Visible light excitable zn2+ fluorescent sensor derived from an intramolecular charge transfer fluorophore and its in vitro and in vivo application

Article abstract of DOI:10.1021/ja806489y

The UV- and sensor-induced interferences to living systems pose a barrier for in vivo Zn²⁺ imaging. In this work, an intramolecular charge transfer (ICT) fluorophore of smaller aromatic plane, 4-amino-7-nitro-2,1,3- benzoxadiazole, was adopted

Full text of DOI:10.1021/ja806489y

Published on Web 01/12/2009
Visible Light Excitable Zn2+ Fluorescent Sensor Derived from
an Intramolecular Charge Transfer Fluorophore and Its in
Vitro and in Vivo Application
Fang Qian,† Changli Zhang,†,§ Yumin Zhang,† Weijiang He,*,† Xiang Gao,‡
Ping Hu,‡ and Zijian Guo*,†
State Key Laboratory of Coordination Chemistry, Coordination Chemistry Institute, School of
Chemistry and Chemical Engineering, Nanjing UniVersity, Nanjing 210093, P. R. China, Animal
Model Research Center, Nanjing UniVersity, Nanjing 210061, P. R. China, and Department of
Chemistry, Nanjing Xiaozhuang College, Nanjing 210017, P. R. China
Received August 15, 2008; E-mail: zguo@nju.edu.cn; heweij69@nju.edu.cn
Abstract: The UV- and sensor-induced interferences to living systems pose a barrier for in vivo Zn2+ imaging.
In this work, an intramolecular charge transfer (ICT) fluorophore of smaller aromatic plane, 4-amino-7-
nitro-2,1,3-benzoxadiazole, was adopted to construct visible light excited fluorescent Zn2+ sensor, NBD-
TPEA. This sensor demonstrates a visible ICT absorption band, a large Stokes shift, and biocompatibility.
It emits weakly (Φ ) 0.003) without pH dependence at pH 7.1-10.1, and the λex and λem are 469 (ε469
)
2.1 × 104 M-1 cm-1) and 550 nm, respectively. The NBD-TPEA displays distinct selective Zn2+-amplified
fluorescence (Φ ) 0.046, ε469 ) 1.4 × 104 M-1 cm-1) with emission shift from 550 to 534 nm, which can
be ascribed to the synergic Zn2+ coordination by the outer bis(pyridin-2-ylmethyl)amine (BPA) and 4-amine.
The Zn2+ binding ratio of NBD-TPEA is 1:1. By comparison with its analogues NBD-BPA and NBD-PMA,
which have no Zn2+ affinity, the outer BPA in NBD-TPEA should be responsible for the Zn2+-induced
photoinduced electron transfer blockage as well as for the enhanced Zn2+ binding ability of 4-amine.
Successful intracellular Zn2+ imaging on living cells with NBD-TPEA staining exhibited a preferential
accumulation at lysosome and Golgi with dual excitability at either 458 or 488 nm. The intact in vivo Zn2+
fluorescence imaging on zebrafish embryo or larva stained with NBD-TPEA revealed two zygomorphic
luminescent areas around its ventricle which could be related to the Zn2+ storage for the zebrafish
development. Moreover, high Zn2+ concentration in the developing neuromasters of zebrafish can be
visualized by confocal fluorescence imaging. This study demonstrates a novel strategy to construct visible
light excited Zn2+ fluorescent sensor based on ICT fluorophore other than xanthenone analogues. Current
data show that NBD-TPEA staining can be a reliable approach for the intact in vivo Zn2+ imaging of zebrafish
larva as well as for the clarification of subcellular distribution of Zn2+ in vitro.
tion on Zn2+ homeostasis and requires a combination of suitable
animal models and fluorescent sensors, is highly demanded. The
transparent zebrafish embryo or larva is a widely used model
in developmental biology, especially for the study of neurode-
Introduction
Zn2+ plays vital roles in cellular metabolism, gene expression,
apoptosis, neurotransmission, and so forth.1 It is also associated
with physical growth retardation and neurological disorders such
as cerebral ischemia and Alzheimer’s disease.2 The spatial and
temporal tracking of in vivo Zn2+ is challenging but essential
to address these issues. Although Zn2+ imaging in living cells
or hippocampus slices has been achieved using diversified Zn2+
sensors,3,4 intact in vivo Zn2+ imaging, which provides informa-
(3) (a) Frederickson, C. J.; Kasarskis, E. J.; Ringo, D.; Frederickson, R. E.
J. Neurosci. Methods 1987, 20, 91–103. (b) Nasir, M. S.; Fahrni, C. J.;
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H.; Urano, Y.; Nagano, T. Angew. Chem., Int. Ed. 2003, 42, 2996–
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† School of Chemistry and Chemical Engineering.
‡ Animal Model Research Center.
§ Nanjing Xiaozhuang College.
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Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3605–3610. (d) Frederickson,
C. J.; Bush, A. I. Biometals 2001, 14, 353–366.
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10.1021/ja806489y CCC: $40.75
2009 American Chemical Society

Visible Light Excitable Zn2+ Fluorescent Sensor
A R T I C L E S
Chart 1. Chemical Structures of NBD-TPEA, NBD-PMA, and
NBD-BPA
velopment, and its fluorescent imaging has been one of the most
frequently used techniques in this field.5 In fact, the intact in
vivo imaging of Ca2+ in zebrafish larva has been successfully
conducted recently using different Ca2+ fluorescent sensors.6 It
is therefore conceivable to use zebrafish larva for in vivo Zn2+
fluorescent imaging to clarify the roles of Zn2+ in the develop-
ment of zabrafish, especially in the development of the
neurosystem.
For the intact in vivo Zn2+ fluorescent imaging of living cells
and zebrafish larvae, reduced UV-induced phototoxicity/autof-
luorescence and sensor-induced interference are essentially
demanded.1c,3c,7 Therefore, visible light excited sensor of
biocompatibility is appealing. The reported visible light excited
Zn2+ fluorescent sensors thus far are mainly derived from bulk
xanthenone fluorophores, such as fluorescein and rhod-
amine.3c-e,4a,b Most of them have been successfully applied in
cell imaging. However, different laser/filter sets of fluorescence
microscopes and the diversified Zn2+ imaging experiments, such
as the complicated colocalization imaging, require visible light
excitable sensors differing in excitation and emission, intracel-
lular distribution, Zn2+ binding constants, and so forth.1c,3b,f-j
Therefore, novel visible light excitable sensors based on
fluorophores other than xanthenone are highly desired. Because
of the intermolecular charge transfer (ICT) effect, ICT fluoro-
phores with conjugated electron donor (D) and receptor (A) are
able to display visible ICT absorption band and large Stokes
shift, although their aromatic plane is smaller.8 In addition to
the visible excitability, sensor based on ICT fluorophore may
even provide the advantage of lower interference to the living
systems because of their small aromatic plane. Moreover, the
larger Stokes shift should be favorable for the improvement of
imaging quality because of the reduced excitation interference.
