A highly selective CHEF-type chemosensor for monitoring Zn2+ in aqueous solution and living cells

Article abstract of DOI:10.1039/c5ra05080d

A new simple quinolone-based chemosensor 1 was synthesized for Zn²⁺. 1 exhibited selective fluorescence enhancement in the presence of Zn²⁺ with a 1:1 stoichiometry in aqueous solution, which was reversible with the addition of ethylenediaminetetraacetic acid (EDTA). The detection limit (0.019 μM) of 1 for Zn²⁺ is far lower than the World Health Organization guideline (76 μM) in drinking water. 1 was successfully applied to quantify and image Zn²⁺ in water samples and living cells. The sensing mechanism of Zn²⁺ by 1via the chelation-enhanced fluorescence (CHEF) effect was supported by theoretical calculations. Therefore, 1 could be used in a practical system for monitoring Zn²⁺ in aqueous solution.

Full text of DOI:10.1039/c5ra05080d

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Graphical Abstract
A new quinolone-based chemosensor was synthesized and successfully applied to quantify
2
+
and image Zn in water samples and living cells.

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2
+
A highly selective CHEF-type chemosensor for monitoring Zn in aqueous solution and
living cells
a
a
a
b
b
a
Jae Jun Lee, Seul Ah Lee, Hyun Kim, LeTuyen Nguyen, Insup Noh, Cheal Kim *
aDepartment of Fine Chemistry and Department of Interdisciplinary Bio IT Materials, Seoul
National University of Science and Technology, Seoul 139-743, Republic of Korea. Fax: +82-
2
-973-9149; Tel: +82-2-970-6693; E-mail: chealkim@seoultech.ac.kr
b
Department of Chemical and Biomolecular Engineering, and Convergence Program of
Biomedical Engineering and Biomaterials, Seoul National University of Science & Technology,
Seoul 139-743, Republic of Korea.
Abstract
2
+
A new simple quinolone-based chemosensor 1 was synthesized for Zn . 1 exhibited the
2
+
selective fluorescence enhancement in the presence of Zn with a 1:1 stoichiometry in aqueous
solution, which was reversible with the addition of ethylenediaminetetraacetic acid (EDTA).
2
+
The detection limit (0.019 μM) of 1 for Zn is far lower than World Health Organization
2
+
guideline (76 μM) in drinking water. 1 was successfully applied to quantify and image Zn in
2
+
water samples and living cells. The sensing mechanism of Zn by 1 via the chelation-enhanced
fluorescence (CHEF) effect was supported by theoretical calculations. Therefore, 1 could be
2
+
used in a practical system for monitoring Zn in aqueous solution.
Keywords: fluorescent chemosensor, zinc ion, CHEF effect, cell imaging, theoretical
calculations
1

