and optical compo-
nents. Recently, we have demonstrated that lanthanide com-
plexes are useful time-gated luminescence probes for ROS.
Three lanthanide complex-based luminescent probes have
been successfully developed for the highly sensitive and se-
lective time-gated luminescence detection of singlet oxygen
(1O2).[9a,10] Based on the above, we have attempted to devel-
op the lanthanide complex-based ratiometric luminescence
probe for specific detection of ONOOꢀ, since three recent
reports have demonstrated that the bis-lanthanide ensemble
Peroxynitrite (ONOOꢀ), a short-lived reactive oxygen spe-
cies (ROS), has attracted much attention, because it can
cause serious damage to living systems.[1] It is generated in
biological systems through the spontaneous coupling reac-
Cꢀ
tion of nitric oxide (NO) and superoxide radical (O2 ). This
peroxide in itself is very reactive; however, its biological
action is particularly notorious under elevated cell/tissue
Cꢀ
rates of NO and/or O2 production. Increasing evidence has
shown that aberrant ONOOꢀ activities may contribute to a
series of human diseases including inflammatory processes,
ischemic reperfusion injury, multiple sclerosis, stroke,
cancer, and neurodegenerative disorders.[2]
Until now, several approaches have been developed for
the detection of ONOOꢀ, including UV/Vis spectroscopy,
chemiluminescence, amperometry and electron spin reso-
nance, and immunohistochemistry.[3] Due to the short life-
time, low concentration, high activity, and elusive nature of
ONOOꢀ in vivo, the precise pathogenic role of ONOOꢀ in
biological systems is still not very clear. To study the physio-
logical role of ONOOꢀ in living cells or tissues, a fluorescent
probe technique was developed as a useful tool, because of
its high sensitivity, selectivity, and experimental conven-
ience.[4] Though a number of fluorescent probes[5] have been
developed and are widely used to monitor ONOOꢀ in vari-
ous biological systems, for example, dihydrorhodamine-123
[a] C. Song, Prof. J. Yuan, Prof. Y. Guan
Department of Instrumentation and Analytical Chemistry
Dalian Institute of Chemical Physics
Chinese Academy of Sciences, Dalian 116023 (China)
Fax : (+86)411-84706293
Fax : (+86)411-84379590
E-mail: jingliyuan@yahoo.com.cn
guanyafeng@dicp.ac.cn
[b] Dr. Z. Ye, Prof. G. Wang, Prof. J. Yuan
School of Chemistry, Dalian University of Technology
Dalian 116012 (China)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201000528.
Chem. Eur. J. 2010, 16, 6464 – 6472
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www.chemeurj.org
6465

J. Yuan, Y. Guan et al.
consisting of a mixture of Eu3+/Tb3+complexes can be used
to generate ratiometric luminescence probes.[11a–c]
In this work, a new nonadentate ligand, [4’-(2,4-dimeth-
a very useful tool for long-term visualization and detection
of ONOOꢀ in intact cells to elucidate the precise role of
ONOOꢀ in various biological processes.
