A highly sensitive fluorescent probe based on simple pyrazoline for Zn 2+ in living neuron cells

Article abstract of DOI:10.1039/c2ob26375k

We develop a pyrazoline-based fluorescent sensor for biological Zn ²⁺ detection. The sensor shows good binding selectivity for Zn ²⁺ over competing metal with 40-fold fluorescence enhancement in response to Zn²⁺. The new probe is cell-permeable and can be used to detect intracellular zinc ions in living neuron cells.

Full text of DOI:10.1039/c2ob26375k

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PAPER
A highly sensitive fluorescent probe based on simple pyrazoline for Zn2+
in living neuron cells†
Zhe Zhang,a Fang-Wu Wang,b Sheng-Qing Wang,a Fei Ge,a Bao-Xiang Zhao*a and Jun-Ying Miao*b
Received 17th July 2012, Accepted 10th September 2012
DOI: 10.1039/c2ob26375k
We develop a pyrazoline-based fluorescent sensor for biological Zn2+ detection. The sensor shows good
binding selectivity for Zn2+ over competing metal with 40-fold fluorescence enhancement in response to
Zn2+. The new probe is cell-permeable and can be used to detect intracellular zinc ions in living neuron
cells.
in toxic organic-containing solution, which does harm to the bio-
logical systems during in vivo tests. What is more, their prep-
Introduction
Zinc ion has attracted much attention due to its biological signifi-
cance. As the second most abundant transition metal ion in the
human body after iron, Zn2+ shows its vital role in catalytic
centers and structural cofactors of many Zn2+-containing
enzymes and DNA-binding proteins.1 It has been reported that
zinc plays an essential part in many biological processes, such as
brain function and pathology, gene transcription, immune func-
tion and mammalian reproduction,2 as well as some pathological
processes, such as Alzheimer’s disease, epilepsy, ischemic stroke
and infantile diarrhea.3 Although most Zn2+ is tightly bound to
enzymes and proteins, free zinc pools exist in some tissues, such
as brain, intestine and retina.4 In addition, while serum zinc
levels are low in the setting of most cancers, tumor tissue in
breast and lung cancer has elevated zinc levels when compared
with the corresponding normal tissues.5 Zinc deficiency may
play an important role in the appearance of diseases, but excess
zinc is toxic for the cell. Therefore, the cellular level of zinc
must be controlled within a suitable range, which is normally
between 0.1 and 0.5 mM.6 The significance of zinc homeostasis
to neuro-physiology and neuropathology has attracted much
interest in devising new ways to detect Zn2+ in biological
samples. Sensitive and selective detection of Zn2+ in biological
systems, therefore, is essential and strongly desired. In the last
decade, numerous scientific endeavors have focused on the
development of fluorescent chemosensors for Zn2+, including
small molecule,7 macrocycle8 and nanoparticles9 for the in vitro
and in vivo detection of Zn2+. Nevertheless, most of them work
aration requires complex multistep organic synthesis that costs a
lot. Up to now, very few Zn2+ probes have been successfully
developed for the imaging of Zn2+ in neuron cells.10 Therefore,
the development of fluorescent probes with high sensitivity and
selectivity for monitoring Zn2+ in living neuron cells remains a
significant challenge.
Recently, we synthesized a novel fluorescent chemosensor
based on pyrazoline and studied the property for detecting zinc
ion in the HEPES (20 mM HEPES, pH = 7.2, 50% (v/v)
CH3CN) buffer solution. However, the sensor was limited by the
poor turn-on response (3-fold) in the presence of excess Zn2+.11
Thus, there is a need to develop a new more sensitive probe for
Zn2+. In this work, a new approach to developing Zn2+ fluore-
scent sensor based on pyrazoline, 5-(2-(allyloxy)phenyl)-3-
(2-hydroxy-5-methylphenyl)-4,5-dihydro-1H-pyrazole-1-carbothio-
amide, is reported. Connected with a group of allyloxy, the lipo-
philic property of the compound is improved, which gives it the
ability to penetrate the membrane easily. Meanwhile, using
the thiosemicarbazide to form pyrazoline not only minimizes the
steric hindrance compared to using other arylhydrazines, but also
adds the group of carbothioamide with more hydrogen bonds in
water solution.
