Highly selective fluorescence imaging of zinc distribution in HeLa cells and Arabidopsis using a naphthalene-based fluorescent probe

Article abstract of DOI:10.1039/c5cc01364j

2-(N,N-Dimethylamino)naphthalene-based probe 1 was found to exhibit a dramatic enhancement in fluorescence upon addition of Zn²⁺, but not with any other metal ions. Probe 1 as a chemoprobe enabled high-resolution fluorescence imaging of zinc ions in HeLa cells and Arabidopsis. This journal is

Full text of DOI:10.1039/c5cc01364j

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Highly selective fluorescence imaging of zinc
distribution in HeLa-cell and Arabidopsis using a
naphthalene-based fluorescent probe
Cite this: DOI: 10.1039/x0xx00000x
Ji Ha Lee,a Jin Hyeok Lee,a Sung Ho Jung,a Tae Kyung Hyun,b,e,* Mingxiao Feng,b
JaeꢀYean Kim,b JaeꢀHong Lee,c Hoyeon Lee,d Jong Seung Kim,c,* Chulhun Kang,d,*
KiꢀYoung Kwona,* and Jong Hwa Junga,*
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
www.rsc.org/
2-(N,N-dimethyamino)naphthalene-based probe 1 was found to
dramatic enhancement fluorescence upon addition of Zn2+, but
not with any other metal ions. Probe 1 as chemoprobe displayed
high-resolution fluorescence imaging for zinc ion in the HeLa-
cell and Arabidopsis.
Zinc ion is the secondꢀmost abundant metal ion in the human
brain and is an active component in growth and reproduction of all
organism.1,2 It plays a key function as a cofactor and a structural
elemental in macromolecules and is required for the activity of an
estimated 300 proteins including transcription factors regulating
gene expression and enzymes involved in metabolism and
detoxification of reaction oxygen species (ROS) in plant
mitochondria.3,4 Furthermore, zinc plays an important role in many
biochemical reactions within the plants.5ꢀ12 Plants such as maize and
sorghum and sugarcane show reduced photosynthetic carbon
metabolism due to zinc deficiency. Zinc modifies and/or regulates
the activity of carbonic anhydrase, an enzyme that regulates the
conversion of carbon dioxide to reactive bicarbonate species for
fixation as carbohydrates in these plants.5ꢀ12 Zinc is also a part of
several other enzymes such as superoxide dismutase and catalase,
which prevent oxidative stress in plant cells.8
With these ideas in mind, we have designed a new fluorescent
probe for Zn2+ derived from 2ꢀ(N,Nꢀdimethylamino)naphthalene as
the receptor, which is soluble in aqueous solution. We report here
highly selective fluorescent probe 1 for Zn2+ which functions well in
the presence of other metal ions such as Na+, Pb2+, Mg2+, Hg2+, Ag+,
Cu2+, Co2+ and Cd2+ in aqueous solution. In practical applications,
we also show highly selective fluorescent images of 1 for Zn2+ in
HeLa cells and in plant tissues.
Although Zn2+ has many important cellular roles,13 its
physiological significance is little understood. Therefore, several
chemical tools for measuring Zn2+ in biological samples have been
developed. A variety of fluorescent probes for Zn2+, based on
benzothiazole, fluorescein, quinolone, and protein, have been
reported.14ꢀ17 Some of these probes can be used under physiological
conditions, but they suffer from problems such as inadequate
selectivity, insufficient sensitivity, dependence of fluorescence upon
the dye concentration, and so forth. In addition, these fluorescent
probes have been used to investigate imaging techniques at the
cellular level. However, these probes have not yet been tested for
their application in plant tissues. Thus, the development of a
selective fluorescent probe for Zn2+ in plants is a worthwhile goal.
Fig. 1 (A) Fluorescence spectra of (a) free ligand
1
(1.0 x10-5 M) and (b) in the
present of Zn(NO3)2 (1.0 Ɛ
10-4 M) in aqueous solution at pH=7. (B) Plot of
fluorescence intensities against concentrations of Zn2+ at 500 nm.