ACHTUNGTRENNUNG
A
R
Results and Discussion
complexes in aqueous buffers are highly luminescent with
the same excitation pattern. After addition of ONOOꢀ, the
Probe design and characterization: The use of fluorescent
probes is one of the most promising methods to analyze and
clarify the roles of ROS in biological processes. Recent ef-
forts in our laboratory have focused on the rational design
of functional lanthanide luminescent probes for ROS. Com-
pared with conventional organic probes, these lanthanide
probes possess a unique advantage in that the luminescence
measurement can be carried out with a time-gated mode to
minimize the effect of background noise.[13] Until now, al-
though some organic-dye-based fluorescent probes for
ONOOꢀ have been developed, a lanthanide-complex-based
probe for ONOOꢀ, to our knowledge, has never been re-
ported. In this work, a new lanthanide chelator—DTTA—
was designed and synthesized by incorporating a 2,4-dime-
thoxyphenyl group into a (2,2’:6’,2’’-terpyridine-6,6’’-diyl)-
bis(methylenenitrilo)tetrakis(acetic acid) moiety, since the
former can specifically respond to ONOOꢀ[6] and the latter
can form highly luminescent and stable complexes both with
Eu3+ and Tb3+ ions in aqueous media.[14]
luminescence of [Tb3+
whereas that of [Eu3+
“cocktail” of [Eu3+/Tb3+
gated luminescence detection of ONOOꢀ, a bi-exponential
correlation between the emission intensity ratio (Eu3+
Tb3+) and the ONOOꢀ concentration was obtained. To in-
vestigate the potential use of [Tb3+(DTTA)] and the [Eu3+
Tb3+(DTTA)] mixture for the detection of ONOOꢀ in living
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
/
A
/
ACHTUNGTRENNUNG
cells, the cell permeable form of DTTA, acetoxymethyl
ester of DTTA (AM-DTTA, Scheme 1), was also synthe-
sized. Both AM-DTTA and Ln3+ (Eu3+ and Tb3+) could be
easily transferred into the cultured cells with ordinary incu-
bation methods, and in the cells, accompanied by the rapid
hydrolysis of AM-DTTA catalyzed by ubiquitous intracellu-
lar esterases,[12a] the stable [Ln3+
ACTHNUTRGNEUNG(DTTA)] complex was
formed (Scheme 1). This process is similar to the cell load-
ing process of the Ca2+ luminescent probe Fura 2.[12b] The
[Tb3+(DTTA)]- and [Eu3+/Tb3+
ACHTUNGTRENNUNG ACHTUNGTRENN(UGN DTTA)]-loaded cells were
used for the luminescence imaging detection of ONOOꢀ.
The results suggest that the Eu3+/Tb3+-complex-based ratio-
metric luminescence probe developed in this work could be
The time-gated excitation and emission spectra of [Eu3+
ACHUTNGRENNUG CAHTUNGTRENNUNG
(DTTA)] and [Tb3+
Both [Eu3+
N
ACHTUNGTRENNUNG
AHCTUNGTERG(NNUN DTTA)] complex shows a typi-
cal Eu3+ emission pattern with
a main emission peak at 612 nm
7
(5D0! F2) and several side
peaks centered at 584, 592, 646,
and 692 nm; the [Tb3+
ACHTUNGTRENUNG(DTTA)]
complex shows a typical Tb3+
emission pattern with a main
emission peak at 541 nm (5D4!
7F5) and several side peaks cen-
tered at 486, 581, and 617 nm.
In an air-saturated 0.05m borate
buffer of pH 9.1, the lumines-
cence quantum yield and life-
time of [Eu3+
10.0% and 1.38 ms, respective-
ly, and those of [Tb3+
(DTTA)]
ACHTUNGTRENUN(NG DTTA)] are
AHCTUNGTRENUNG
are 9.9% and 0.26 ms, respec-
tively. The shorter luminescence
lifetime of the Tb3+ complex is
considered to be caused by the
dissolved oxygen. In an argon-
saturated buffer, the lumines-
cence lifetime of the Tb3+ com-
plex was measured to be
Scheme 1. Schematic diagram of the cell loading process of [Ln3+
ACTHNUTRGNE(NUG DTTA)] complex.
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Cell Imaging
FULL PAPER
dination sphere of Eu3+ ion was 0.05, calculated by the
equation q=1.2(1/tH Oꢀ1/tD Oꢀ0.25)[16] (tD O and tH O are lu-
2
2
2
2
minescence lifetimes of [Eu3+
ACHTNUTRGNE(NUG DTTA)] in D2O and H2O
buffers, respectively). The effects of pH on the luminescence
intensity and lifetime of [Ln3+(DTTA)] (Ln3+: Eu3+ or
Tb3+) were investigated by using 1.0 mm of [Ln3+
(DTTA)] in
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
0.05m Tris-HCl buffers at different pHs ranging from 2 to 10
(Figure 2). The results show that both luminescence intensi-
ty and lifetime of [Ln3+
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
cent probe in weakly acidic, neutral, and weakly basic buf-
fers.
Figure 1. Top: Time-gated excitation and emission spectra of [Eu3+
ACHTUNGTRENNUNG ACHTUNGTRENNUNG
(DTTA)] (2.0 mm, solid lines) and [Tb3+
[Eu3+/Tb3+
ACHTUNGTRENNUNG(DTTA)] mixtures (total concentration of 2.0 mm) with differ-
ent molar ratios of Eu3+/Tb3+ in 0.05m borate buffer of pH 9.1.
