A high sensitive fluorescence turn-on probe for imaging Zn2+ in aqueous solution and living cells

Article abstract of DOI:10.1039/c4ra00584h

A new pyrazoline-based fluorescent probe was designed and synthesized. The probe can induce a 60-fold fluorescence enhancement after 5 equiv. of Zn ²⁺ was added, which is superior to a previous report (13-fold), and exhibits high sensitivity and selectivity for response to Zn²⁺ in aqueous solution. The detection limit and association constant were calculated as 1.23 × 10^-7 M and 3.89 × 10³ M^-1 by a fluorescence titration experiment. The 1:1 binding stoichiometry of L-Zn²⁺ complex was analysed by Job's plot. The probe showed good reversibility upon addition of EDTA or TPEN. Furthermore, the probe L showed good membrane permeability and can be applied to monitor Zn²⁺ ions in living HeLa cells.

Full text of DOI:10.1039/c4ra00584h

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A high sensitive fluorescence turn-on probe for
imaging Zn2+ in aqueous solution and living cells†
Cite this: RSC Adv., 2014, 4, 16973
a
b
a
Ting-Ting Zhang,‡ Xin-Peng Chen,‡ Jin-Ting Liu, Liang-Zhong Zhang,a
*
a
b
a
*
*
Jia-Ming Chu, Le Su and Bao-Xiang Zhao
A new pyrazoline-based fluorescent probe was designed and synthesized. The probe can induce a 60-fold
fluorescence enhancement after 5 equiv. of Zn2+ was added, which is superior to a previous report (13-fold),
and exhibits high sensitivity and selectivity for response to Zn2+ in aqueous solution. The detection limit and
association constant were calculated as 1.23 ꢀ 10ꢁ7 M and 3.89 ꢀ 103 Mꢁ1 by a fluorescence titration
experiment. The 1 : 1 binding stoichiometry of L–Zn2+ complex was analysed by Job's plot. The probe
showed good reversibility upon addition of EDTA or TPEN. Furthermore, the probe L showed good
membrane permeability and can be applied to monitor Zn2+ ions in living HeLa cells.
Received 21st January 2014
Accepted 27th March 2014
DOI: 10.1039/c4ra00584h
www.rsc.org/advances
excellent blue uorescence property, the high uorescence
quantum yield and electroluminescence elds.17 A number of
zinc probes based on pyrazoline have been reported.18
Introduction
Zinc is the second most abundant transition metal in the
human body aer iron, and it has attracted much attention due
to its biological signicance. Zinc ion plays vital roles in
numerous biological processes, such as muscle contraction,1
cellular metabolism,2 gene transcription, immune function,
metallo enzyme function,3 DNA-binding proteins4 and so forth.
In addition, the disorder of zinc homeostasis relates to some
pathological processes, like Alzheimer's disease, epilepsy,
ischemic stroke, and infantile diarrhea.5 Therefore, the quan-
tication and qualication of zinc is of great importance in the
biological or environmental systems.
We have been attempting to develop uorescence probe for
monitoring metal ions.18a–d,19 In continuation with our previous
work, we report a new and easily synthesized pyrazoline deriv-
ative which can induce prominent uorescence enhancement
upon complexation with Zn2+ in aqueous media, which is
superior to previous reported.18b,d The excitation wavelength of
the probe was 410 nm which is in the visible light region. The
probe shows high sensitivity and selectivity for monitoring Zn2+
and can be applied to microscopic imaging Zn2+ in HeLa cells
because of its good membrane-permeable property. We also
performed the cell viability assay, and found the probe has no
cytotoxicity to the cultured cell line under experimental condi-
tions. In contrast with the previous work, present new probe
make the test projects more perfect.
Fluorescence probes are powerful tools to monitor metal
ions owing to their simplicity and high response speed, and
have attracted considerable interest to apply in biological
system with high selectivity and sensitivity.6 Up to now, a variety
of uorescent probes based on Schiff bases,7 BODIPY,8 terpyr-
idine,9
quinoline,10
naphthalimide,11
nanoparticles,12
Results and discussion
Synthesis of the probe
coumarin,13 bipyridine,14 TPEA(N,N,N0-tri(pyridine-2-ylmethyl)-
ethane-1,2-diamine)15 and other uorescein16 have been repor-
ted for detecting Zn2+. Although these probes can be used to
detect zinc in vitro and in vivo, many uorescent probes have
disadvantages such as poor solubility in water, working in toxic
solvents like acetonitrile, complicated preparation process, and
have no better sensitivity or reversibility. Recently, pyrazoline
derivatives have attracted much attention because of their
The general synthetic route of compound 4 (probe L) is given in
Scheme 1. The probe was easily synthesized by two steps.