The possible two-photon excitability of their D-π-A motif
may even offer additional advantage of deeper sectioning in in
vivo imaging.4c Therefore, the development of novel fluorescent
Zn2+ sensors based on typical ICT fluorophore is an attractive
and promising strategy.
biocompatibility.11 Different from the normal photoinduced
electron transfer (PET) fluorophores, such as anthracene, this
typical ICT fluorophore possesses larger Stokes shift (∼80 nm)
and visible absorption band due to the ICT effect from 4-amine
to the conjugated 7-NO2,9 yet it possesses a smaller aromatic
plane. On the other hand, Hg2+ sensors, formed by bridging
ANBD with sulfur-containing ionophore, displayed not only the
emission enhancement but also the emission shift, which
demonstrated the possibility to construct a sensor of both PET
and photoinduced charge transfer (PCT) sensing behaviors.12
Since the reported Cu2+ sensor 4-bis(pyridin-2-ylmethyl)amino-
7-nitro-2,1,3-benzoxadiazole (NBD-BPA) with Zn2+ ionophore
bis(pyridin-2-ylmethyl)amine acting as 4-amino donor group (4-
BPA) displayed very poor Zn2+ affinity owing to the ICT effect
from 4-amine to 7-nitro,13 in this work, an additional BPA
moiety is introduced to replace one of the pyridine of NBD-
BPA to construct a new Zn2+ sensor, NBD-TPEA. The
additional BPA is expected to coordinate to Zn2+ in a synergic
manner with the retained (pyridin-2-yl)methylamine (PMA)
motif. To clarify the emission and sensing behavior of NBD-
TPEA, its simple analogue NBD-PMA without the outer BPA
motif has also been studied for comparison. Chart 1 shows the
chemical structures of NBD-TPEA, NBD-PMA, and NBD-
BPA.
In this work, a novel approach in developing visible light
excitable Zn2+ fluorescent sensor based on the typical ICT
fluorophore, 4-amino-7-nitro-2,1,3-benzoxadiazole (ANBD, λex
≈ 460 nm), is reported.9 The ANBD is closely associated with
the frequently used amine labeling agents, 4-chloro- or 4-fluoro-
7-nitro-2,1,3-benzoxadiazole (4-ClNBD or 4-FNBD). The two
labeling agents emit intensively when their 4-chloro- or 4-fluoro-
is substituted by amine to form ANBD fluorophore.10 More
importantly, ANBD derivatives have been frequently affixed
to different biomolecules for cell imaging, displaying favorable
Results and Discussion
Synthesis. NBD-TPEA and NBD-PMA were prepared by
reacting commercially available 4-ClNBD with N,N,N′-tri(py-
ridin-2-ylmethyl)ethane-1,2-diamine (TPEA) or PMA in the
presence of base in THF. The electron-withdrawing effect of
7-nitro group makes 4-Cl substitutable by nucleophilic agents
such as amino- or mercapto- group in ambient condition via an
SN2 pathway. The instant appearance of green emission after
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A R T I C L E S
Qian et al.
Figure 1. Emission spectra of 5 µM NBD-PMA (a) and NBD-TPEA (b) in water containing 10% DMSO (v/v) at different pH. λex ) 469 nm. The pH value
is shown above each individual spectrum. Inset in (a): The fluorescent pH titration profile of NBD-PMA according to F/FpH6.9 at 542 nm. Inset in (b): The
fluorescent pH titration profile of NBD-TPEA according to F/FpH7.1 at 534 nm.
mixing 4-ClNBD with the amines suggests the reaction is quite
efficient. In fact, the nonemissive 4-ClNBD is an effective
fluorescent labeling agent for amine-containing compounds in
variable chromatographic detection.10c,d
Fluorescence of NBD-TPEA and Its pH Dependence. NBD-
PMA and NBD-TPEA can be readily dissolved in water
containing 10% DMSO, and their fluorescence spectroscopic
study was carried out in HEPES buffer (pH 7.4) containing 10%
DMSO except for the fluorescent pH-dependence determination.
For NBD-PMA, intensive emission with λex and λem at 469 and
542 nm was observed at neutral condition. This compound
displays stable emission at pH 3.9-8.9, whereas the lower pH
at 3.1 induces the minor emission blue shift from 542 to 534
nm. The protonation of 4-amine, which is the only amino group
in NBD-PMA, alters the PCT process, resulting in the blue
shift.14
Figure 2. UV spectra of NBD-TPEA (40 µM) obtained during the titration
by Zn(NO3)2 (1.5 mM) in HEPES buffer (1:9, DMSO/water, v/v; 50 mM
HEPES, 100 mM KNO3; pH ) 7.40). Inset is the titration profile according
to the absorbance at 496 nm.
However, NBD-TPEA fluoresces weakly in neutral aqueous
solution (pH 7.4) with λex and λem at 469 and 550 nm,
respectively. The Stokes shift of 81 nm satisfies the requirement
for a practical sensor providing high quality imaging and is
larger than the reported values of many visible light sensors
derived from xanthenone analogues (their Stokes shifts are
normally ∼30 nm). Its emission intensity does not change
significantly at pH 7.1-10.1. However, a distinct emission
enhancement (enhancement factor of ∼13) accompanied by the
emission blue shift from 550 to 534 nm can be observed when
reducing pH from 7.1 to 2.0 (Figure 1). The stable fluorescence
of NBD-TPEA at around pH 7.0 is favorable for its in vivo
application. In fact, many of the reported visible light excitable
Zn2+ sensors based on xanthenone structures showed evident
fluorescent pH dependence at this condition.15
The comparison of the emission behavior between NBD-
PMA and NBD-TPEA reveals that the latter functions mainly
as a PET sensor yet possesses some degree of the PCT
property.14 This emission behavior should be attributed to the
presence of two amine groups. The BPA (outer amine) con-
necting to the ANBD fluorophore via an ethylene group is able
to quench the emission of ANBD fluorophore via a through-
space PET process. However, the 4-amino group (inner amine)
functions as the donor of the ICT fluorophore. Both amines can
be prontonated at lower pH. At neutral condition, the quantum
yield of NBD-TPEA accounts for only 1/25 of that of NBD-
PMA (Figure S1 in the Supporting Information). This should
be due to the presence of the additional outer amine group in
NBD-TPEA, which quenches the emission of ANBD fluoro-
phore via a through-space PET process. The protonation of outer
amine at pH e 7.1 blocks the PET process and results in the
emission enhancement of NBD-TPEA. Moreover, the emission
shift of NBD-TPEA can be observed at pH 7.4-6 (from 550
to 542 nm) and at pH 3-2 (from 542 to 534 nm). The latter
change is identical to the protonation effect of 4-amine observed
for NBD-PMA. It seems that both 4-amine and pyridine
protonation contribute to the emission shift.13
Both NBD-TPEA and NBD-PMA demonstrate that the
4-amine protonation of ANBD fluorophore only leads to minor
blue shift in emission. Therefore, metal coordination at 4-amine
in ANBD is not expected to induce large emission shift. This
is supported by the fact that the reported Hg2+ and Cu2+ sensors
based on ANBD fluorophore did not show any distinct emission
shift in their sensing process.12,13,16
Zn2+ Binding Behavior of NBD-TPEA. As a Zn2+ fluorescent
sensor, the Zn2+ binding behavior of NBD-TPEA was inves-
tigated in detail. The UV-vis titration experiment demonstrates
that free NBD-TPEA has two main absorption bands centering
at 496 and 350 nm, which can be assigned to ICT and π-π*
transition bands, respectively (Figure 2). When Zn2+ was added,
distinct reduction of the former band can be observed ac-
(14) Jiang, P.; Guo, Z. Coord. Chem. ReV. 2004, 248, 205–229.