0.68 ms, while that of the Eu3+ complex was almost un-
changed (1.33 ms). Since DTTA can form highly lumines-
cent complexes both with Eu3+ and Tb3+ ions, the [Eu3+
/
/
ACHTUNGTRENNUNG
Tb3+(DTTA)] mixtures with different molar ratios of Eu3+
Tb3+ show different luminescence emission spectra when ex-
cited at the same wavelength (335 nm). Thus, by adjusting
the relative amount of Eu3+ and Tb3+, a variety of emission
spectra were obtained (Figure 1, bottom). These results indi-
cate that DTTA is an ideal Eu3+/Tb3+ chelator for a two-
emission-wavelength signaling approach.
The [Eu3+
high stability in aqueous media, because of the polyacid
structure of the ligand. When [Eu3+(DTTA)] and [Tb3+
(DTTA)] were exposed to fivefold excess of ethylenedi-
amine tetraacetic acid, the conditional stability constants,
measured by Verhoevenꢁs method,[15] were found to be 7.2ꢂ
1020 and 1.6ꢂ1020 for [Eu3+(DTTA)] and [Tb3+
(DTTA)],
respectively. In addition, no significant changes in the lumi-
nescence intensities of the two complexes were observed
after several weeks at room temperature. The average
number (q) of coordinated water molecules in the first coor-
ACHTUNGTRENNUNG ACHTUNGTRNE(NUGN DTTA)] complexes show a
(DTTA)] and [Tb3+
AHCTUNGTRENNUNG
Figure 2. Effects of pH on the luminescence intensity (black square) and
ACHTUNGTRENNUNG
lifetime (circle) of [Eu3+(DTTA)] (top) and [Tb3+
ACTHUNTGRNENUG ACHTUNTGERN(NUGN DTTA)] (bottom).
ACHTUNGTRENNUNG
A
U
ACTHNGUTERNNUG
Reaction between [Ln3+(DTTA)] and ONOOꢀ: The effects
of ONOOꢀ on the optical properties including luminescence
intensity, lifetime, and time-gated luminescence intensity of
the [Ln3+
ACTHNUGRTENUNG(DTTA)] complexes were investigated. Figure 3
ACTHNUTRGNEUNG
shows the optical responses of [Eu3+(DTTA)] and [Tb3+
Chem. Eur. J. 2010, 16, 6464 – 6472
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J. Yuan, Y. Guan et al.
Figure 4. Time-gated excitation and emission spectra of [Eu3+
(2.0 mm) (top) and [Tb3+
(DTTA)] (2.0 mm) (bottom) in the presence of
different concentrations of ONOOꢀ.
ACHTUNGTRENNUNG(DTTA)]
Figure 3. Optical responses (square: luminescence intensity; triangle: lu-
minescence lifetime; circle: time-gated luminescence intensity) of [Eu3+
AHCTUNGTRENNUNG
ACHTUNGTRENNUNG ACHTUNGTRENNUNG
(DTTA)] (2.0 mm) (top) and [Tb3+
5
excited state energy level of the D4 for Tb3+ (20400 cmꢀ1)
5
G
is higher than that of the D0 for Eu3+ (17200 cmꢀ1).[11a,17]
Thus, the quenching of the [Tb3+
ACTHNUTRGNE(UNG DTTA)] luminescence by
R
ONOOꢀ can be considered to occur by a charge-transfer
mechanism, which has been examined more recently and
shown to involve interaction of the electron-rich quenching
species with the excited chromophore (exciplex).[11d]
AHCTUNGTRENNUNG
was decreased gradually. Moreover, the luminescence life-
time and time-gated luminescence intensity of [Tb3+
A
The different quenching behaviors of [Eu3+
A
tial decays) with the increase of ONOOꢀ concentration. The
results of luminescence titration experiments with ONOOꢀ