We conducted an investigation of the recognition capabilities of 1
for Zn2+ in a series of UVꢀVis and fluorescence spectroscopy
studies. The first observation of the zinc ion detection capability
revealed a correlation between the absorption properties of 1 and the
concentration of Zn2+ in aqueous solution (Fig. S1). The absorbance
of 1 (ε= 9.30 x 104 M−1 cm−1) at ~ 370 nm increased upon addition
of Zn2+. The fluorescence emission of 1 (excitation at 370 nm) at
500 nm increased significantly upon addition of Zn2+, with a
fluorescence enhancement of up to ~10ꢀfold (Φ= 0.089) with the
addition of 10 equivalent of Zn2+ (Fig. 1A). This “turnꢀon”
mechanism can be attributed to the blocking of the photoinduced
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electron transfer process18 and the decrease in the electronꢀdonating was unaffected between pH 3ꢀ11 (Fig. S9), a sign of the strong
ability of the amino group by the complexation of 1 with Zn2+. The complex formation between the receptor motif of 1 and Zn2+.
addition of Zn2+ resulted in a linear increase in the fluorescence
We performed two types of cell imaging for Zn2+ by using 1 as a
intensities of 1 (Figs. 1B and S2), indicating that Zn2+ was chemoprobe for practical applications. In Fig. S10, HeLa cells that
quantitatively bound to 1. Probe 1 also showed excellent sensitivity were incubated with 5 x 10ꢀ6 M of 1 for 30 min showed fluorescence
with detection of 3.2 ppb of Zn2+, a level which is sensitive enough emission through
a twoꢀphoton absorption by its 2ꢀ(N,Nꢀ
for practical applications in measurements of Zn2+ in biological dimethylamino)naphthalene moiety at λex = 740 nm. However, after
fluids. In addition, the Job plot using the absorption changes preꢀincubation with pyrithionꢀZn2+ 1:1 complex (2 x 10ꢀ5 M) for 40
indicated 1:1 binding for 1 with Zn2+ (Fig. S3). We further
min, the fluorescence intensity was dramatically increased, which
was likely due to Zn2+ chelation of 1, in a similar manner to that in
Fig. 1. This result indicates that compound 1 can selectively sense
Zn2+ ions in a cell. The conclusion was confirmed by the observation
that addition of 0.1 mM TPEN (N,N,N′,N′ꢀtetrakis(2ꢀ
pyridylmethyl)ethylenediamine), a Zn2+ ionꢀselective chelator,22 to
the Zn2+ꢀtreated cells reduced the fluorescence intensity.
investigated the interaction between 1 and Zn2+ by using ESIꢀmass
spectroscopy (Fig. S4). The ESIꢀmass spectrum of 1 upon addition
of Zn2+ corresponded to the [1-+Zn2+ꢀH2O]+ at m/z ~ 545.84
indicating that the binding between 1 and Zn2+ led to a 1:1
stoichiometric ratio. The association constant (Ka) of 1 with Zn2+
was calculated for 1:1 stoichiometry on the basis of a Benesieꢀ
Hildebrand plot,19 and it was calculated to be 7.51 x 104 Mꢀ1 (Fig.
S5). In general, Zn2+ acts as soft acid by HSAB theory.20ꢀ21
Therefore, Zn2+ is strongly bound to the nitrogen and oxygen atoms
of N,Nꢀbis(acetic acid)aniline moiety of 1 (Fig. S6).
Fig. 3 (A) Fluorescence images of plants treated with different concentration of
ZnSO4. Five-day-old seedlings pre-treated with different concentration of ZnSO4
(a) Mock (b) 50 µM (c) 100 µM (d) 500 µM and (e) 1mM for 6 h were incubated
with probe
1 (100 µM). (B) The specificity of probe 1 (100 µM) in plant. Six hours
(a) before and (b~e) after treatment of (a) Mock (b) AgNO3 (1 mM), (c) CuSO4 (1
mM), (d) MnSO4 (1 mM) and (e) ZnSO4 (1 mM), seedlings were treated with
probe 1 (100 µM) for 5 min. Fluorescence was visualized using confocal laser
scanning microscope.
To identify the cellular location of Zn2+ accumulation, a series of
colocalization experiments with organelle selective trackers was
performed for the fluorescence image of compound 1 in HeLa cells
(Fig. 2). In the panels, the image of the mitochondrial tracker mostly
overlapped that of 1 where Pearson’s correlation coefficient was
0.85, significantly higher than obtained for other trackers. The
mitochondrial targeting of 1 is very interesting considering that this
probe has two carboxylate groups, in contrast with general motifs for
other molecules that track mitochondria which typically have large
hydrophobic cation structures. In this study, we think that probe 1
spreads over the whole cell without preference to mitochondria and
its emission increases when it forms a complex with Zn2+ ion,
Likewise, the apparent mitochondrial preference is due to the
localization of Zn2+ in mitochondria.
Fig. 2 Confocal fluorescent images of HeLa cell incubated with 5 x 10-6 M of 1 with
Zn(NO3)2 (1.0 ×10-4 M) for 30 min, λ = 740 nm. The excitation wavelengths were 740
ex
nm, 543 nm and 633 nm for ER tracker Red, Lyso Tracker Red DND-99 and Mito Tracker
Deep Red FM, respectively.