(15) Nolan, E. M.; Ryu, J. W.; Jaworski, J.; Feazell, R. P.; Sheng, M.;
Lippard, S. J. J. Am. Chem. Soc. 2006, 128, 15517–15528.
(16) Boiocchi, M.; Fabbrizzi, L.; Licchelli, M.; Sacchi, D.; Va´zquez, M.;
Zampa, C. Chem. Commun. 2003, 1812–1813.
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Visible Light Excitable Zn2+ Fluorescent Sensor
A R T I C L E S
Chart 2. Proposed Zn2+ Binding Mode of NBD-TPEA
density of the meta-carbon atom. A similar observation was
reported by Sakamoto and co-workers in the ANBD-based Hg2+
sensor.12 All the signals for the substituting TPEA undergo
significant changes during Zn2+ titration process, suggesting that
all N atoms of pyridine and amine participate in Zn2+ coordina-
tion directly. The proposed Zn2+ binding mode is shown in Chart
2.
Figure 3. 1H NMR spectra of NBD-TPEA obtained during the titration
with Zn2+ in CD3OD. (a) Spectrum of free sensor. (b) Spectrum obtained
when the ratio of [Zn2+
]total/[sensor] equals 0.5:1. (c) Spectrum obtained
when the ratio of [Zn2+
]total/[sensor] is 1:1. The signals marked with + and
In aqueous medium, NBD-PMA does not display any evident
g are for the protons from free sensor and zinc-bound sensor, respectively.
1
change in its UV-vis and H NMR spectra when titrated by
Zn2+. The results indicate that its 4-PMA motif solely has poor
affinity to Zn2+. However, when the additional BPA is intro-
duced to 4-amine via an ethylene bridge in NBD-TPEA, the
PMA especially its 4-amine is also involved in Zn2+ coordina-
tion in a synergic manner and alters both the π-electron density
and ICT effect of ANBD fluorophore. From the comparison of
Zn2+ binding behaviors between NBD-BPA and NBD-TPEA,
it can be concluded that the additional outer BPA in NBD-TPEA
connecting to ANBD via ethylene enhances the Zn2+ affinity,
whereas the inner BPA (4-BPA) directly connected to NBD
ring has poor Zn2+ affinity.13
1
Table 1. Assignments of H NMR Spectra of NBD-TPEA during
Zn2+ Titration in CD3OD
protons
signals for free NBD-TPEA (ppm)
signals for Zn2+/NBD-TPEA (ppm)
Hc′
8. 49 (d, 1H, J ) 4.0 Hz)
8.39 (d, 2H, J ) 4.0 Hz)
8.35 (d, 1H, J ) 9.0 Hz)
7.79 (t, 1 H, J ) 8.0 Hz)
7.70 (t, 2H, J ) 7.0 Hz)
7.55 (d, 2H, J ) 7.5 Hz)
7.34 (m, 2H)
8.37 (s, 1H)
Hc
8.58 (s, 2H)
Hb
He′
8.35 (d, 1H, J ) 9.0 Hz)
7.92 (m, 1H)
He
Hf
Hd′, Hf′
8.22 (t, 2H, J ) 7.0 Hz)
7.82 (d, 2H, J ) 7.5 Hz)
7.53 (d, 1H, J ) 7.5 Hz) Hf′
7.39 (m, 1H) Hd′
7.67 (m, 2H)
6.34 (d, 1H, J ) 9.0 Hz)
5.17 (s, 2H)
4.42 (s, 2H)
Hd
Ha
Hg
Hh
Hj
7.20 (t, 2H, J ) 6.0 Hz)
6.21 (d 1H, J ) 9.0 Hz)
5.34 (br, 2H)
Zn2+ Fluorescent Response of NBD-TPEA. Fluorescence
titration of NBD-TPEA by Zn2+ in HEPES buffer exhibits a
linear emission enhancement with [Zn2+
]total, and an enhance-
4.20 (br, 2H)
3.92 (s, 4H)
ment factor of ∼14 is observed when the [Zn2+]/[NBD-TPEA]
4.62, 4.31 (2d, J ) 16 Hz)
3.23 (m, 2H)
Hi
3.06 (t, 2H, J ) 6.0 Hz)
ratio attains 1:1. Then, its fluorescence turns out to be stable
and higher [Zn2+
]
total
does not lead to any further evident
enhancement, suggesting that there is no additional complex
species formed in addition to the 1:1 Zn2+/NBD-TPEA complex
(Figure 4). As a PET fluorescent compound, the Zn2+-induced
emission enhancement of NBD-TPEA should mainly be caused
by the PET blocking effect induced by the Zn2+ coordination
to the outer amine. Emission shift from 550 to 534 nm induced
by Zn2+ addition is similar to that induced by protonation.
Therefore, the shift process could be correlated to the Zn2+
coordination of 4-amine, which reduces the donating ability of
4-amino group of ANBD. The Zn2+-induced change in ICT
effect can be confirmed by the ICT band shift from 496 to 478
nm in UV-vis Zn2+ titration.
companied by the evident hypsochromic shift to 478 nm,
suggesting the decreased electron-donating ability of 4-amino
group induced by Zn2+ binding. Similar change was also
observed for the latter band, yet the band shift is much less
pronounced. The clear isosbestic points at 453, 375, and 323
nm imply the undoubted conversion of free NBD-TPEA to a
zinc complex. The titration profile based on the former band
shows that the absorbance descends linearly with [Zn2+
]
total
at
the [Zn2+ total/[NBD-TPEA] ratio e1:1. Higher [Zn2+
] ]total does
not lead to any further evident change, suggesting a 1:1
stoichiometry for the zinc complex.