also indicate that the time-gated excitation and emission
[Tb3+
[Eu3+
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
metric luminescence probe for ONOOꢀ. Compared with lu-
minescence-intensity-based probes, ratiometric luminescence
probes allow the emission intensities at two different wave-
lengths to be measured; this process provides a built-in cor-
rection for environmental effects and increases the selectivi-
ty and sensitivity of the measurement. Accordingly, by using
spectra of [Eu3+
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
N
a “cocktail” of [Eu3+/Tb3+
ACTHNGUTER(NNUG DTTA)], the emission intensity
G
ratio of the Eu3+/Tb3+ should be a direct function of the
ONOOꢀ concentration. Figure 5 shows the time-gated emis-
based on the d-PET mechanism. Early work has demon-
strated that the lanthanide excited state can be deactivated
by some electron-rich species, and the quenching of Tb3+
emission is greater than that of Eu3+ emission because the
sion spectra of [Eu3+/Tb3+
ACTHNUTRGNE(NUG DTTA)] mixture in the presence
of different concentrations of ONOOꢀ. As expected, the lu-
minescence intensity of Tb3+ emission was remarkably de-
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FULL PAPER
Figure 5. Time-gated emission spectra of [Eu3+/Tb3+
ACHTNUTRGNEUNG(DTTA)] mixture
(total concentration of 2.0 mm, Eu3+/Tb3+ =1:2) in the presence of differ-
ent concentrations of ONOOꢀ (the inset shows the intensity ratio of F612
F541 as a function of ONOOꢀ concentration).
/
creased upon addition of ONOOꢀ, but the response for
Eu3+ emission was weak. The inset in Figure 5 shows the
variation of the emission intensity ratio of Eu3+ (612 nm) to
Tb3+ (541 nm) as a function of ONOOꢀ concentration. The
curve, fitted to a double exponential correlation with a de-
tection limit of ꢁ7 mm, is similar to that of the reported lan-
thanide-complex-based ratiometric probe for uric acid.[11a]
The specificity of luminescence response of the complex
towards ONOOꢀ was also investigated. As shown in
Figure 6 (top), the luminescence intensity changes of [Tb3+
Figure 6. Time-gated luminescence intensity of [Tb3+
ACHTUNGTRENNUNG
(top) at 541 nm and F612/F541 ratio of [Eu3+/Tb3+
ACHTUNGTRENNUNG
ꢀ
1
ꢀ
Cꢀ
concentration of 2.0 mm, Eu3+/Tb3+ =1:2) (bottom) in the presence of
various ROS (200 mm) at room temperature.
ACHTUNGTRENNUNG(DTTA)] in the presence of ClO , O2, O2 , H2O2 and NO3
are very small (<5%) compared with the remarkable de-
crease (98%) in the presence of ONOOꢀ. It is notable that
ꢀ
C
NO, OH and NO2 also induced ꢁ20, ꢁ30, and ꢁ50% de-
crease, respectively, in the luminescence intensity of [Tb3+
(DTTA)]. It seems likely that NO, NO2 and other decom-
The luminescence quenching kinetics of ONOOꢀ–[Tb3+
ꢀ
A
ACHTUNGTREN(NUGN DTTA)] system was determined by real-time recording the
position products of ONOOꢀ are also quenching species for
the luminescence of [Tb3+
(DTTA)]. However, when the
[Eu3+/Tb3+
(DTTA)] mixture was used as a ratiometric
probe, the high specificity towards ONOOꢀ was obtained.