Results and discussion
The synthesis of probe L (compound 4) from chalcone (com-
pound 3) is described in Scheme 1. The structures of 3 and L
were determined by X-ray analysis (Fig. S1, S2, ESI†). The
structure of L with 1 equiv. cavities capable of chelating Zn2+
possesses a tridentate chelating conformation by the group of
phenolic oxygen, pyrazoline N and allyloxy double bond. The
formation of L–Zn(II) can enhance the immobility of the rotata-
ble bonds, leading to a fairly rigid structure with considerably
stronger fluorescence properties than free L.
aInstitute of Organic Chemistry, School of Chemistry and Chemical
Engineering, Shandong University, Jinan 250100, P. R. China.
E-mail: bxzhao@sdu.edu.cn
bInstitute of Developmental Biology, School of Life Science, Shandong
University, Jinan 250100, P. R. China. E-mail: miaojy@sdu.edu.cn
†Electronic supplementary information (ESI) available: X-ray crystallo-
graphic data of compound 3 and 4. CCDC 891147 and 891148. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c2ob26375k
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Scheme 1 The synthesis of compound 4 (L).
Fig. 1 UV-vis spectra of L (100 μM) upon the titration of Zn2+ (0.2,
0.4, 0.6, 0.8, 1.0, 1.2, 1.5, 2.0, 3.0, 4.0, 5.0 equiv.) in HEPES buffer
solution (20 mM HEPES, pH 7.2, EtOH : H2O = 1 : 1).
Fig. 2 Fluorescence intensity changes ((I − I0)/I0) of free L (10−5 M)
at 476 nm in the HEPES buffer solution (20 mM HEPES, pH = 7.2,
EtOH : H2O = 1 : 1) upon addition of various metal ions (10−4 M). I and
I0 denote fluorescence intensity of L in the presence and absence of
metal ions. Excitation wavelength: 395 nm.
To verify the practical applications in a biological environ-
ment, the spectroscopic properties of L and with different metal
ions were measured under simulated physiological conditions
(20 mM HEPES buffer at pH 7.2, EtOH : H2O = 1 : 1). As
shown in the ESI (Fig. S3, S4, ESI†), during the UV-vis spec-
metal ions in living cells, such as Na+, K+ and Ca2+, is necess-
ary. In our research, the concentration of these metal ions were
40 times over Zn2+ and showed a negligible effect on the fluor-
escence response for L. The existence of other important tran-
sition metal ions may also affect the detection ability of L. Iron
as the most abundant transition metal ion shows no interference,
even in the presence of 10 equiv. iron ions (Fig. S7, ESI†). Com-
pared with some available Zn2+ sensors, which exhibit some
enhancement of the fluorescence for Cd2+,12 our sensor has a
selective response to Zn2+ without the interference of Cd2+ in
the buffered solution, whereas, the existence of copper obviously
quenched the fluorescence intensity, which is consistent with
previous reports.7a,13 Thus, this probe can detect Zn2+ with
minimum interference from other competing metal ions and
should have good selectivity for Zn2+ in the biological studies.
In addition, the intensity of L–Zn(II) at 476 nm showed a
decrease with the addition of Cu2+, which indicated that Cu2+
replaced Zn2+ and formed L–Cu(II). This property might make it
useful as a good ON–OFF sensor for Cu2+.
trum of
L with different concentrations, the absorption
maximum is at 341 nm and the ε is 2.5 × 104 M−1 cm−1. In the
UV-vis titration spectra of L with Zn2+ (0–5 equiv.) (Fig. 1), a
decrease of the absorbance at 341 nm and an increase of a new
absorption band centered at 395 nm were observed with a dis-
tinct isobestic point at 366 nm.
