Novel pyrazoline-based selective fluorescent sensor for Hg2+

Article abstract of DOI:10.1007/s10895-013-1339-y

This paper presents the preparation of a pyrazoline compound and the properties of its UV-Vis absorption and fluorescence emission. Moreover, this compound can be used to determine Hg²⁺ ion with selectivity and sensitivity in the EtOH:H₂O=9:1 (v/v) solution. This sensor forms a 1:1 complex with Hg²⁺ and shows a fluorescent enhancement with good tolerance of other metal ions. This sensor is very sensitive with fluorometric detection limit of 3.85×10^-10 M. In addition, the fluorescent probe has practical application in cells imaging.

Full text of DOI:10.1007/s10895-013-1339-y

J Fluoresc
DOI 10.1007/s10895-013-1339-y
SHORT COMMUNICATION
Novel Pyrazoline-Based Selective Fluorescent Sensor for Hg2+
Sheng-Qing Wang & Shu-Yan Liu & Hao-Yan Wang &
Xiao-Xin Zheng & Xun Yuan & Yi-Zhi Liu &
Jun-Ying Miao & Bao-Xiang Zhao
Received: 3 September 2013 /Accepted: 27 November 2013
#
Springer Science+Business Media New York 2013
Abstract This paper presents the preparation of a pyrazoline
compound and the properties of its UV–Vis absorption and
fluorescence emission. Moreover, this compound can be used
to determine Hg2+ ion with selectivity and sensitivity in the
EtOH:H2O=9:1 (v/v) solution. This sensor forms a 1:1 complex
with Hg2+ and shows a fluorescent enhancement with good
tolerance of other metal ions. This sensor is very sensitive with
fluorometric detection limit of 3.85×10−10 M. In addition, the
fluorescent probe has practical application in cells imaging.
fluorescent chemosensors for Hg2+ including small molecules
[2–7], conjugated polymers [8, 9], nanoparticles [10–13], and
biomolecules [14–16]. However, many of these systems suffer
from practical use, such as cross-sensitivities toward other
metal ions, narrow pH span, and delayed response, etc. Ac-
cordingly, developing new and practical, sensitive and selective
chemosensor for Hg2+ is still a challenge.
Pyrazolines are important nitrogen containing 5-membered
heterocyclic compounds with stronger fluorescence, have
higher hole-transport efficiency and excellent emitting blue-
ness property. Therefore, pyrazoline derivatives have widely
been used as whitening or brightening reagents for synthetic
fibers, fluorescent chemosensors for recognition of transition
metal ions, hole-transport materials in the electrophotography
and electroluminescence fields [17–22]. Moreover, pyrazoline
with membrane permeability, low toxicity, and high quantum
yield render the fluorophore attractive for biological applica-
tions [23, 24]. However, a few pyrazoline derivatives as
effective “turn on” fluorescent sensors for metal ions were
reported [25, 26] and only one paper presented the pyrazoline
probe to detect mercury ion [27]. As an extension of our work
on the development of fluorescent probe for monitoring metal
ions [28–32], herein, we developed a new pyrazoline com-
pound as a selective and sensitive fluorescent sensor for Hg2+
in aqueous solution. The structure of the compound was
.
.
Keywords Pyrazoline Fluorescent probe Mercury ion
.
detection Selective
Introduction
As a dangerous and widespread global pollutant, mercury ion
can easily pass through skin, respiratory, and gastrointestinal
tissues into the human body, and damage the central nervous
and endocrine systems [1]. Thus, mercury pollution has sparked
interest in the design of new tactics to monitor Hg2+ in biolog-
ical and environmental samples. In the last decade, numerous
scientific endeavors have focused on the development of
1
Sheng-Qing Wang and Shu-Yan Liu with equal contribution
characterized by IR, H NMR and HRMS. The association
constant Ka measured for coordination of the sensor with
Hg2+ was 3.03×104 M−1, and the detection limit of the sensor
toward Hg2+ was 3.85×10−10 M.
Electronic supplementary material The online version of this article
(doi:10.1007/s10895-013-1339-y) contains supplementary material,
which is available to authorized users.
:
:
:
:
:
S.<Q. Wang H.<Y. Wang X.<X. Zheng X. Yuan Y.<Z. Liu
B.<X. Zhao (*)
School of Chemistry and Chemical Engineering, Shandong
University, Jinan 250100, People’s Republic of China
e-mail: bxzhao@sdu.edu.cn
Experimental Details
Apparatus
:
S.<Y. Liu J.<Y. Miao (*)
School of Life Science, Shandong University, Jinan 250100, People’s
Republic of China
e-mail: miaojy@sdu.edu.cn
Thin-layer chromatography (TLC) was conducted on silica
gel 60 F254 plates (Merck KGaA). H NMR spectra were
1