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Introduction
Zinc ion is one of the most abundant metal ions present in living organism, owing to its rich
coordination chemistry. 1-4 Although most of the Zn2+ ions in a cell are tightly bound to
metalloproteins, free forms are also found throughout the cell. In the brain, 5-20% of the total
2
+
2+
Zn is stored in presynaptic vesicles, and the highest intracellular free Zn is found in the
hippocampus.5-6 Zn modulates brain excitability and plays a key role in synaptic plasticity.
2+
7-
8
On the other hand, the deficiency of zinc causes unbalanced metabolism, which in turn can
induce retarded growth in children, brain disorders and high blood cholesterol, and also be
implicated in various neurodegenerative disorders such as Alzheimer’s disease, epilepsy,
ischemic stroke, and infantile diarrhea.9-15 In addition, an excess of zinc in the environment
may reduce the soil microbial activity which results in phytotoxic effect.16-21 Therefore,
selective detection and quantification for zinc ion is very important object of increasing
investigation.22-24
A variety of traditional methods (e.g. inductively coupled plasma atomic emission
spectrometry, atomic absorption spectroscopy, and electrochemical methods) have been
developed for analyses of zinc in environmental samples and for diagnosis of their deficiencies
in body tissue.25-28 However, these methods require sophisticated instrumentations, tedious
sample preparation procedures, and trained operators. By contrast, chemosensors are highly
estimable means for the selective recognition of chemicals and biological species in
2
9
environmental chemistry and biology. The design of chemosensors with high selectivity and
sensitivity for zinc, is currently of great importance as they allow nondestructive and prompt
detection of metal ions by simple fluorescence enhancement (turn-on) responses.22-24
2
+
2+
Especially, the development of chemosensors that can discriminate Zn from Cd is still a
huge challenge as they are in the same group of the Periodic table and have similar properties.30-
3
3
Thus, low cost and easily-prepared Zn2+ fluorescence chemsensors are needed for
convenience.34-41
In view of this requirement and as part of our research effort devoted to zinc ion recognition,
we have considered the combination of a quinoline moiety and a 2-(ethyl(pyridin-2-
yl)amino)ethan-1-ol (2, see Scheme 1) unit.22,42-45 In particular, we expected that the ethanol
group would make water-solubility of 1 increase. Additionally, we theoretically expected that
fluorescence enhancement was originated from the stabilization of quinoline group. Finally,
we synthesized a new type of water-soluble chemosensor 1 (Scheme 1) by combining the
2

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quinolone group with 2, and tested its sensing properties towards various metal ions.
Herein, we report a new chemosensor 1, composed of the quinoline and 2-(ethyl(pyridin-2-
yl)amino)ethan-1-ol. 1 showed an intense fluorescence enhancement in the presence of zinc
2
+
2+
2+
ions in aqueous solution, could distinguish Zn from Cd , and sensed quantitatively Zn in
living cells for practical application. Additionally, the experimental data and the sensing
mechanism were supported by theoretical calculations.
Experimental section
Materials and instrumentation
All the solvents and reagents (analytical grade and spectroscopic grade) were obtained
commercially and used as received. NMR spectra were recorded on a Varian 400 spectrometer.
Chemical shifts (δ) were reported in ppm, relative to tetramethylsilane Si(CH
3
4
) . Absorption
spectra were recorded at 25°C using a Perkin Elmer model Lambda 2S UV/Vis spectrometer.
The emission spectra were recorded on a Perkin-Elmer LS45 fluorescence spectrometer.
Electrospray ionization mass spectra (ESI-MS) were collected on a Thermo Finnigan (San Jose,
CA, USA) LCQTM Advantage MAX quadrupole ion trap instrument. Elemental analysis for
carbon, nitrogen, and hydrogen was carried out by using a Flash EA 1112 elemental analyzer
(
thermo) in Organic Chemistry Research Center of Sogang University, Korea. 3-Chloro-N-
quinolin-8-yl)propanamide was obtained from the previous study.34
(
Synthesis of sensor 1
2
-Aminoethanol (0.97 mL, 10 mmol) and picolinaldehyde (0.61 mL, 10 mmol) were
dissolved in ethanol (15 mL) and stirred for 3 h. Then, NaBH (0.38 g, 10.2 mmol) was added,
4
and the reaction solution was cooled in an ice bath. It was stirred for 2 h and the solvent was
removed under reduced pressure to obtain colorless oil (2) (Scheme 1). Yield of 2 was 1.05 g
(
69 %). The colorless oily residue (2) was dissolved in methylene chloride and then the solution
was washed twice with water. Organic layer was dried over anhydrous Na SO and the solvent
was evaporated under vacuo. The resultant product 2 (0.30 g, 2 mmol), 3-chloro-N-(quinolin-
-yl)propanamide (0.49 g, 2.2 mmol) and triethylamine (TEA, 0.28 mL, 2 mmol) were
2
4
8
dissolved in tetrahydrofuran (THF, 30 mL), stirred and refluxed for 1 d. The mixture was cooled
down to room temperature and the solvent was removed under reduced pressure to obtain
brown oil, which was purified by silica gel column chromatography (9 : 1 v/v CHCl
3
-CH OH).
3
3