The Zn2+ binding behavior of NBD-TPEA was further
studied by 1H NMR titration in CD3OD (Figure 3). Upon Zn2+
addition, another set of signals for the Zn2+/NBD-TPEA
complex appears in the spectrum besides the set of signals for
The Zn2+-specific amplified fluorescence of NBD-TPEA was
verified by screening against major biologically relevant metal
cations. As can be noted from Figure 4b, only Zn2+ and Cd2+
induce an emission enhancement; all other tested metal cations
do not induce any notable emission change. Although Cd2+ also
induces an emission enhancement, its effect is much less
pronounced than that of Zn2+ and it will not interfere with Zn2+
detection in living cells because of its scarcity. On the other
hand, K+, Na+, Ca2+, and Mg2+, which are abundant in cells,
do not affect Zn2+ response even when the [M]/[Zn2+] ratio
attains 1000:1. These properties make NBD-TPEA a candidate
as selective sensor for intracellular Zn2+ imaging.
free NBD-TPEA. When the [Zn2+
]total/[NBD-TPEA] ratio is
0.5:1, two sets of signals display almost identical intensity,
wheras when the ratio reaches 1:1, only the signals of zinc
complex can be observed. More Zn2+ does not lead to any
further evident change in the 1H NMR spectrum. The result also
confirms that the Zn2+ binding ratio of NBD-TPEA is 1:1. The
detailed assignments of the signals are shown in Table 1. Zn2+
coordination triggers a clear downfield shift of Ha from 6.21
to 6.34 ppm, wheras the signal for Hb at 8.35 ppm remains
unchanged. It suggests that the 4-amino group may be involved
in direct Zn2+ coordination which decreases the π-electron
It should be noted that Zn2+ binding does not change the
excitation maximum of NBD-TPEA. The extinction coefficients
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A R T I C L E S
Qian et al.
Figure 4. (a) Emission spectra of 5 µM NBD-TPEA in HEPES buffer (50 mM HEPES, 0.1 M KNO3, pH 7.4; DMSO/H2O, v/v 1:10) titrated by Zn(NO3)2
(1.25 mM) solution. The [Zn2+
]
values in the solution are 0, 1.04, 2.08, 3.13, 4.17, 5.21, 6.25, 7.29, 8.33, and 11.25 µM (from bottom to top). Inset is
total
the titration profile according to F/F0 at 544 nm. (b) Histogram of F/F0 at 544 nm induced by 1 equiv of transition-metal cations or 1000 equiv of Na+, Ca2+
and Mg2+ in the same buffer. λExex ) 469 nm.
,
dim image with only the former luminescent area displaying
even lower brightness. When the focal plane for imaging is
changed, scattering bright dots surrounding the dim nucleolus
are displayed in the images (Figure S3 in the Supporting
Information). The exogenous addition of Zn2+ to HeLa cells
leads to a sharp emission enhancement. The followed TPEN
treatment makes the cells show a recovered image with even
lower luminescence. These results indicate that NBD-TPEA is
an effective intracellular Zn2+ imaging agent of cell permeability
and visible excitability. Moreover, NBD-TPEA may be pref-
erentially accumulated in certain organelles of HeLa cells.
Localization studies of NBD-TPEA in HeLa cells were
carried out by costaining with lysosome maker Red DND-99,
mitochondria marker Red CMXRos (a rhodamine derivative),
and Golgi maker BODIPY TR ceramide, respectively (Figure
6).10e,15 There is no colocalization with Red CMXRos no matter
Figure 5. Confocal fluorescence images of HeLa cells upon irradiation at
458 (a,b,c) and 488 nm (d,e,f). (a,d) HeLa cells preincubated in 5 µM NBD-
TPEA solution at room temperature (20 min). (b,e) Rinsed HeLa cells (1
× PBS, three times) in (a,d) were further incubated in 5 µM ZnSO4/
pyrithione (1:1) solution, followed by being rinsed with 5 µM NBD-TPEA
solution. (c,f) HeLa cells in (b,e) rinsed with 25 µM TPEN solution.
whether with or without exogenous introduction of Zn2+
.
However, fine colocalization is observed when Red DND-99 is
costained with NBD-TPEA, and scattered orange dots are
observed in the overlaid images. Similary, fine colocalization
is also found when BODIPY TR ceramide is costained with
NBD-TPEA (Figures 6k-o and S4 in the Supporting Informa-
tion). These results imply that NBD-TPEA is preferentially
accumulated in lysosome and Golgi apparatus. The successful
costaining experiment with mitochondria marker Red CMXRos
also suggests that NBD-TPEA may be able to function
simultaneously with rhodamine-derived Zn2+ sensors such as
X-RhodZin and RhodZin to track Zn2+ in different organelles.3d
The intracellular Zn2+ imaging behaviors of NBD-TPEA on
other cells such as HepG2, PC12, and A549 were also
investigated. Because of the visible excitation, all the imaging
displays clear images without apoptosis evidence in the imaging
process (Figures S5-S7 in the Supporting Information), while
the fluorescence images of HeLa cells dyed by PBITA (a UV-
excitable Zn2+ sensor developed in our laboratory) display
evident cell apoptosis and autofluorescence (Figure S8 in the
Supporting Information). In fact, the reported intracellular Zn2+
imaging with UV-excited sensors such as Zinquin normally gave
faint images providing no details in the cells.18 The intracellular
temporal Zn2+ tracking ability of the sensor was also investigated
on PC12 cells (Figure 7). When dyed by NBD-TPEA, the
of free NBD-TPEA and its zinc complex at excitation maximum
are 2.1 × 104 and 1.4 × 104 M-1 cm-1, respectively. Their
quantum yields in HEPES buffer are 0.003 and 0.046, when
determined with N-methyl ANBD in acetonitrile as the reference
(Supporting Information).17 The Kd of Zn2+/NBD-TPEA com-
plex was determined to be ca. 2 nM from the emission changes
of 30 µL of NBD-TPEA solution (1 mM) mixed with a series
of Zn2+-buffered solutions (Supporting Information).4d
Intracellular Zn2+ Imaging with NBD-TPEA as Imaging
Agent. The intracellular Zn2+ imaging ability of NBD-TPEA
was confirmed first. Since its excitation maximum is 469 nm,
both the laser beams of 458 and 488 nm were adopted as
excitation light for the confocal imaging (Figure 5). Both
excitations give almost identical Zn2+ images, and the images
under irradiation at 458 nm are obtained at higher output power.