Binding affinities of compound L with different metal ions
were also evaluated by UV-vis spectroscopy. Upon addition of
the metal ions, the absorption spectra vary in different manners,
as shown in the ESI (Fig. S5†). In the case of Co2+, Ag+, Cu2+,
Ni2+, Hg2+ and Zn2+, the addition of the metal ions caused
changes in shape. Especially, stronger absorptions appeared in
the range of 375–450 nm due to the binding of L with Cu2+,
Ni2+ Co2+ and Ag+, whereas the others did not change. The
binding of L with Cu2+, Ni2+ Co2+ and Ag+ can further quench
the fluorescence intensity of L.
In the fluorescence titration spectra of L with Zn2+ (0–50
equiv.) excited at 395 nm (Fig. 3), L alone shows a weak fluor-
escence emission band at 476 nm with a quite weak fluorescence
quantum yield (0.0023). When adding the Zn2+ to the buffer solu-
tion, a gradual increase in fluorescence (fluorescence quantum
yield 0.091) with a conspicuous color change from colorless to
yellow is found. From the photo in the ESI (Fig. S8†), we can
see the stronger blue emission of L with addition of 10 μM Zn2+
ion under the irradiation at 365 nm than without the addition of
Ion selectivity is an important property of the fluorescence
probes. Thus, we evaluated the fluorescent response of L with
different metal ions, including Ag+, Ba2+, Ca2+, Cd2+, Co2+,
Cr3+, Cu2+, Fe3+, K+, Mg2+, Na+, Ni2+ and Pb2+ in HEPES
buffer solution and found the perfect selectivity for Zn2+ with a
considerable signal output (Fig. 2). The Job plots were
implemented a 1 : 1 stoichiometry for L–Zn(II) (Fig. S6, ESI†).
To apply L as a probe in a biological environment, determin-
ing the tolerance to the interference of physiologically important
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Fig. 3 Fluorescence emission spectra of L (10−5 M) was titrated with
Zn2+ (0–50 × 10−5 M) in the HEPES buffer solution (20 mM HEPES,
pH = 7.2, EtOH : H2O = 1 : 1). Inset: changes of fluorescence intensity
of L (10−5 M) with different equivalents of Zn2+ at 476 nm.
Fig. 5 Fluorescence microscope images of living PC12 cells. (a1–a3):
images of cells incubated with 10 μM probe L for 30 min at 37 °C.
Cells were exposed to pyrithione (pyr, concentration equivalent of 1/2
added Zn2+) in the presence of increased concentrations of extracellular
Zn2+ as (b1–b3) 10, (c1–c3) 25 and (d1–d3) 50 μM following incubation
with probe L for 30 min. (a1–d1) Bright-field, (a2–d2) fluorescent,
(a3–d3) overlay image. Scale bar = 10 μm.
of L contains a phenol hydroxyl with acidity and a nitrogen
atom with basicity. In the condition of strong acidity, nitrogen
might be protonated to suppress the binding ability to Zn(II), so
that the fluorescence decreases. Whereas, when pH > 11,
hydroxyl in phenol may undergo deprotonation, which might
influence the binding model to lower fluorescence. The strong
and stable fluorescence of L–Zn(II) in the biologically relevant
pH range indicated its practicality in physiological conditions.
The intracellular Zn2+ imaging behaviour of L was studied on
PC12 neuronal cells by a fluorescence microscope. After incu-
bation with DMSO-containing L (10 μM, DMSO : water =
1 : 100) for 30 min at 37 °C, the cells exhibited very faint fluor-
escence. However, fluorescence became clearly visible in the
cytoplasm of PC12 cells when exogenous Zn2+ was introduced
into the cells in the presence of a zinc-selective ionophore, pyri-
thione (2-mercaptopyridine N-oxide), providing visual evidence
of the probe L readily entering cells and information on the
intracellular existence of Zn2+ (Fig. 5). Moreover, with increas-
ing concentration of Zn2+ incubated, the fluorescence intensity
enhanced obviously in a concentration-dependent manner. These
results demonstrate that probe L has the potential for biological
applications.