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1
Yield: 0.49 g (73 %). H NMR (400 MHz, DMSO-d , 25 °C): δ = 10.88 (s, 1H), 8.86 (d, J =
6
8
7
.9 Hz, 1H), 8.65 (d, J = 8.7 Hz, 1H), 8.43 (d, J = 8.4 Hz, 1H), 8.40 (d, J = 8.4 Hz, 1H), 7.66-
.51 (m, 2H), 7.57 (d, J = 7.6 Hz, 1H), 7.53 (m, 2H), 7.49 (t, J = 7.1 Hz, 1H), 4.45 (t, J = 4.4
Hz, 1H), 3.89 (s, 2H), 3.58 (t, J = 3.6 Hz, 2H), 2.93 (t, J = 3.0 Hz, 2H), 2.49 (m, J = 2.9 Hz,
1
3
4
1
1
C
H) ppm. C NMR (100 MHz, DMSO-d , 25 °C): δ = 171.7 (1C), 156.7 (2C), 147.1 (2C),
6
39.9 (1C), 138.8 (1C), 129.8 (1C), 129.0 (1C), 127.7 (1C), 127.5 (1C), 126.0 (2C), 124.8 (1C),
22.8 (2C), 121.8 (1C), 121.5 (1C), 110.2 (1C), 21.8 (1C) ppm. LRMS (ESI): m/z calcd for
+
2+
20
H
22
N
4
O
2
-H +Zn : 413.095; found 413.267. Elemental analysis calcd (%) for C20
H
22
N
4
2
O :
C, 68.55; H, 6.33; N, 15.99; found: C, 68.65; H, 5.95; N, 15.75.
Fluorescence titration of 1 with Zn2+
Sensor 1 (0.5 mg, 0.0015 mmol) was dissolved in MeOH (0.5 mL) and 10 μL of the sensor 1
(3 mM) were diluted to 2.990 mL bis-tris buffer solution (10 mM, pH 7.0) to make the final
concentration of 10 μM. Zn(NO
3
2
) (0.01 mmol) was dissolved in bis-tris buffer solution (1 mL)
2
+
and 0.3-4.8 μL of the Zn solution (10 mM) were added to the sensor 1 solution (10 μM, 3
mL) prepared above. After mixing them for a few seconds, fluorescence spectra were obtained
at room temperature. Identical results were also obtained in pure water.
UV-vis titration of 1 with Zn2+
Sensor 1 (0.5 mg, 0.0015 mmol) was dissolved in MeOH (0.5 mL) and 10 μL of the sensor 1
(3 mM) were diluted to 2.990 mL bis-tris buffer solution (10 mM, pH 7.0) to make the final
concentration of 10 μM. Zn(NO (0.01 mmol) was dissolved in bis-tris buffer solution (1 mL)
3 2
)
2
+
and 0.3-4.8 μL of the Zn solution (10 mM) were transferred to separate sensor solutions (10
μM, 3 mL). After mixing them for a few seconds, UV-vis spectra were taken at room
temperature.
1H NMR titration of 1 with Zn2+
Five NMR tubes of 1 (0.7 mg, 0.002 mmol) dissolved in in D
2
O/CD OD (9:1, v/v) were
3
prepared, and five different equivalents (0, 0.3, 0.5, 1 and 1.5 equiv) of zinc nitrate dissolved
in D
2
O (0.5 mL) were added separately to the solutions of 1. After shaking them for a few
1
seconds, the H NMR spectra were taken.
4