As shown in Figure 6 (bottom), a 5.6-fold increase in F612
F541 ratio can be observed when [Eu3+/Tb3+
(DTTA)] is re-
acted with ONOOꢀ, whereas the reactions of [Eu3+/Tb3+
time-gated luminescence intensity change of [Tb3+
ACHTUNGTRENUNG(DTTA)]
A
after addition of ONOOꢀ. As shown in Figure 7, upon addi-
tion of different concentrations of ONOOꢀ, the lumines-
ACHTUNGTRENNUNG
cence intensity of [Tb3+
ACTHNUTRGNE(NUG DTTA)] decreased rapidly, and
/
achieved a steady value in a very short time (<3 s). This
U
result indicates that the luminescence quenching of the
ONOOꢀ–[Tb3+
ACTHNUGRTENUNG(DTTA)] system is very fast, which is favor-
1
ꢀ
ꢀ
Cꢀ
C
U
able for the detection of short-lived ONOOꢀ in neutral
ACHTUNGTRENNUNG
can not induce the change of F612/F541 ratio (the change
<0.1). Although a ꢁ0.7-fold increase is observed in the
aqueous solutions (t50 <1 s).[18]
ꢀ
presence of NO2 , this response is much weaker than that in
Luminescence imaging of ONOOꢀ in living cells: The cul-
tured HeLa cells were used to investigate the potential of
the new probe for the luminescence imaging detection of
ONOOꢀ in living systems. When the cells were co-incubated
the presence of ONOOꢀ. The specificity increase of the ra-
tiometric probe is considered to be attributed to the over-
lapping of the Tb3+ emission at 617 nm and the Eu3+ emis-
sion at 612 nm, which causes the F612/F541 ratio of the ratio-
metric probe to be also related to the F617/F541 ratio of the
Tb3+ luminescence. It is quite evident that the Tb3+ emis-
sion at 617 nm is beneficial to the specificity increase of the
ratiometric probe.
with [Tb3+
ACHTUNGTRENNUNG
the time-gated mode, indicating that [Tb3+
AHCTUNGTRENNUNG
permeate through the cell membrane into the cells. After
co-incubating with AM-DTTA and Tb3+ ions, bright cyan
(mixture of blue and green from cell components and the
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6469

J. Yuan, Y. Guan et al.
intracellular esterases, the stable and cell-membrane-imper-
meable [Tb3+
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
used as a probe for the time-gated luminescence imaging de-
tection of ONOOꢀ in living cells.
The [Eu3+/Tb3+
ACTHNUTRGNEU(GN DTTA)]-loaded HeLa cells were also
prepared by co-incubating the cells with AM-DTTA and
Eu3+/Tb3+ (Eu3+/Tb3+ =1:2) mixture. In this case, the
strong hybrid luminescence (pale blue) from the cells was
observed (Figure 9A). After treatment with different con-
centrations of SIN-1 for 30 min, the color changes of the
cells from pale pink to red were observed (Figure 9B to
9D). This phenomenon can be explained as follows. Upon
addition of SIN-1, the luminescence of [Tb3+
ACHTUNGTRENUN(NG DTTA)] was
gradually quenched, while that of [Eu3+
AHCTUNGTRENNUNG
which caused the increase of the emission intensity ratio of
the Eu3+/Tb3+ in the cells, yielding a series of luminescence
Figure 7. Luminescence quenching kinetic curves of [Tb3+
ACTHNUTRGNEU(GN DTTA)]
(2.0 mm) at different concentrations of ONOOꢀ in 0.05m Tris-HCl buffer
of pH 7.4.
Tb3+ complex) luminescence and a strong green time-gated
luminescence (from the Tb3+ complex) from the cells were
observed (Figure 8A). When the [Tb3+
ACHTNUTRGNEUNG(DTTA)]-loaded
HeLa cells were treated with a ONOOꢀ donor, 3-morpholi-
nosydnonimine (SIN-1), for 30 min, both normal lumines-
cence and time-gated luminescence intensities of the cells
gradually decreased with increasing SIN-1 concentration
(Figure 8B and 8C). The above results demonstrate that
AM-DTTA and Tb3+ ions can be easily transferred into the
cultured HeLa cells by an ordinary incubation method.
After hydrolysis of the acetoxymethyl ester by ubiquitous
Figure 8. Bright-field (left), normal luminescence (middle), and time-
gated luminescence (right) images of the [Tb3+
N
Figure 9. Bright-field (left) and normal luminescence (right) images of
cells in the presence of ONOOꢀ. The [Tb3+
G
ACTHNGUTERNNUG
the [Eu3+/Tb3+(DTTA)]-loaded HeLa cells in the presence of ONOOꢀ.