Confocal fluorescent imaging of normal HeLa cells stained by
NBD-TPEA displays a feint luminescent area close to the dim
nucleolus. When exogenous Zn2+ is introduced into the cells
via incubation in ZnSO4/pyrithione solution (5 µM), the former
luminescent area becomes brighter and additional scattering
bright dots are also observed. When the cells are treated further
by membrane-permeable chelator, N,N,N′,N′-tetrakis(2-pyridyl-
methyl)ethylenediamine (TPEN),3,4 the cells show a recovered
(18) (a) Zalewski, P. D.; Forbes, J.; Seamark, R. F.; Borlinghaus, R.; Betts,
W. H.; Lincoln, S. F.; Ward, A. D. Chem. Biol. 1994, 1, 153–161. (b)
Zalewski, P. D.; Forbes, I. J.; Betts, W. H. Biochem. J. 1993, 296,
403–408.
(17) Uchiyama, S.; Matsumura, Y.; de Silva, A. P.; Iwai, K. Anal. Chem.
2003, 75, 5926–5935.
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Visible Light Excitable Zn2+ Fluorescent Sensor
A R T I C L E S
Figure 6. Confocal fluorescence images of HeLa cells preincubated with 50 nM MitoTracker Red CMXRos (or 1 µM LysoTracker Red DND-99, 5 µM
BODIPY TR ceramide) and 5 µM NBD-TPEA. (a) Fluorescence image visualized by red emission of Red CMXRos, (b) fluorescence image of cells visualized
by green emission of NBD-TPEA, (c) green fluorescence image of cells after further incubation with 5 µM ZnSO4/pyrithione (1:1) solution, (d) overlay
between (a) and (c), (e) green fluorescence image of HeLa cells experienced (c) followed by the treatment with 25 µM TPEN solution, (f) fluorescence
image visualized by red emission of Red DND-99, (g) fluorescence image visualized by green emission of NBD-TPEA, (h) fluorescence image of cells after
incubation with 5 µM ZnSO4/pyrithione (1:1) solution, (i) overlay between (f) and (h), (j) green fluorescence image of HeLa cells experienced (h) followed
by the treatment with 25 µM TPEN solution, (k) fluorescence image visualized by BODIPY TR ceramide, (l) fluorescence image of cells visualized by green
emission of NBD-TPEA, (m) green fluorescence image of cells after incubation with 5 µM ZnSO4/pyrithione (1:1) solution, (n) overlay between (k) and
(m), (o) green fluorescence image of HeLa cells experienced (m) followed by the treatment with 25 µM TPEN solution. For the NBD-TPEA imaging,
irradiation at 488 nm, band path 500-600 nm. For the imaging of Red CMXRos, Red DND-99 and BODIPY TR ceramide, irradiation at 543 nm, band path
555-650 nm.
Figure 7. Confocal fluorescence images of PC12 cells preincubated with 5 µM NBD-TPEA. (a-e) Fluorescence images in the course of incubation with
5 µM ZnSO4/pyrithione (1:1) solution. (f-j) Fluorescence images of the cells experienced (a-e) obtained in the followed course of the incubation with 25
µM TPEN solution. Red bar ) 10 µm.
confocal images display a faint image almost identical to that
in Figure 7a. If the cells are further incubated with ZnSO4/
pyrithione solution, fast luminescence enhancement can be
observed just as shown in Figure 7a-e. The luminescence
becomes stable in 5 min, and a bright image similar to that in
Figure 7f can be obtained. However, when the cells were further
treated with TPEN, the confocal imaging displayed a reverse
process and the luminescence disappeared in 104 s. The results
suggest that NBD-TPEA staining is reversible and NBD-TPEA
can be an effective sensor for temporal Zn2+ tracking.
conceivable. The preliminary in vivo Zn2+ imaging of intact
4-day-old zebrafish larva with NBD-TPEA staining was per-
formed first using fluorescence microscope (Figure S9 in the
Supporting Information). Although the nonstained zebrafish
larva does not exhibit any evident luminescent areas under
irradiation, the preincubation of larva with 5 µM NBD-TPEA
solution at 28.5 °C for 20 min results in two zygomorphic
luminescent areas (ZLAs) around its ventricle, accompanied by
a few bright dots on the forehead (Figure S9 in the Supporting
Information). The development of zebrafish embryo was tracked
by NBD-TPEA staining. A faint chain composed of green dots
close to the bottom of the venter was observed in the initial
development stage (Figure 8a). The chain becomes brighter and
Intact in Vivo Zn2+ Imaging of Zebrafish with NBD-TPEA. The
favorable intracellular Zn2+ imaging ability on living cells makes
the intact in vivo Zn2+ imaging on living zebrafish larva
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A R T I C L E S
Qian et al.
Figure 8. Fluorescence microscopic images of NBD-TPEA-stained zebrafish larva incubated at 28.5 °C. 1-Phenyl-2-thiourea (PTU) was added in the
incubation media to depress the development of pigment after 8 h of incubation. The final concentration is 0.003%. (a) Fluorescence image of the embryo
after 18 h of incubation, (b) bright-field transmission image of (a), (c) fluorescence image after 25 h of incubation, the total length is ∼1.9 mm, (d) bright-
field image of (c), (e) fluorescence image after 36 h of incubation, the total length is ∼2.7 mm, (f) fluorescence image after 54 h of incubation, the total
length is ∼3.2 mm, (g) bright-field image of (f), (h) fluorescence image of 5-day-old zebrafish larva, the total length is ∼3.5 mm, (i) fluorescence image of
7-day-old zebrafish larva, the total length is ∼4 mm, (j) fluorescence image of 5-day-old zebrafish larva after being treated by TPEN (1 h, 100 µM).
moves to the upside of the venter after 25 h of incubation. The
bright chain moves to the top of the venter after 36 h and
becomes necklace-like (Figure 8e). Further incubation makes
the necklace move to the zebrafish ventricle, displaying as the
ZLAs (Figure 8f, 54 h). There is no evident change in the
fluorescent image in the succedent development course. How-
ever, the ZLAs are no longer observable in the image of 7-day-
old zebrafish, only scattered bright dots are distributed around
the ventricle and forehead. The localization of ZLAs with phase
contrast image for larva after 48 h of incubation was given also
in Figure S10 in the Supporting Information.