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Methods of cell test of 1 with Zn2+
Human dermal fibroblast cells in low passage were cultured in FGM-2 medium (Lonza,
Switzerland) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin in
the in vitro incubator with 5% CO
2
at 37 °C. Cells were seeded onto a 8 well plate (SPL
5
Lifesciences, Korea) at a density of 2 × 10 cells per well and then incubated at 37 °C
for 4 h after addition of various concentrations (0-100 μM) of Zn(NO
MeOH. After washing with phosphate bu ered saline (PBS) two times to remove the
remaining Zn(NO , the cells were incubated with (20 μM) dissolved in MeOH at
3
)
2
dissolved in
ff
3
)
2
1
room temperature for 1 h. The cells were observed using a microscope (Olympus, Japan).
The fluorescent images of the cells were obtained using a fluorescence microscope
(Leica DMLB, Germany) at the excitation wavelength of 425 nm. The relative
fluorescence intensity of fluorescence microscopy images was evaluated by Icy
software.46 The four cell-body regions in visible field (green light source) were
randomly selected as the region of interest to determine the average fluorescence
intensity.
2
+
Determination of Zn in water samples
2
+
Fluorescence spectral measurement of water samples containing Zn were carried by adding
2
0 μL of 3 mmol/L stock solution of 1 and 0.60 mL of 50 mmol/L bis-tris buffer stock solution
◦
to 2.38 mL sample solutions. After well mixed, the solutions were allowed to stand at 25 C for
2
min before the test.
2
+
Method for theoretical calculations of 1 and 1-Zn complex
4
6
All theoretical calculations were performed by using the Gaussian 03 suite. The singlet
2
+
ground states (S
0
) of 1 and 1-Zn complex were optimized by DFT methods with Becke’s three
parametrized Lee-Yang-Parr (B3LYP) exchange functional47-48 with 6-31G** basis set49-50. In
2
+
vibrational frequency calculations, there was no imaginary frequency for 1 and 1-Zn complex,
2
+
suggesting that the optimized 1 and 1-Zn complex represented local minima.
Results and discussion
Synthesis of 1
The compound 2 (2-((pyridin-2-yl)methylamino)ethanol) was synthesized by condensing
5