Chalcone 3, easily prepared from compound 1 and 2, reacted
with hydrazine hydrate to afford the probe. The structure of the
1
probe was characterized by IR, H NMR, 13C NMR and HRMS
spectra.
aSchool of Chemistry and Chemical Engineering, Shandong University, Jinan 250100,
P.R. China. E-mail: jintliu@sdu.edu.cn; bxzhao@sdu.edu.cn
bSchool of Life Science, Shandong University, Jinan 250100, P.R. China. E-mail: sule@
sdu.edu.cn
UV-vis absorption studies
To verify the practical applications in biological environment,
the absorption spectral property of L toward different metal
ions was measured in EtOH–HEPES (1 : 1, v/v, pH 7.2) solution.
As shown in Fig. S1,† L alone (50 mM) exhibits a broad band
centered at 320 nm and 355 nm. Upon addition of 5 equiv.
† Electronic supplementary information (ESI) available: Supplementary
Fig. S1–S21. See DOI: 10.1039/c4ra00584h
‡ Equal contribution.
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Scheme 1 Synthesis of compound 4 (L).
Fig. 1 UV-vis spectra of L (50 mM) upon the titration of Zn2+ (0–10
equiv.) in HEPES buffer solution (20 mM HEPES, pH 7.2, EtOH–H2O ¼
1 : 1). The inset shows the titration profile evaluated from the
absorption at 410 nm.
metal ions, such as Ag+, Al3+, Fe3+, Ba2+, Ca2+, Cd2+, Cr3+, K+,
Mg2+, Na+, Hg2+ did not induce signicant changes in absorp-
tion curves. In contrast, Pb2+, Co2+, Ni2+, Zn2+, Cu2+, caused a
new band at 390–450 nm, accompanied by a decrease of
absorbance at 320 nm and 355 nm.
when the two metal ions co-exist, Cu2+ is easier to complex with
The UV-vis spectra of L with different concentration were
also explored (Fig. S2 and S3, ESI†). The absorption maximum
shows at 355 nm and the molar extinction coefficient 3 is 1.2 ꢀ
104 Mꢁ1 cmꢁ1. To get more insight on the binding behavior of L
and Zn2+, the absorption spectra titration experiment was
further investigated. Upon addition of increasing concentration
of Zn2+ to the buffered solution of L (50 mM), absorbance at 355
nm gradually decreased and a new absorption band at 410 nm
probe L. Obviously uorescence quenching was observed while
complex with some paramagnetic metal ions, such as Co2+
,
Fe3+, Ni2+, Cu2+, which is always encountered in the other metal
ion probes.7d,11,20 Therefore, probe L possesses good selectivity
for response to Zn2+ in EtOH–H2O (1 : 1, v/v, pH 7.2) solution.
In order to address the sensitivity of L toward Zn2+, the
uorescence titration of L (10 mM) with Zn2+ was carried out in
EtOH–H2O (1 : 1, v/v, pH 7.2) (Fig. 4). With the increase addition
of Zn2+ (0–50 equiv.), the uorescent intensity increased
prominent. These changes in the uorescence spectra became
negligible when more Zn2+ was added. The limit of detection
(LOD) is evaluated using 3sbi/m,21 the detection limit calculated
from the uorescence titration curves is 1.23 ꢀ 10ꢁ7 M (Fig. S5,
ESI†). The association constant (Ka) of L with Zn2+ was calcu-
lated to be 3.89 ꢀ 103 Mꢁ1 (Fig. S6, ESI†). In titration experi-
ments, it reached the maximum value upon addition of 50
equiv. of Zn2+. So we also measured the interference experiment
upon addition of 50 equiv. of other anions and found that the
interference of other anions at 50 equiv. concentration was
similar to that at 5 equiv. concentration (Fig. S7, ESI†).
signicantly enhanced which indicate
formed. A distinct isobestic point at 378 nm was observed
a
complex L–Zn2+
(Fig. 1). The ratio of absorbance at 354 nm and 410 nm (A354
/
A410) reached minimum when 5 equiv. Zn2+ was added (Fig. S4,
ESI†), so this probe can be used as a ratiometric in colorimetric
to detect Zn2+. These phenomena are similar to our previous
reports,18d but absorption maximum red shied (25 nm).