Fig. 4 The proposed binding mode of L with Zn2+ in aqueous
solution.
Zn2+ ion, presumably due to the blocking of the photoinduced
electron transfer (PET) process upon complexation with Zn2+.
The fluorescence enhancement was approximately 40-fold in the
presence of excess Zn2+.
A proposed binding mode is shown in Fig. 4. The fact that the
hydrogens bound to the double bond of the allyloxy group were
disordered by X-ray analysis indicated that the π-electrons were
delocalized. The interactions of delocalized π-electron systems
with metal centers are of fundamental importance in organo-
metallic chemistry and have received considerable attention. The
allyl group represents the simplest delocalized π-electron system
and allyl metal complexes are therefore ideal model systems.14
To get an insight into the binding mode, 1H NMR titration
experiments were also performed. Fig. S9† in the ESI shows the
change in 1H NMR of L in the absence and presence of
0.2 equiv. Zn2+. As a result of metal chelation, all protons in the
allyl and aryl groups showed an upfield or downfield shift,
which are attributed to the changes in electron density. The
double bond in the allyloxy moiety as a ligand contributes an
electron to a zinc orbital.
Conclusions
In summary, a new fluorescent sensor for Zn2+ has been
designed and synthesized by taking advantage of the pyrazoline
derivative. This sensor exhibits good sensitivity and selectivity
toward Zn2+ in aqueous solution. Moreover, the sensor can
monitor intracellular Zn2+ level in living neuronal cells.
Following a Benesi–Hildebrand-type analysis,15 the associ-
ation constant Ka was determined to be 4.83 × 104 M−1. The flu-
orescent titration profile of L with Zn2+ demonstrated that the
detection limit of Zn2+ is 6.1 × 10−7 M (Fig. S10, ESI†).
For practical application, the appropriate pH conditions for
successful operation of the probe were explored. It is interesting
to observe the bell-shaped profile (Fig. S11, ESI†). The structure
Experimental section
Materials and characterization
Deionized water was used throughout the experiment of absorp-
tion and fluorescence determination. All of the reagents were
8642 | Org. Biomol. Chem., 2012, 10, 8640–8644
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purchased from commercial suppliers and used without further
purification. The solutions of metal ions were prepared
from NaNO3, Mg(NO3)2·6H2O, Al(NO3)3·9H2O, KNO3, Ca-
(NO3)2·4H2O, Cr(NO3)3·9H2O, Fe(NO3)3·9H2O, Co(NO3)2·6H2O,
Ni(NO3)2·6H2O, Cu(NO3)2·3H2O, Zn(NO3)2·6H2O, AgNO3,
Cd(NO3)2·4H2O, Ba(NO3)2 and Pb(NO3)2 with deionized water.
All samples were prepared at room temperature, shaken for 10 s
and left to stand for 18 h before UV-vis and fluorescence
determination. Thin-layer chromatography (TLC) was conducted
on silica gel 60 F254 plates (Merck KGaA). HEPES buffer
solutions (pH 7.2) were prepared using 20 mM HEPES and the
appropriate amount of aqueous sodium hydroxide using a
pH meter. 1H NMR (300 MHz) and 13C NMR (75 MHz) spectra
were recorded on a Bruker Avance 300 spectrometer using
CDCl3 as the solvent and tetramethylsilane (TMS) as the
internal standard. Melting points were determined on a XD-4
digital micro melting point apparatus. IR spectra were recorded
with an IR spectrophotometer VERTEX 70 FT-IR
(Bruker Optics). HRMS spectra were recorded on a Q-TOF6510
spectrograph (Agilent). UV-vis spectra were recorded on a
U-4100 UV-Vis-NIR Spectrometer (Hitachi). Fluorescent
measurements were recorded on a Hitachi F-4500 fluorescence
spectrophotometer.
compound 3 was also determined by X-ray analysis and is
shown in Fig. S1† (CCDC no. 891148).