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2
-aminoethanol and picolinaldehyde in ethanol (Scheme 1). Subsequently, the substitution
reaction of 2 to 3-chloro-N-(quinolin-8-yl)propanamide afforded 1, which was characterized
1
13
by H NMR, C NMR, ESI-mass, spectrometry and elemental analysis.
Fluorescence and absorption spectroscopic studies of 1 toward Zn2+
The fluorescent behavior of 1 toward various metal ions was studied in bis-tris buffer solution
(10 mM, pH 7.0). When excited at 355 nm, 1 exhibited a weak fluorescence emission (max =
2
+
5
21 nm) compared to that (37-folds) in the presence of Zn (Fig. 1). In contrast, upon addition
+
+
2+
2+
3+
3+
3+
2+
2+
3+
2+
2+
of other metal ions such as Na , K , Mg , Ca , Al , Ga , Cr , Mn , Fe , Fe , Co , Ni ,
2
+
2+
3+
2+
Cu , Zn , Ga and Pb , no increase in intensity was observed. These results indicated that
2
+
sensor 1 could be used as a fluorescence chemosensor for Zn . The selective fluorescence
2
+
2+
enhancement response to Zn might be due to the effective coordination of Zn with 1 over
other metal ions, which might be attributed to the operation of CHEF (chelation enhanced
fluorescence) mechanism.31,51
To further investigate the chemosensing properties of 1, fluorescence titration of the sensor
2
+
1
with Zn ion was performed. As shown in Fig. 2, the emission intensity of 1 at 521 nm
2
+
steadily increased until the amount of Zn reached 1.2 equiv. The photophysical properties of
1
were also examined using UV-vis spectrometry. UV-vis absorption spectrum of 1 showed
2+
two absorption bands at 237 and 302 nm (Fig. S1). Upon the addition of Zn ions to a solution
of 1, the two bands have red-shifted to 253 and 351 nm, respectively. Meanwhile, three clear
isosbestic points were observed at 243, 280 and 325 nm, suggesting that only one product was
2
+
produced from the binding of 1 with Zn .
52
2+
The Job plot showed a 1:1 complexation stoichiometry between 1 and Zn (Fig. S2),
which was further confirmed by ESI-mass spectrometry analysis (Fig. 3). The positive-ion
2
+
+
2+
mass spectrum of 1 upon addition of 1 equiv of Zn showed the formation of the 1-H +Zn
m/z: 413.267; calcd, 413.095]. Based on pK values (11.9 vs 15.8) of the acetamide and
ethanol moieties reported in the literatures , we proposed that the proton of the amide moiety
[
a
5
3
2
+
might be deprotonated when 1 coordinated to Zn as shown in the Fig. 3. From the UV-vis
2
+
7
-1
titration data, the association constant for 1 with Zn was determined as 3.3 (±0.1) x 10 M
5
4
using Benesi-Hildebrand equation (Fig. S3). This value was within the range of those (1.0 ~
1
2
2+
55-58
1
1
.0 x 10 ) reported for Zn sensing chemosensors.
The detection limit was estimated as
9 nM based on the 3σ/slope (Fig. S4), which was about ten thousand folds lower than the
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2
+
35,59
WHO guideline (76 μM) for Zn ions in drinking water.
2
+
To check the practical applicability of 1 as Zn selective sensor, fluorescence competition
2
+
experiments were carried out in the presence of Zn mixed with various metal ions. When 1
2
+
was treated with 1.5 equiv of Zn in the presence of the same concentrations of other metal
ions (Fig. 4), other background metal ions had no obvious interference with the detection of
Zn2+ ion, except for Co2+ and Cu2+ which showed about 50 % and 70% interferences,
respectively. The interferences with Co2+ and Cu2+ might be due to the strong quenching
properties to fluorescence.60,61 Nevertheless, they still showed sufficient turn-on fluorescence.
2
+
3+
In the presence of excess other metal ions (15 equiv), Hg and Fe interfered with some, and
2
+
2+
2+
Co and Cu did completely (Fig. S5). In particular, Cd ion which is in the same group with
2
+
2+
Zn didn’t affect the emission intensity of 1-Zn . These results indicated that 1 could be a
2
+
2+
2+
good Zn sensor which could distinguish Zn from Cd commonly having similar properties.
1H NMR spectroscopic studies of 1 toward Zn2+
1
The H NMR titration experiments were studied in D
2
O/CD
3
OD (9:1, v/v) to further
2
+
2+
examine the binding mode between 1 and Zn ion (Fig. 5). Upon addition of Zn to sensor 1,
1
all of the aromatic and the aliphatic protons showed downfield shift. Moreover, H NMR
titration in CD CN showed that the proton signal of the amide moiety gradually disappeared
3
2
+
according to the increase of Zn (Fig. S6). These chemical shifts suggested that the oxygen
atom of the ethanol moiety and the four nitrogen atoms might coordinate to Zn ion. This
coordinative behavior of potentially penta-dentate ligand 1 with a zinc ion was previously
3
6
observed in the similar type of zinc complexes. There was no shift in the position of proton
2
+
signals on further addition of Zn (>1.5 equiv). Based on Job plot, ESI-mass spectrometry
1
analysis, H NMR titration, and the crystal structures of similar types of zinc complexes, we
2
+
proposed the structure of 1-Zn complex as shown in scheme 2.
pH effect of 1 toward Zn2+
To study the practical applicability of 1, the effects of pH on the fluorescence response of
2
+
Zn were investigated at pH ranging from 2 to 12 (Fig. 6). The fluorescence intensity of 1 in
the presence of Zn2+ showed a significant response between pH 5 and 12. These results
2
+
indicated that Zn could be clearly detected by the fluorescence spectra measurement using 1
6
2
over the environmentally and physiologically relevant pH range (pH 7.0-8.4), especially for
7