Fluorescence property
The uorescence response behavior of L with respective metal
ions was investigated in buffered EtOH–HEPES solution. As
shown in Fig. 2, the solution of L displayed a very weak emis-
sion, while upon addition of 5 equiv. Zn2+ created a signicant
uorescence enhancement almost 60-fold at 475 nm. However,
other metal ions like Na+, Mg2+, K+, Ca2+, Al3+, Cu2+, Cr3+, Cd2+,
Fe3+, Co2+, Ni2+, Ba2+, Ag+, Pb2+ and Hg2+, did not induce obvi-
ously uorescence changes.
Quantum yield was determined by the relative comparison
procedure, using quinine sulfate dehydrate ($99.0%) in 0.1 N
H2SO4 as the main standard.22 And it was calculated by the
2
general equation: F ¼ Fs(IAs/IsA)(h2/hs ).23 The uorescence
quantum yield (F) of probe L is 0.0093 and L–Zn2+ complex was
measured to be 0.58. Additionally, the effects of anionic ions on
the sensing behavior of probe L towards Zn2+ ion was investi-
gated (Fig. S8, ESI†). There were negligibly changes in the
uorescence responses of L to ZnSO4, Zn(AcO)2, ZnCl2, and
Zn(NO3)2.
Furthermore, competition experiments for Zn2+ in the pres-
ence of other ions were carried out in the same condition. As
displayed in Fig. 3, Al3+, Co2+, Fe3+, Ni2+, Cr3+ can partly quench
uorescence of L–Zn2+, whereas Cu2+ completely quenched the
uorescence of L–Zn2+. This may be due to that Cu2+ still binds
to probe L in the presence of Zn2+. To conrm our speculation,
the DFT calculations were performed. Aer calculated, the sum
of electronic and zero-point energies of L with Zn2+ and L with
Cu2+ were ꢁ1595.407841 and ꢁ1725.662761 respectively. So
Job plot analysis of the uorescence data shows that the
stoichiometric ratio was 1 : 1 for the L–Zn2+ complex (Fig. S9,
ESI†). A proposed binding mode is shown in Fig. S10 (ESI†),
which should be similar to our previous report.18d To further
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the probe L with Zn2+, the 1H NMR experiment was investigated.
Upon addition of 5 equiv. of Zn2+, the spectra revealed an
upeld or downeld shi of pyrazoline and aryl protons
(Fig. S14, ESI†).
To obtain a better understanding of the response of probe L
towards Zn2+, the effect of EDTA on the uorescence behavior of
the binding of Zn2+ to L was examined. As depicted in Fig. S15
(ESI†), upon addition of 10 mM EDTA to the mixture of L (10 mM)
and Zn2+ (10 mM) the uorescent intensity was almost quenched
to the uorescent intensity of probe L alone. However, the
uorescence was almost completely recovered when 1 equiv. of
Zn2+ was added to the mixture of L–Zn2+–EDTA (10 mM). The
results indicated that probe L was reversible for Zn2+
.
For practical applications, the effects of pH and time are
important factors for the probe. The relative uorescence
intensity for probe L alone was not sensitive to pH from 4.0 to
9.0. However, the uorescence of L–Zn2+ was strongly depen-
dent on pH, while pH from 6 to 7.6, uorescence of L–Zn2+
increased sharply and peaked at pH around 7.6, and then
decreased with the increase of pH value from 7.6 to 9 (Fig. S16,
ESI†). In addition, the time course of L to 5 equiv. of Zn2+
showed that the probe L takes less than 10 min to interact with
Zn2+ (Fig. S17, ESI†). The results showed that the probe can fast
response Zn2+ in physiological condition.