The [Eu3+/Tb3+
ACTHNUTRGNE(NUG DTTA)]-loaded cells were prepared by incubating HeLa
a
cells in the culture medium (1.0 mL) containing a freshly prepared solu-
tion of AM-DTTA (0.5 mm of total DTTA) and Eu3+/Tb3+ mixture
(0.5 mm, Eu3+/Tb3+ =1:2) for 2 h at 378C in a 5% CO2/95% air incuba-
tor, and were further incubated in the isotonic saline solution containing
different concentrations of SIN-1 (A: negative control; B: 100 mm of SIN-
1; C: 200 mm of SIN-1; D: 500 mm of SIN-1) for another 30 min, and then
used for the luminescence microscopy imaging detection. Scale bars:
10 mm.
DTTA) and Tb3+ (0.5 mm) for 2 h at 378C in a 5% CO2/95% air incuba-
tor, and were further incubated in the isotonic saline solution containing
different concentrations of SIN-1 (A: negative control; B: 500 mm of SIN-
1; C: 1.0 mm of SIN-1) for another 30 min, and then used for the lumines-
cence microscopy imaging detection. Scale bars: 10 mm. The time-gated
luminescence images are shown in pseudo-color (wavelength of 545 nm)
treated by a SimplePCI software.[9a]
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Chem. Eur. J. 2010, 16, 6464 – 6472

Cell Imaging
FULL PAPER
color changes of the cells. Since the luminescence color
change is easier observed than the luminescence intensity
change, the ratiometric luminescence probe [Eu3+/Tb3+
ric luminescence probe specific for ONOOꢀ was successfully
developed. This probe can specifically and rapidly recognize
ONOOꢀ, resulting from remarkable luminescence quench-
(DTTA)] is more favorable to be used for the sensitive and
ACTHNGUTRENNUG ACTHNUGTREN(NGNU DTTA)].
ing for [Tb3+(DTTA)], but no response for [Eu3+
selective detection of ONOOꢀ in living cells compared with
other intensity-based luminescent probes. Due to the lack of
a true-color time-gated luminescence microscope, the per-
formance of this ratiometric luminescence probe for imaging
ONOOꢀ with time-gated mode to eliminate the effect of au-
tofluorescence from the cells was not determined.
Compared with previously reported ONOOꢀ luminescence
probes, the new probe has the advantages of good specifici-
ty, sensitivity, kinetic and thermodynamic stabilities, water
solubility, wide pH available range, and the applicability for
ratiometric and time-gated measurements. The results of lu-
minescence imaging by monitoring ONOOꢀ in living cells
demonstrated the utility of the probe for in vivo ONOOꢀ
detection. The new luminescence probe, with fine ratiomet-
ric and time-gated capacities, provides a novel strategy for
visualizing the temporal and spatial distribution of ONOOꢀ
in cells and biological tissues, which would be a useful tool
for investigating the pathogenic role of ONOOꢀ in biologi-
cal systems.
To examine the intracellular retention of the probe, [Eu3+
ACHTUNGTRENNUNG(DTTA)]-loaded HeLa cells were prepared. After washing
twice with the isotonic saline solution, luminescence images
of the cells were determined over a period of 30 min at
10 min intervals (Figure 10). As expected, there were no
Experimental Section
Materials: The ONOOꢀ solution was prepared according to the literature
method,[19] and was stored at ꢀ308C. The concentration was determined
by using its molar extinction coefficient of 1670mꢀ1 cmꢀ1 at 302 nm before
use.[7a] 1-Hydroxy-2-oxo-3-(3-aminopropyl)-3-methyl-1-triazene (NOC-
13) was synthesized by using a reported method.[20] Bromomethyl acetate
and 3-morpholinosydnonimine (SIN-1) were purchased from Sigma–Al-
drich. HeLa cells were obtained from Dalian Medical University. The iso-
tonic saline solution consisting of 140 mm NaCl, 10 mm glucose, and
3.5 mm KCl was prepared in our laboratory. Deionized and distilled
water was used throughout. Unless otherwise stated, all chemical materi-
als were purchased from commercial sources and used without further
purification.
Physical measurements: The 1H NMR spectra were measured on
a
Figure 10. Luminescence images of the [Eu3+
in the isotonic saline solution within 30 min. The [Eu3+
AHCTUNGTRENNUNG
cells were prepared by incubating HeLa cells in the culture medium
(1.0 mL) containing freshly prepared solution of AM-DTTA (0.5 mm of
total DTTA) and Eu3+ (0.5 mm) for 2 h at 378C in a 5% CO2/95% air in-
cubator. Scale bars, 10 mm.