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2
+
monitoring Zn in water samples and living cells.
2
+
Reversible test of 1 toward Zn by using EDTA
To examine the reversibility of sensor 1 toward Zn2+ in buffer solution,
ethylenediaminetetraacetic acid (EDTA, 1.5 equiv) was added to the complexed solution of
2
+
sensor 1 and Zn . As shown in Fig. S7, a fluorescence signal at 521 nm was immediately
quenched. Upon addition of Zn2+ again, the fluorescence was recovered. The fluorescence
emission changes were almost reversible even after several cycles with the sequentially
2
+
alternative addition of Zn and EDTA. These results indicated that sensor 1 could be recyclable
simply through treatment with a proper reagent such as EDTA. Such reversibility and
2
+
regeneration could be important for the fabrication of chemosensor to sense Zn .
Biological application for Zn2+
2
+
To further demonstrate the potential of 1 to monitor Zn in living matrices, fluorescence
imaging experiments were carried out in living cells (Fig. 7a). Adult human dermal fibroblasts
2
+
were first incubated with various concentrations of aqueous Zn solutions (0, 5, 10, 40, 80 and
00 μM) for 4 h and then exposed to 1 for 1 h before imaging. The experimental results showed
1
2
+
that the fibroblast cells without either Zn or 1 showed negligible intracellular fluorescence,
2
+
2+
while those cultured with both Zn and 1 exhibited fluorescence. With an increase in Zn
concentration from 5 to 100 μM, the relative fluorescence intensity of the cells with 1 increased
Fig. 7b). These observations confirmed that 1 could be cell membrane permeable and thus
(
2
+
successfully applied for the determination of Zn in living cells.
Determination of zinc ion in water samples
In order to examine the practical properties of the chemosensor 1 in environmental samples,
the chemosensor 1 was applied for the determination of Zn2+ in water samples, using a
2
+
calibration curve of 1 toward Zn (Fig. S8). Tap water and artificial polluted water samples
were chosen. As shown in Table 1, a suitable recovery and R.S.D. values of the water samples
were obtained.
Theoretical calculations
2
+
To obtain a deeper insight into the interaction of 1 with Zn , theoretical calculations were
8

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carried out in parallel to the experimental studies. We performed all theoretical calculations by
a 1:1 stoichiometry, based on Job plot and ESI-mass analysis. As the proton of the amide group
2
+
2+
in 1 was deprotonated by Zn , we calculated the deprotonated product for 1-Zn complex.
Energy-minimized structures (S
) for 1 and 1-Zn2+ complex were optimized by applying
0
density functional theory (DFT/B3LYP/6-31G**). The significant structural properties of the
energy-minimized structures were indicated in Fig S9. Based on the molecular orbitals (MOs)
2
+
2+
for the ground state of 1 and 1-Zn complex (Fig. 8 and S10), the chelation of Zn with 1
rendered the HOMO to LUMO energy gap of 1 decrease, which is consistent with the red shift
2
+
2+
in the UV-vis spectrum of 1-Zn complex. Thus, the fluorescence enhancement of 1-Zn
complex could be explained by CHEF effect.63,64
Conclusion
We have developed a new ethyl alcohol-functionalized chemosensor 1, which detected Zn2+
ion in aqueous solution. 1 showed highly selective fluorescence response to Zn2+ with the
2
+
detection limit at the nanomolar level (19 nM). Importantly, 1 can clearly distinguish Zn from
2
+
2+
2+
Cd , where such distinction of Zn from Cd is a well-known challenge. The binding of the
2
+
sensor 1 and Zn was chemically reversible with EDTA. In addition, 1 could be successfully
applied to real samples with the satisfactory recovery and R.S.D. values. The living cell
experiments demonstrated its value in the practical application. Moreover, the sensing
2
+
mechanism of Zn by 1 via CHEF effect was be explained by DFT calculations. Therefore,
we believe that sensor 1 will be an excellent prototype for the feasible system for monitoring
2
+
Zn concentrations in biological and environmental environments.
Acknowledgements
Financial support from Basic Science Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology
(NRF-2014R1A2A1A11051794, 2012001725 and 2012008875) are gratefully acknowledged.
We thank Nano-Inorganic Laboratory, Department of Nano & Bio Chemistry, Kookmin
University to access the Gaussian 03 program packages.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://????.
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1
3