Fig. 2 Fluorescence spectra of 10 mM L in buffered EtOH–HEPES
solution with 5 equiv. of metal ions (excitation wavelength: 410 nm).
understand the binding mode of L and Zn2+, we also prepared
compound 6 without phenolic hydroxyl group. In contrast to L,
compound 6 did not induce obviously change in the absence
and presence of 5 equiv. of Zn2+ in the HEPES buffer solution
(Fig. S11, ESI†). In addition, we also used the MS method to
conrm our hypothesis. The [(L–H)/Zn]+ complex was calculated
at m/z 393.0581 and found at 393.0567 (Fig. S12, ESI†). To verify
the mechanism of L–Zn2+, rst-principles density functional
theory calculations were performed using the Gaussian 09
program. The exchange and correlation interactions were
modelled using the generalized gradient approximation (GGA)
with the PW91 functional. The 6-311++G(d,p) (Lanl2dz on Zn)
basis set was used for geometric optimization of the complex.
And the calculation results support proposed binding mode
(Fig. S13, ESI†). To better understand the binding behaviour of
Cell culture and imaging of intracellular Zn2+
Finally, to investigate the potential biological application of the
probe L in living cells, imaging inside HeLa cells was monitored
by uorescence microscopy. The cells showed very faint uo-
rescence aer incubation with 5 mM of the probe for 30 min at
37 ꢂC. However, uorescence became clearly visible when
exogenous Zn2+ (10, 25, 50 mM) was introduced into the cells in
the present of zinc-selective ionophore (concentration equiva-
lent of 1/2 added Zn2+), pyrithione(2-mercaptopyridine N-oxide),
providing visual evidence of the probe L readily entering cells
Fig. 3 Fluorescence emission spectra of L–Zn(II) (1.0 ꢀ 10ꢁ5 M) in the Fig. 4 Fluorescence emission spectra of L (10 mM) upon the titration of
presence of Al3+, Cr3+, Fe3+, Co2+, Cu2+, Ni2+, Ba2+ and Pb2+ (5 ꢀ 10ꢁ5 Zn2+ (0–50 equiv.) in HEPES buffer solution (20 mM HEPES, pH 7.2,
M) and Na+, Mg2+, K+, and Ca2+ (20 ꢀ 10ꢁ5 M) in the HEPES buffer EtOH–H2O ¼ 1 : 1). The inset shows fluorescence intensity of Zn2+
solution (20 mM HEPES, pH ¼ 7.2, EtOH–H2O ¼ 1 : 1).
concentration at 475 nm (excitation wavelength: 410 nm).
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and information on the intracellular existence of Zn2+ (Fig. 5A).
In addition, uorescence intensity signicantly decreased aer
the addition of N,N,N0,N0-tetrakis (2-pyridylmethy1) ethylenedi-
amine (TPEN, 200 mM) for 30 min (Fig. 5B). The results indi-
cated that probe
L should be potential for biological
applications. We also analyzed the uorescence intensity
quantitation using the ImageJ image analysis tool and found
that the uorescence intensity increased as the concentration of
exogenous Zn2+ (Fig. 5C).
To investigate the biocompatibility of the probe, the cyto-
toxicity of the probe was evaluated using HeLa cell line by SRB
assay, and the results were shown in Fig. S18 (ESI).† The probe
showed a cell viability of 100% contrasting to the control group,
which clearly demonstrated that the probe had no cytotoxicity
to the cultured cell line under experimental conditions at the
concentration of 5 mM aer treating cells for 6 h.
Conclusions
In summary, we have developed a simple pyrazoline-based
uorescent probe for the detection of Zn2+ in aqueous solution.
The probe displayed a 60-fold uorescence enhancement upon
complexation with Zn2+ under physiological conditions. And
the addition of EDTA or TPEN can quench the uorescence of
L–Zn2+ which shows that L is a reversible probe. The detection
limit of probe L for Zn2+ was calculated to be 1.23 ꢀ 10ꢁ7 M and
the association constant was 3.89 ꢀ 103 Mꢁ1. Additionally, we
have demonstrated that the probe can be successfully used to
the microscopic imaging for detection of intracellular Zn2+ in
HeLa cells.