The TPEN treatment of 5-day-old larva results in the complete
elimination of the ZLAs, suggesting that the ZLAs and the
former luminescent necklace should be correlated to the presence
of Zn2+ (Figure 8j). Inductively coupled plasma mass spec-
trometry (ICP-MS) data confirm that zinc content in the
separated ZLAs of one larva accounts for 74% of the total zinc
amount of intact larva (4-day-old), although its volume is less
Figure 9. Confocal fluorescence images of the head of a 4-day-old zebrafish
than 1% of the intact larva. Therefore, the disappearance of
larva that was fed in Zn2+ solution (5 µM) at 28.5 °C for 12 h. (a)
ZLAs in the later development stages suggests that the ZLAs
Colocalization of bright-field and fluorescence images for the head (dorsal
view). (b) Zoomed bright-field image of the left part of the head. (c)
Colocalization of bright-field and fluorescence images for the left part of
the head. (d) Zoomed fluorescence image of the left part of the head.
correspond to the Zn2+ storage for the development of zebrafish.
Moreover, 12 h incubation of the 4-day-old larva with Zn2+
-
containing solution (5 µM) induces the numerous bright dots
dispersing from the cephalon to venter. Intriguingly, the former
ZLAs on the chest are no longer observable. The exact Zn2+
feeding effect on the zebrafish development is still unknown
(Figure S9c in the Supporting Information).
suggests the presence of a high concentration of Zn2+. The
position of the bright dots is very close to the neuromasts of
the anterior lateral-line system (ALL system) of zebrafish larva
that relates to the perception and analysis of the water flow.5c,19
It is therefore possible to achieve spatial information on Zn2+
homeostasis of intact zebrafish embryos via optical sectioning
imaging upon NBD-TPEA staining. The sensor may also be
applied for exploring the neurodevelopment of zebrafish
embryos,5c,d,19 where enriched Zn2+ abundance can be easily
observed with simple staining procedure.
Compared with the fluorescence image of zebrafish larva
stained by NBPEA-I (a new visible light excitable Zn2+
fluorescent sensor developed in our laboratory), NBD-TPEA
staining displays images of higher quality because of the lower
background fluorescence (Figures S9 and S11 in the Supporting
Information). The special in vivo distribution pattern of NBD-
TPEA in zebrafish larva may contribute to the lower background
fluorescence. All the results suggest that NBD-TPEA can be
used for in vivo Zn2+ imaging of zebrafish larva and has the
potential to become a useful sensor for clarifying the role of
Zn2+ in developmental biology.
Conclusions
Novel visible light excited Zn2+ fluorescent sensor, NBD-
TPEA, was designed from small ICT fluorophore, ANBD,
Confocal fluorescence imaging of the head of Zn2+-fed
zebrafish larva (4-day-old) via NBD-TPEA staining exhibits
symmetrically distributed bright dots between the two eyes
(Figure 9). The exact organ of the bright dots is not known yet,
but colocalization of the fluorescence and bright-field images
(19) Ghysen, A.; Dambly-Chaudie`re, C. Bioessays 2005, 27, 488–494.
(20) (a) Kawabata, E.; Kikuchi, K.; Urano, Y.; Kojima, H.; Odani, A.;
Nagano, T. J. Am. Chem. Soc. 2005, 127, 818–819. (b) Horner, O.;
Girerd, J. J.; Philouze, C.; Tchertanov, L. Inorg. Chim. Acta 1999,
290, 139–144.
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A R T I C L E S
(t, 1H, J ) 7.9 Hz), 7.39 (m, 1H), 7.35 (m, 1H), 6.27 (d, J ) 8.4
Hz), 4.80 (s, 2H). 13C NMR (Bruker DRX-500, CD3Cl, 500 MHz,
δ, ppm): 156.2, 149.2, 147.3, 144.2, 137.9, 137.1, 122.7, 121.6
102.6, 99.9 (aromatic C), 48.2 (aliphatic C). Anal. Calcd for
C12H9N5O3: C, 53.14; H, 3.34; N, 25.82%. Found: C, 53.43; H,
3.66; N, 25.88%. ESMS (positive mode, m/z): 272.1 [M + H]+,
294.1 [M + Na]+.
Spectroscopic Study. The emission and excitation spectra of
NBD-TPEA and NBD-PMA (5 µM) were determined in a HEPES
buffer (1:9, DMSO/water, v/v; 50 mM HEPES, 100 mM KNO3;
pH ) 7.40) using AMINCO Bowman series 2. Its pH dependence
of emission was determined in aqueous DMSO solution (5 µM,
DMSO/water, 1:9 v/v) at different pH values adjusted by 5 M HNO3
and 5 M NaOH.
Zn2+ Titration of NBD-TPEA and NBD-PMA Solution Deter-
mined by UV, Fluorescence, and 1H NMR Spectroscopy. The UV
titration experiment of NBD-TPEA was carried out by adding
aliquots of 10 µL of Zn(NO3)2 aqueous solution (1.5 mM) to 3 mL
of NBD-TPEA solution (40 µM, 1:9, DMSO/water v/v; 50 mM
HEPES, 100 mM KNO3; pH ) 7.40) in a cuvette. The spectra were
recorded after the solution was completely mixed. The fluorescence
titration experiment was investigated in a similar procedure. The
concentrations of NBD-TPEA and Zn(NO3)2 solutions were 5 µM
and 1.25 mM, respectively. Aliquots of 2.5 µL of Zn2+ solution
were added to 3 mL of NBD-TPEA solution (5 µM, 1:9, DMSO/
water v/v; 50 mM HEPES, 100 mM KNO3; pH ) 7.40). The
excitation wavelength was 469 nm. 1H NMR study of Zn2+ titration
was carried out on Bruker DRX-500 (500 MHz) at 25 ( 1 °C in
CD3OD. Chemical shift was referenced to an external sample of
TMS (δ, 0.00 ppm). The concentration of NBD-TPEA was 20 mM,
and the concentration of Zn(NO3)2 ·6H2O varied from 0 to 30 mM.
All Zn2+ titration experiments of NBD-PMA solution were
carried out in procedures similar to those described above.