Synthesis of 5-(2-(allyloxy)phenyl)-3-(2-hydroxy-5-
methylphenyl)-4,5-dihydro-1H-pyrazole-1-carbothioamide (4)
Compound 4 was prepared by the reaction of chalcone (3) and
thiosemicarbazide. Briefly, NaOH and thiosemicarbazide was
added to a stirred solution of chalcone (3) in ethanol and
refluxed for 4 h (monitored by TLC). After reaction, the mixture
was cooled to room temperature and hydrochloric acid was
added in droplets to neutralize it to pH 7. The crude product was
obtained as a yellow solid, which was recrystallized from ethanol
to obtain compound 4. White solid; yield: 52%; mp:
222–223 °C; IR (KBr, cm−1): 3436.6, 3326.8, 1600.4, 1481.1,
1337.7, 1250.9, 816.8, 746.7; 1H NMR (300 MHz, CDCl3):
2.27 (s, 3H, CH3), 3.25 (dd, 1H, J = 18, 3.6 Hz, CHH in pyrazo-
line moiety), 3.90 (dd, 1H, J = 18, 11.4 Hz, CHH in pyrazoline
moiety), 4.57 (d, 2H, J = 5.4 Hz, CH2 in allyloxy moiety), 5.23
(d, 1H, JABcis = 10.5 Hz, vCHH), 5.37 (d, 1H, JACtrans = 17.1,
vCHH), 5.93–6.06 (m, 1H, vCH–), 6.24 (dd, 1H, J = 11.4,
3.6 Hz, CH in pyrazoline moiety), 6.26–6.60 (m, 2H, NH2),
6.88–6.96 (m, 3H, Ar–H), 7.00 (s, 1H, Ar–H), 7.09–7.26
(m, 3H, Ar–H), 9.53 (s, 1H, OH); 13C NMR (75 MHz, CDCl3):
175.7, 159.2, 155.0, 154.5, 133.2, 132.4, 128.7, 128.5, 128.4,
128.1, 126.4, 120.3, 117.2, 116.3, 114.0, 111.8, 68.6, 58.5, 41.8,
19.9; HRMS: calcd for [M + H]+ C20H22N3O2S: 368.1433;
found: 368.1424. The X-ray structure is shown in Fig. S2†
(CCDC no. 891147).
Cell culture and imaging
PC12 cells were grown in Dulbecco’s Modified Eagle’s Medium
(DMEM, Gibco), containing 10% fetal bovine serum (Gibco),
5% donor horse serum (Biological Industries) and 1% PSN anti-
biotic, at 37 °C in a humidified incubator with 5% CO2. For the
experimental study, cells were grown to 80–90% confluence, har-
vested with 0.05% trypsin (Sangon Biotech) in phosphate-
buffered saline (PBS) and plated at the desired cell concentration.
For fluorescence imaging, the cells were seeded into 24-well
plates and experiments to assay Zn2+ uptake were performed in
the same media supplemented with different concentrations of
zinc nitrate with pyrithione (zinc ionophore, concentration equiv-
alent of 1/2 added Zn2+). The cells were rinsed twice with PBS
and treated with DMSO-containing L (10 μM, DMSO : water =
1 : 100) for 30 min at 37 °C. After washing twice with PBS, the
cells were imaged under an inverted fluorescence microscope
(Nikon TE2000-S).
Acknowledgements
This study was supported by 973 Program (2010CB933504) and
National Natural Science Foundation of China (90813022 and
20972088).
Notes and references
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