Experimental section
Materials and characterization
Fig. 5 (A) Fluorescence microscope images of living HeLa cells. Cells
of control groups were incubated with 5 mM probe L for 30 min at 37
ꢂC. Cells were exposed to pyrithione (pyr, concentration equivalent of
1/2 added Zn2+) in the presence of 10, 25 and 50 mM extracellular Zn2+
for 30 min. After rinsed twice with PBS, cells were treated with 5 mM
probe L for 30 min at 37 ꢂC. (a) Fluorescent, (b) bright-field, (c) overlay
image. (B) Fluorescent image after TPEN treatment. Intensity of fluo-
rescence was significantly diminished after addition of 200 mM TPEN
for 30 min. (d) Fluorescent, (e) bright-field, (f) overlay image. (C)
Fluorescence intensity quantitation. Results are presented as means ꢃ
SE (n ¼ 3; *p < 0.05 vs. control; **p < 0.01 vs. control).
Deionized water was used throughout the experiment. All the
reagents were purchased from commercial suppliers and used
without further purication. The aqueous solutions of metal
ions were prepared from KNO3, NaNO3, Ca(NO3)2$4H2O,
Mg(NO3)2$6H2O, Fe(NO3)3$9H2O, AgNO3, Co(NO3)2$6H2O,
Al(NO3)3$9H2O, Ba(NO3)2, Cr(NO3)3$9H2O, Ni(NO3)2$6H2O,
Cd(NO3)2$4H2O, Pb(NO3)2, Cu(NO3)2$3H2O, Zn(NO3)2$6H2O
and HgCl2, respectively. All samples were prepared at room
temperature, shaken for 10 s and stood for 18 h before UV-vis
and uorescence determination. Thin-layer chromatography
(TLC) was conducted on silica gel 60F254 plates (Merck KGaA). on a U-4100 (Hitachi). Fluorescent measurements were recor-
HEPES buffer solutions (pH 7.2) were prepared using 20 mM ded on an F-7000 (Hitachi) luminescence spectrophotometer.
HEPES, and the appropriate amount of aqueous sodium
hydroxide using a pH meter. 1H NMR spectra were recorded on
a Bruker Avance 300 (300 MHz) spectrometer, using DMSO as
Cell culture and imaging
solvent. 13C NMR spectra were recorded on a Bruker Avance 300 HeLa cells were cultured in Dulbecco's modied Eagle's
(75 MHz) spectrometer, using DMSO as solvent, and tetrame- medium (DMEM, Gibco) containing 10% calf bovine serum 50
thylsilane (TMS) as internal standard. Melting points were (HyClone) at 37 ꢂC in humidied air with 5% CO2. For uo-
determined on an XD-4 digital micro melting point apparatus. rescence imaging, the cells (2 ꢀ 104 mLꢁ1) were seeded into 24
IR spectra were recorded with an IR spectrophotometer VERTEX well plates overnight, and then experiments to assay Zn2+
70 FT-IR (Bruker Optics). HRMS spectra were recorded on a uptake were performed in the same media supplemented with
QTOF6510 spectrograph (Agilent). UV-vis spectra were recorded different concentrations of zinc nitrate with pyrithione (zinc
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ionophore, concentration equivalent of 1/2 added Zn2+). The CHH in pyrazoline moiety), 4.09 (dd, 1H, J ¼ 11.8, 18.4 Hz, CHH
cells were washed twice with PBS buffer and incubated with the in pyrazoline moiety), 5.58 (dd, 1H, J ¼ 4.6, 11.8 Hz, CH in
probe (5 mM) for 30 min in the incubator. Aer washing twice pyrazoline moiety), 7.25–7.65 (m, 9H, Ar–H), 7.91 (dd, 1H, J ¼
with PBS, the cells were imaged under an inverted uorescence 2.7, 7.2 Hz, Ar–H), 8.32 (m, 1H, Ar–H), 11.30 (s, 1H, OH)
microscope (Nikon TE2000-S) with a 20ꢀ objective lens.