Selective Fluorescent Response of NBD-TPEA to Different
Metal Cations. The fluorescent response of NBD-TPEA to
different metal cations was determined in 5 µM NBD-TPEA
buffered solution (1:9, DMSO/water, v/v; 50 mM HEPES, 100 mM
KNO3; pH ) 7.40). Metal cation solution (12.5 µL, 1.2 mM) was
added to 3 mL of this solution, and the fluorescence spectra were
determined after complete mixing. The excitation wavelength was
469 nm.
utilizing its ICT-induced visible ICT absorption and large Stokes
shift. Its distinct selective Zn2+-amplified fluorescence and the
Zn2+-induced minor emission shift in aqueous medium can be
rationalized and attributed to the synergic Zn2+ coordination
by its outer amine (BPA) and inner amine, respectively. The
sensor displays a selective distribution in lysosome and Golgi
as shown from the intracellular Zn2+ imaging. In confocal
imaging, both 488- and 458-nm laser beams can be used to
excite the fluorescence of NBD-TPEA effectively, which
facilitates its costaining experiments with other dyes. This study
demonstrates a novel strategy to construct visible light excitable
Zn2+ sensors via suitable ICT fluorophore modification, which
is different from those based on xanthenone analogues. The
smaller aromatic core of NBD-TPEA may reduce the sensor-
induced interference to the living systems in imaging. Its larger
Stokes shift is also helpful for reducing the influence of
excitation light. Consequently, NBD-TPEA possesses several
unique properties including reduced UV-induced interference,
dual excitability in confocal imaging, fluorescence pH inde-
pendence at neutral conditions, and so forth. Intact in vivo Zn2+
fluorescence and confocal fluorescence imaging of zebrafish
larva with NBD-TPEA revealed some interesting phenomena
associated with Zn2+ distribution, which appears to have never
been observed before. Such a sensor could become valuable in
revealing the roles of Zn2+ in biological systems under either
in vitro or in vivo conditions.
Experimental Section
Materials and Methods. 4-ClNBD and PMA were purchased
from Aldrich. TPEA was prepared according to the reported
procedure.20 All other reagents were commercially available and
of analytical grade. 1H NMR and 13C NMR spectra were determined
by a Bruker DRX-500 spectrometer. All the UV-vis spectra were
recorded by a Shimadzu UV-3100 spectrophotometer. The emission
spectra were obtained using an AMINCO Bowman series 2
fluorescence spectrometer. The pH values of sample solutions were
monitored by a PHS-3 system.
Synthesis of NBD-TPEA. 4-ClNBD (907 mg, 4.59 mmol),
K2CO3 (571 mg, 4.13 mmol), and TPEA (1377 mg, 4.13 mmol)
were mixed in 100 mL of THF. The mixture was stirred overnight
at room temperature. Then the solids were filtered off and washed
with CHCl3. After combining the filtration and the CHCl3 solution,
we removed the solvents by evaporation in vacuo. The hygroscopic
product was obtained by purifying the resulting mixture with silica
gel chromatography. Ethyl acetate/methanol (v/v, 8:1) was used as
the eluent. Yield, 63%.
To clarify whether the coexisting alkaline and alkaline earth metal
cations affect its Zn2+ response, 15 µL of 1 M Ca2+, Mg2+, or Na+
solution was added following the Zn2+ addition (12.5 µL of 1.2
mM Zn(NO3)2), and the emission was determined after complete
mixing. The final concentration of alkaline or alkaline earth metal
cation was 1000 times as high as that of Zn2+
.
Cell Culture Methods and Staining Procedure. The NBD-
TPEA working solution for cell staining was prepared from a 5
mM aqueous stock solution (containing 10% DMSO) of NBD-
TPEA by diluting with 1 × PBS to a final concentration of 5 µM.
HeLa, A549, PC12, and HepG2 cells were cultured in glass
bottom dishes following the same procedure. The culture medium
was Dulbucco’s modified Eagle medium supplemented with 10%
fetal bovine serum, 100 units/mL of penicillin, 100 µg/mL of
streptomycin, and 3.7 mg/mL of NaHCO3.
1H NMR (Bruker DRX500, CD3OD, 500 MHz, δ, ppm): 8.49
(d, 1H, J ) 4.0 Hz), 8.39 (d, 2H, J ) 4.0 Hz), 8.35 (d, 1H, J ) 9.0
Hz), 7.79 (t, 1 H, J ) 8.0 Hz), 7.70 (t, 2H, J ) 7.0 Hz), 7.55 (d,
2H, J ) 7.5 Hz), 7.34 (m, 2H), 7.20 (t, 2H, J ) 6.0 Hz), 6.21 (d
1H, J ) 9.0 Hz), 5.34 (br, 2H), 4.20 (br, 2H), 3.92 (s, 4H), 3.06 (t,
2H, J ) 6.0 Hz). 13C NMR (Bruker DRX500, CD3OD, 500 MHz,
δ, ppm): 160.97, 151.44, 150.59, 147.87, 147.05, 146.89, 139.89,
139.57, 137.64, 126.16, 125.13, 124.79, 124.25, 123.84, 123.82,
104.85 (aromatic C), 62.73, 60.93, 54.56, 52.94 (aliphatic C). Anal.
Calcd for C26H24N8O3: C, 62.89; H, 4.87; N, 22.57%. Found: C,
62.61; H, 5.19; N, 22.30%. ESMS (positive mode, m/z): 497.2 [M
+ H]+.
For intracellular Zn2+ imaging of cells solely stained by NBD-
TPEA, the incubation media was removed and the cells were rinsed
three times with 1 × PBS. Then the cells were incubated in the
NBD-TPEA working solution for 20 min at room temperature.
After removing the solution, we washed the dish three times with
PBS. The confocal images of the cells were obtained using a Leica
TCS-SL microscope equipped with a 63× oil-immersion objective.
The samples were excited at 458 or 488 nm with an Ar laser. A
band-pass from 500 to 600 nm was adopted for observation. For
intracellular Zn2+ imaging of cells with exogenous Zn2+, the
exogenous Zn2+ was introduced by incubating the cells with 5 µM
ZnSO4/2-mercaptopyridine-N-oxide solution, which was prepared
by diluting 5 mM ZnSO4 and 2-mercaptopyridine-N-oxide stock
Synthesis of NBD-PMA. PMA (54 mg, 0.5 mmol) and K2CO3
(76 mg, 0.55mmol) were mixed in 5 mL of THF, then 4-ClNBD
(100 mg, 0.5 mmol) dissolved in 10 mL of THF was added to the
mixture dropwise with stirring at room temperature in 0.5 h. The
solids were removed via filtration after 3 h. Then the product in
the filtrate was purified by silica gel chromatography (dichlo-
romethane/ethyl acetate v/v, 10:1). Yield, 40%.
1H NMR (Bruker DRX-500, CD3Cl, 500 MHz, δ, ppm): 8.70
(d, 1H, J ) 4.8 Hz), 8.55 (d, 1H, J ) 8.7 Hz), 7.97 (b, 1H), 7.80
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solutions with 1 × PBS. Then the cells were dyed with NBD-TPEA
solution in a procedure similar to that described above and imaged.
Zn2+ deprival of the NBD-TPEA dyed by HeLa cells with
exogenous Zn2+ by TPEN solution was also investigated. After
incubation with 5 µM ZnSO4/2-mercaptopyridine-N-oxide solution,
the cells were treated with TPEN solution (TPEN stock solution in
DMSO was diluted with 1 × PBS and the final concentration was
25 µM) followed by washing with 1 × PBS and then the confocal
images were obtained.