ACHTUNGTRENNUNG
Bruker DRX 400 spectrometer (400 MHz). The MS spectra were record-
ed on a HP1100 LC/MSD electrospray ionization mass spectrometry
(ESI/MS). Elemental analysis was carried out on a Vanio-EL CHN ana-
lyzer. Melting points were determined on a WRS-1B digital melting-
point apparatus. Absorption spectra were measured on a Perkin–Elmer
Lambda 35 UV/Vis spectrometer. Time-gated luminescence spectra were
measured on a Perkin–Elmer LS 50B spectrofluorometer with the set-
tings of delay time, 0.2 ms; gate time, 0.4 ms; cycle time, 20 ms; excitation
slit, 10 nm; and emission slit, 5 nm. Luminescence quantum yields were
measured by using the previously reported methods.[10] HPLC analysis
was carried out on a SinoChrom ODS-BP 5 mm (4.6ꢂ250 mm) column
by using an HPLC system consisting of two pumps (Elite P230) and a de-
tector (Elite UV 230+). All normal luminescence imaging and time-
gated luminescence imaging measurements were carried out on a labora-
tory-use luminescence microscope.[9a] The microscope (TE2000-E;
Nikon), equipped with a 100 W mercury lamp, a UV-2A filters (Nikon,
excitation filter, 330–380 nm; dichroic mirror, 400 nm; emission filter,
>420 nm) and a color CCD camera system (RET-2000R-F-CLR-12-C,
Qimaging) was used for the normal luminescence imaging measurement
with an exposure time of 20 s. The microscope, equipped with a 30 W
xenon flash-lamp (Pulse300, Photonic Research Systems), UV-2A filters
and a time-gated digital black-and-white CCD camera system (Photonic
Research Systems) was used for the time-gated luminescence imaging
measurement with the conditions of delay time, 50 ms; gate time, 1000 ms;
lamp pulse width, 6 ms; and exposure time, 90 s.
changes of the luminescence images in the period of 30 min,
indicating that the probe [Eu3+
ACTHNUTRGNE(UNG DTTA)] in the cells could
not be transferred to the outside solution. This result dem-
onstrates that, compared with conventional organic fluores-
cent probes, the new [Ln3+
ACTHNUTRGNE(NUG DTTA)] probe has superior in-
tracellular retention and is more favorable to be used for
the visualization and continuous observation of ONOOꢀ in
living systems. In addition, the result of a trypan blue ex-
periment shows that the [Ln3+
ACTHNUTRGNE(UNG DTTA)]-loaded HeLa cells
still retain their activity, which indicates that the effect of
the probe on the viability of the cells is low.
Conclusion
Synthesis of the ligand DTTA: The details of the DTTA synthesis are
shown in Supporting Information.
By incorporating a 2,4-dimethoxyphenyl moiety into a lan-
thanide
bis(methylenenitrilo)tetrakis
Tb3+
(DTTA)], the first lanthanide-complex-based ratiomet-
luminophore,
(2,2’:6’,2’’-terpyridine-6,6’’-diyl)-
Synthesis of the acetoxymethyl ester of DTTA (AM-DTTA): A solution
of DTTA (18.5 mg, 0.025 mmol), dry triethylamine (14.6 mg, 1.30 mmol),
and bromomethyl acetate (37.5 mg, 0.25 mmol) in dry dimethyl sulfoxide
A
E
,
/
ACHTUNGTRENNUNG
Chem. Eur. J. 2010, 16, 6464 – 6472
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
6471

J. Yuan, Y. Guan et al.
(0.5 mL) was stirred at room temperature overnight. The resulting brown
mixture containing AM-DTTA and un-esterified DTTA was used for the
cell imaging experiment without further purification. ESI-MS (m/z) for
AM-DTTA: m/z (%): 948.3 (25) [M+H]+.