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Scheme 1. Synthesis of 1.
1
4

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Scheme 2. Fluorescence enhancement mechanism and proposed structure of 1 in the presence
2
+
of Zn .
1
5

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Table 1. Determination of Zn(II) in water samples
Sample
Zn(II) added
Zn(II) found
Recovery
(%)
R.S.D. (n = 3)
(μmol/L)
(μmol/L)
(%)
Tap water
0.00
0.00
5.9
6
.00
0.00
.00
98.5
97.3
95.2
3.1
4.2
8.8
Water
[
a]
5.8
7.7
sample
2
[
2
a] Prepared by deionized water, 6.00 μmol/L Zn(II), 10 μmol/L Cd(II), Pb(II), Na(I), K(I), Ca(II), Mg(II). Conditions: [1] =
0 μmol/L in 10 mM bis-tris buffer-MeOH solution (999:1, pH 7.0).
1
6

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Figure Captions
Figure 1. Fluorescence spectral changes of 1 (10 µM) in the presence of different metal ions
+
+
2+
2+
3+
3+
3+
2+
2+
3+
2+
2+
2+
(
1.5 equiv) such as Na , K , Mg , Ca , Al ,Ga , Cr , Mn , Fe , Fe , Co , Ni , Cu ,
2
+
3+
2+
Zn , Ga and Pb with an excitation of 355 nm in buffer solution (10 mM bis-tris, pH 7.0).
Figure 2. Fluorescence spectral changes of 1 (10 μM) in the presence of different
2
+
concentrations of Zn ions in buffer solution (10 mM bis-tris, pH 7.0). Inset: Fluorescence
2
+
intensity at 521 nm versus the number of equiv of Zn added.
Figure 3. Positive-ion electrospray ionization mass spectrum of 1 (20 μM) upon addition of
Zn(NO
3
2
) (1.5 equiv).
2
+
Figure 4. Competitive selectivity of 1 (10 μM) toward Zn (1.5 equiv) in the presence of other
metal ions (1.5 equiv) with an excitation of 355 nm in buffer solution (10 mM bis-tris, pH 7.0).
1
Figure 5. H NMR titration of 1 with Zn(NO
3
)
2
in D
2
O/CD
3
OD solution (D
2
O/CD OD, 9:1,
3
v/v).
2
+
Figure 6. Fluorescence intensity (at 521 nm) of 1 in the presence of Zn at different pH values
2-12) in buffer solution (10 mM bis-tris, pH 7.0).
(
2
+
Figure 7. (a) Fluorescence images of fibroblasts cultured with Zn and 1. Cells were exposed
to 0 (A and G), 5 (B and H), 10 (C and I), 40 (D and J), 80 (E and K) and 100 μM (F and L)
Zn(NO
3
2
) for 4 h and then later with 1 for 1 h. The top images (A-F) were observed with the
light microscope and the bottom images (G-L) were taken with a fluorescence microscope. The
scale bar is 50 μm. (b) Quantification (green light source) of mean fluorescence intensity in the
bottom images (G-L).
Figure 8. Molecular orbital diagrams participating in the singlet state for the 1 and 1-Zn2+
complex.
1
7

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Fig. 1.
1
8

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Fig. 2.
1
9

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Fig. 3.
2
0

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Fig. 4.
2
1

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Fig. 5.
2
2

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Fig. 6.
2
3

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(a)
(b)
Fig. 7.
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Fig. 8.
2
5