NBE-TPEA solution. After being washed by PBS three times, the
larvae were also imaged by a fluorescence stereomicroscope in
similar conditions.
Four-day-old zebrafish larvae cultured similarly but without PTU
were also investigated by fluorescence imaging (Supporting Infor-
mation). After incubation with 5 µM NBD-TPEA solution at 28.5
°C for 20 min, the larvae were washed with 1 × PBS three times
and then embedded in methyl cellulose for imaging. The imaging
of Zn2+-fed zebrafish larvae was also performed, and non-PTU
cultured larvae were used. The 4-day-old zebrafish larvae were fed
with 5 µM Zn2+ solution at 28.5 °C for 12 h. Then the larvae were
washed with 1 × PBS three times followed by NBD-TPEA staining
in a procedure similar to that described above. Then the larvae were
imaged by the fluorescence stereomicroscope in similar conditions.
After removing the NBD-TPEA solution, we rinsed the Zn2+-fed
zebrafish larvae three times with 1 × PBS. Then, the Zn2+-fed
zebrafish were incubated further with 25 µM TPEN solution. After
removing the TPEN solution and rinsing with PBS three times, we
dyed the larvae by NBD-TPEA solution and imaged them.
For the costaining experiments with NBD-TPEA and organelle
marker, the PBS-rinsed cells were dyed first by the marker in their
individual standard procedure followed by NBD-TPEA staining
in the procedure described above. Then, the confocal images were
obtained respectively at the imaging conditions of the marker or
NBD-TPEA. After that, the cells were imposed further with
exogenous Zn2+ in the procedure described above and imaged.
Finally, the cells with exogenous Zn2+ were deprived with TPEN
in the procedure described above. For the lysosome staining by
LysoTracker Red DND-99 (from Invitrogen), the HeLa cells were
incubated in 1 µM Red DND-99 at room temperature for 5 min,
followed by being rinsed with 1 × PBS (two times). For the
mitochondria staining with MitoTracker Red CMXRos (Invitrogen),
the HeLa cells were incubated in 50 nM of Red CMXRos solution
at room temperature for 15 min, followed by being rinsed with 1
× PBS (two times). For the Golgi staining with BODIPY TR
ceramide (Invitrogen), the HeLa cells were incubated in 5 µM
BODIPY TR ceramide solution at 4 °C for 30 min, then the cells
were washed with 1 × PBS two times followed by incubation with
1 × PBS for 30 min. Finally, the cells were washed with 1 × PBS
two times before imaging. The excitation wavelength for NBD-
TPEA in costaining experiments was 488 nm, and the band path
was 500-600 nm. The excitation wavelength for LysoTracker Red
DND-99, MitoTracker Red CMXRos, and BODIPY TR ceramide
was 543 nm. The band path for the marker imaging was from 555
to 650 nm.
The confocal imaging of 4-day-old Zn2+-fed zebrafish larva (non-
PTU cultured) embedded in methyl cellulose was performed on
the head with a Leica TCS-SL confocal microscope equipped with
a 20× objective at the same conditions given in cell confocal
imaging.
ICP-MS Determination of Zinc in Zebrafish Larva and in
the Separated Zygomorphic Luminescent Areas Visualized
by NBD-TPEA Staining around Its Ventricle. Four-day-old
zebrafish larvae were washed with pure water from a Milli-Q system
three times, followed by anaesthetization with tricaine. Then every
two intact larvae or the separated zygomorphic luminescent areas
(separated with microdissecting tweezer under microscope) of three
larvae were put respectively into the polyethylene tube (acid-rinsed)
filled with 100 µL of pure water. Then all the tubes were added
with 50 µL of HNO3 and incubated at 95 °C for 2 h. After the
addition of 20 µL of H2O2, the mixtures were incubated at 95 °C
for 1.5 h. Finally, 20 µL of HNO3 was added and the samples were
incubated at 37 °C for an additional 0.5 h. After the digestion, the
volume of every sample was adjusted to 2 mL with pure water for
ICP-MS determination. Zinc analysis was performed with ICP-MS
using a standard Plasma-Quad II instrument (VG Elemental, Thermo
Optek Corp). The most abundant isotope of zinc (m/z 65) was
measured. Blank samples were also determined as control.
Temporal Zn2+ tracking was investigated on high differential
PC12 cells. Thus, the cells were incubated with 5 µM NBD-TPEA
solution (5 min) and imaged. After removing the NBD-TPEA
solution, we added 5 µM ZnSO4/2-mercaptopyridine-N-oxide
solution and immediately imaged the cells. The imaging lasted for
5 min. Then the Zn2+ solution was removed, and 25 µM TPEN
solution was added. The imaging was started immediately upon
TPEN addition and lasted 5 min.
Intact in Vivo Zn2+ Imaging of Zebrafish Larva with
NBD-TPEA Staining. Zebrafish embryos or larvae were incubated
in pure water from MilliQ system at 28.5 °C. PTU (Sigma) was
added into the incubation media to depress the development of
pigment after 8 h. The final concentration was 0.003%. The
incubation course lasted for more than 7 days. The fluorescence
images of the larvae at certain development stage were obtained
after incubation with 5 µM NBD-TPEA solution at 28.5 °C for 20
min followed by being embedded in methyl cellulose. A Leica
MZ16F fluorescence stereomicroscope was adopted for the obser-
vation. Light from the mercury lamp through GFP2 filter was used
as the excitation light, and the exposure time was 1.0 s. TPEN
treatment to zebrafish larvae was also investigated and imaged. The
5-day-old zebrafish larvae were incubated with 25 µM TPEN
solution at 28.5 °C after being rinsed with 1 × PBS three times.
Then the larvae were washed with PBS three times and dyed by
[Zn2+
]
is 11 ( 2 ppb, [Zn2+] of two intact larva is 20 ( 1
control
ppb, and [Zn2+] of the separated ZLAs of three larvae is 21 ( 4
ppb. ICP-MS data disclose that zinc content in the separated ZLAs
of one larva accounts for 74% of the total zinc amount of intact
larva, although its volume is only less than 1% of the intact larva.
Acknowledgment. Financial support from the National Natural
Science Foundation of China (Nos. 20571043, 30370351, 90713001,
and 20721002) and the Natural Science Foundation of Jiangsu
Province (BK2008015) is gratefully acknowledged.
Supporting Information Available: Quantum yield and dis-
sociation constant determination, Figures S1-S11. This material
is available free of charge via the Internet at http://pubs.acs.org.
JA806489Y
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1468 J. AM. CHEM. SOC. VOL. 131, NO. 4, 2009