(Fig. S19, ESI†); 13C NMR (75 MHz, DMSO): d ¼ 166.8, 157.0,
153.8, 142.0, 134.7, 128.7 (2C), 128.1, 127.6, 127.3, 126.0, 125.5
(2C), 125.0, 123.9, 122.6, 119.3, 108.9, 58.1, 43.0, 21.9 (Fig. S20,
Cell viability assay
+
ESI†); HRMS: calcd for C21H19N2O2 [M + H]+ 331.1447, found:
Cells (2 ꢀ 104 mLꢁ1) were seeded in 96-well plates. Aer 24 h,
cells were treated with 0.1% DMSO (as control) or the probe at
working concentration for 6 h. The probe was dissolved in
DMSO as stock solution and applied to cells such that the nal
concentration of DMSO in the culture medium was below 0.1%
(v/v). DMSO at a concentration of 0.1% (v/v) did not affect the
viability of the cells. Cell viability was evaluated by sulforhod-
amine B (SRB) assay. Briey, the medium was poured off and
the cells were xed by adding 100 mL of cold 10% trichloroacetic
acid (TCA) and incubating for 1 h at 4 ꢂC. The supernatant was
discarded and then the plates were washed 5 times with
deionized water. 50 mL of 0.4% (w/v) SRB solution in 1% acetic
acid was added to each well and shaken for 5 min on a titer plate
shaker. The plates were washed 5 times with 1% acetic acid and
subsequently 100 mL of 10 mM unbuffered Tris base (pH 10.5)
was added to dissolve the bound dye. Aer being mixed for 5
min on a microtiter plate shaker and optical densities were read
at the wavelength of 540 nm using a SpectraMAX 190 microplate
spectrophotometer (GMI Co., USA).
331.1439 (Fig. S21, ESI†).
Synthesis of 1-(3-naphthalen-2-yl)-5-phenyl-4,5-dihydro-1H-
pyrazol-1-yl))ethanone
Chalcone 5 was easily prepared according to the literature24 with
an 86% yield. Compound 6 was prepared in the same method
with probe 4. White solid; yield: 70%; mp: 136–138 ꢂC. 1H NMR
(400 MHz, DMSO-d6): d ¼ 2.37 (s, 3H, CH3), 3.27 (dd, 1H, J ¼ 4.6,
17.9 Hz, CHH in pyrazoline moiety), 3.95 (dd, 1H, J ¼ 11.8, 17.9
Hz, CHH in pyrazoline moiety), 5.60 (dd, 1H, J ¼ 4.6, 11.8 Hz,
CH in pyrazoline moiety), 7.22–7.35 (m, 5H, Ar–H), 7.55–7.60
(m, 2H, Ar–H), 7.94–8.07 (m, 4H, Ar–H), 8.18 (s, 1H, Ar–H); 13
C
NMR (100 MHz, DMSO): d ¼ 167.9, 154.6, 142.9, 134.1, 133.2,
129.1 (3C), 128.9, 128.8, 128.2, 128.0, 127.8, 127.7, 127.3, 125.9
(2C), 123.5, 60.0, 42.6, 22.2; HRMS: calcd for C21H19N2O+ [M +
H]+ 315.1497, found: 315.1494.
Acknowledgements
Synthesis of 1-(3-hydroxynaphthalen-2-yl)-5-phenyl-4,5-
dihydro-1H-pyrazol-1-yl))ethanone
This study was supported by 973 Program (2010CB933504) and
National Natural Science Foundation of China (91313303).
A mixture of 1-(1-hydroxynaphthalen-2-yl)ethanone (933 mg, 5
mmol), benzaldehyde (540 mg, 5 mmol), 25% sodium hydroxide
aqueous solution (15 mL) and ethanol (30 mL) was stirring at
room temperature for about 10 h. Aer then, 80 mL of water was
added to the reaction mixture and hydrochloric acid (6 M) was
added to adjust pH to 5–6, then a red solid was precipitated. The
crude product was obtained by ltration. Desired compound 3
was obtained by recrystallization from ethanol in 71% yield.
mp: 124–126 ꢂC. 1H NMR (400 MHz, DMSO-d6): d ¼ 7.48 (d, 1H, J
¼ 15.4 Hz), d ¼ 7.49–7.52 (m, 3H), 7.62 (t, 1H, J ¼ 8.0 Hz), 7.75 (t,
1H, J ¼ 7.4 Hz), 7.79–8.00 (m, 4H), 8.20 (d, 1H, J ¼ 15.4 Hz), 8.33
Notes and references
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A mixture of chalcone 3 (182 mg, 0.66 mmol) and 80%
hydrazine hydrate (166 mg, 3.3 mmol) in acetic acid (15 mL) was
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