We also investigated the practical applicability of 1 as a Zn2+
probe to function within living systems like plants. Fiveꢀdayꢀold
Arabidopsis seedlings treated with different concentrations of ZnSO4
for 6 h were incubated with 100 µM of fluorescent probe 1, and the
fluorescence signal was observed using a confocal laser scanning
microscope. As shown in Fig. 3, 1 gave a sufficiently vigorous
change in emission intensity under Zn2+ preꢀtreatment conditions,
and fluorescence intensity increased in a doseꢀdependent manner
with increasing Zn2+ concentrations in Arabidopsis seedlings.
Although a low background signal was observed in mockꢀtreated
seedlings due to a basal signal from probe 1, the increasing signal
with Zn2+ addition indicated that autofluorescence from a variety of
plant biomolecules including chlorophyll, carotene, and phenolic
compounds did not appear to have any effect on probe 1ꢀmediated
fluorescence emission in plants. The high level of fluorescence
signal was observed in vascular tissue and epidermal cells,
We also investigated the binding ability of 1 to serve as an ionꢀ
selective fluorogenic probe upon the binding of other metal ions
including Na+, Pb2+, Mg2+, Hg2+, Ag+, Cu2+, Co2+, Ca2+, Fe3+ and
Cd2+. However, no significant spectral changes were observed upon
addition of any of these metal ions (Fig. S7), indicating that complex
1 is a highly selective chemoprobe for the detection of Zn2+.
To determine if the specificity of detection for Zn2+ by 1 was still
functional in the presence of an excess of other metal ions, we
carried out competition experiments (Fig. S8). The addition of
various metal ions (100 equivalents) in aqueous solution at pH 7 to
the solution of 1 containing 10 equivalents of Zn2+ did not induce
any significant changes. Hence, 1 may serve as a selective
chemoprobe for Zn2+ even in the presence of other relevant metal
ions. Comparison of this response across a large pH range revealed
that this characteristic fluorescence enhancement by Zn2+ exposure
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suggesting that Zn accumulates in both cells of the cotyledons under
the Zn2+ treatment condition (Fig. 3A). The loading of Zn2+ into the
apoplastic xylem was required for translocation of Zn2+ from the root
to the shoot and leaf.23 In addition, trichomes and epidermal cells are
known as a site which accumulate the highest concentrations of
Zn2+, although Zn2+ distribution between cells is not fully
understood.24Therefore, these results indicate that the high level of
probe 1ꢀmediated fluorescence emission in vascular tissue and
epidermal cells is due to the Zn2+ movement to the major storage
site. Furthermore, the fluorescence intensity from probe 1 remained
constant over 2 hours (Fig. S11), indicating that probe 1 formed
stable complex with Zn2+ under physiological conditions.
In vitro tests suggested that probe 1 displayed high specificity for
binding to Zn2+ in preference to other metal ions. In order to confirm
the specificity of probe 1, probe 1ꢀmediated fluorescence emission
from Zn2+ꢀtreated seedlings was compared with seedlings preꢀtreated
with different metal cations. As expected, probe 1 was not influenced
by other metal ions, such as Ag+, Cu2+ and Mn2+, which exist in
nature in concentrations as high as 1 mM (Fig. 3B). Taken together,
the stability and specificity of probe 1 in physiological conditions
indicate its great potential for biological applications.
In conclusion, we have developed a highly selective “turnꢀon”
fluorescent probe for Zn2+ in the presence of other metal ions, with
3.2 ppb sensitivity and pH insensitivity in the biologically relevant
range. Zn2+ imaging by observing the ion interaction with the
fluorescent probe 1 was demonstrated using probe 1 in HeLaꢀcell
and plants as practical applications. In particular, probe 1 exhibited a
strong fluorescence imaging for Zn2+ accumulated in the
mitochondrial part of HeLa cells. Application of fluorescent probe 1
for plant cell imaging suggested that Zn2+ absorbed from growth
media might be sequestered in the vacuole in Arabidopsis. These
results highlight the capability of 1 for visualization of Zn2+
accumulated in plants at the organelle level. Thus, the use of 1
enables micrometerꢀlevel analysis of Zn2+, and we anticipate that this
capability will soon be extended to the nanoscale.
†
Electronic Supplementary Information (ESI) available: Experimental
details and spectroscopic analysis. See DOI: 10.1039/c000000x/
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(2014M2B2A9030338 and 2012R1A4A1027750) supported from the
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(NRF) grant funded by the Korea government (MSIP)
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