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Reactions of [Ln3+
out in the air-saturated 0.05m Tris-HCl buffer of pH 7.4 with the same
concentration of [Tb3+(DTTA)] (2.0 mm) or [Eu3+/Tb3+
(DTTA)] (total
ACHTUNGTRENNUNG(DTTA)] with ROS: All the reactions were carried
A
ACHTUNGTRENNUNG
concentration of 2.0 mm, Eu3+/Tb3+ =1:2) for 30 min at room tempera-
ture. Various ROS were generated with the following procedures. Hydro-
gen peroxide and hypochlorite were delivered from 30 and 10% aqueous
solutions, respectively. Prior to use, hydrogen peroxide was assayed by
using its molar absorption coefficient of 43.6mꢀ1 cmꢀ1 at 240 nm.[21] Super-
Cꢀ
oxide solution (O2 ) was prepared by dissolving solid KO2 in dry dimeth-
yl sulfoxide and the mixture was stirred vigorously for 10 min before use.
C
Hydroxyl radical ( OH) was generated by the Fenton reaction between
ferrous ammonium sulfate and hydrogen peroxide.[9a] NO was generated
by using NOC-13 as an NO donor.[20] Singlet oxygen (1O2) was generated
by the reaction of hypochlorite with hydrogen peroxide.[22] The stock so-
lution of ONOOꢀ was used throughout.
Detection of reaction kinetics of [Tb3+
tion kinetics between [Tb3+(DTTA)] and ONOOꢀ was determined in the
air-saturated 0.05m Tris-HCl buffer of pH 7.4 at room temperature. After
the ONOOꢀ solution was added to [Tb3+
(DTTA)] solution (2.0 mm), the
(DTTA)] with ONOOꢀ: The reac-
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
AHCTUNGTRENNUNG
kinetic curve was recorded immediately on a Perkin–Elmer LS 50B lumi-
nescence spectrometer with a time-gated luminescence mode with the
settings of delay time, 0.2 ms; gate time, 0.4 ms; cycle time, 20 ms; excita-
tion wavelength, 335 nm; emission wavelength, 541 nm; excitation slit,
10 nm; emission slit, 5 nm; and data interval, 0.1 s.
Detection of ONOOꢀ in aqueous media: The reactions of [Eu3+
ACHTUNGTRENNUNG ACHTUNGTRENNUNG ACTHNUGTRENNGUN
(DTTA)], [Tb3+(DTTA)], and [Eu3+/Tb3+(DTTA)] with ONOOꢀ were
carried out in the air-saturated 0.05m Tris-HCl buffer of pH 7.4 at room
temperature. A series of ONOOꢀ solutions at different concentrations
were added to the buffer solutions containing 2 mm of [Eu3+
ACHTUNGTRENNUNG
[Tb3+(DTTA)], or [Eu3+/Tb3+
ACHTUNGTRENNUNG ACHTUNGTRENNUNG
for 15 min, the time-gated excitation and emission spectra were mea-
sured.
Luminescent cell imaging: HeLa cells were cultured in RPMI-1640
medium, supplemented with 10% fetal bovine serum, 1% penicillin, and
1% streptomycin at 378C in a 5% CO2/95% air incubator. For ONOOꢀ
imaging detection, the [Tb3+(DTTA)]- or [Eu3+/Tb3+
ACTHNUTRGENNUG ACHUTNTGREN(NUGN DTTA)]-loaded
cells were prepared by adding AM-DTTA and Tb3+ or AM-DTTA and
Eu3+/Tb3+ into the culture medium of HeLa cells (4ꢂ105 cellsmLꢀ1) ac-
cording to the conditions as described in the legend of Figures 8–10.
After incubation for 2 h at room temperature, the cells were separated
by centrifugation at 1000 rpm, washed three times with the isotonic
saline solution, and confirmed with the trypan blue experiment. The cells
were further incubated in the isotonic saline solution containing the
ONOOꢀ donor SIN-1 for another 30 min, and then spotted on a glass
slide for luminescence microscopy imaging detection.
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Acknowledgements
Financial supports from the National Natural Science Foundation of
China (Nos. 20835001, 20975017) and the Specialized Research Fund for
the Doctoral Program of Higher Education of China (No. 200801410003)
are gratefully acknowledged.
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Received: March 1, 2010
Published online: May 19, 2010
6472
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Chem. Eur. J. 2010, 16, 6464 – 6472