J Fluoresc
recorded on a Bruker Avance 300 (300 MHz) spectrometer,
using DMSO-d6 as solvent and tetramethylsilane (TMS) as
internal standard. Melting points were determined on an XD-4
digital micro melting point apparatus. IR spectra were record-
ed with an IR spectrophotometer VERTEX 70 FT-IR (Bruker
Optics). HRMS spectra were recorded on a Q-TOF6510 spec-
trograph (Agilent). UV–Vis spectra were recorded on a U-
4100 (Hitachi). Fluorescent measurements were recorded on a
Perkin-Elmer LS-55 luminescence spectrophotometer. All pH
measurements were made with a Model PHS-3C pH meter
(Shanghai, China) and operated at room temperature about
298 K.
Analytical Procedure
A 1.0×10−3 M of stock solution of compound L was prepared
in ethanol. The cationic stocks were all in H2O with a concen-
tration of 10−1 M for UV–Vis absorption and fluorescence
spectra analysis. For all measurements of fluorescence spectra,
excitation was at 330 nm with 10 nm of excitation slit width and
scan speed was set at 600 nm min−1. UV–Vis and fluorescence
titration experiments were performed using 5×10−5 M and 1×
10−5 M of compound L in the EtOH:H2O=9:1, respectively.
For Hg2+ ion absorption and fluorescence titration experiments,
a 3 mL solution of compound L (1×10−5 M) were filled in the
quartz cell of 1 cm optical path length, and each time 1.0 μL
solution of Hg2+ (3×10−3 M) were added into the quartz cell
gradually by using a micro-syringe, respectively. After each
addition of Hg2+ ion, the solution was stirred for 3 min. The
volume of cationic stock solution added was less than 100 μL
with the purpose of keeping the total volume of testing solution
without obvious change.
Reagents
Deionized water was used throughout the experiment. All the
reagents were purchased from commercial suppliers and used
without further purification. The salts used in stock aqueous
solutions of metal ions were NaNO3, Fe(NO3)3 · 9H2O, Ag-
NO3, KNO3, Co(NO3)2 · 6H2O, Mg(NO3)2 · 6H2O, Ca(NO3)2
· 4H2O, 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.
Fluorescence Quantum Yield
The ability for the molecules to emit the absorbed light energy
is characterized quantitatively by the fluorescence quantum
yield (ΦF). Quantum yield was determined by the relative
comparison procedure, using quinine sulfate dehydrate (≥
99.0 %) in 0.1 N H2SO4 as the main standard. The corrected
emission spectra were measured for the quinine sulfate dehy-
drate standard (λex=330 nm; A (Absorption) <0.01; ΦF=
0.560) [33]. For all the measurements of fluorescence spectra,
scan speed was 900 nm min−1 using a quartz cell of 1 cm
optical path length. The UV–Vis absorption spectra were
recorded in a standard 1 cm path length quartz cell in range
250–600 nm with spectral resolution 1 nm. The general equa-
tion used in the determination of relative quantum yields from
earlier research was given in Eq. (1) [34].
General Procedure for the Synthesis of Compound L
To a stirred solution of chalcone 1 (0.248 g, 1.0 mmol) and
NaOH (0.116 g, 3.0 mmol) in ethanol (15 mL) was added
hydrazinecarbothioamide 2 (0.116 g, 1.2 mmol). After 12 h at
refluxing, the reaction mixture was allowed to cool to room
temperature. Subsequently, water (30 mL) was added to the
reaction mixture and the solution was neutralized with dilute
hydrochloric acid. The crude product was filtrated and recrys-
tallized with ethanol to give the products L, (Fig. 1) Pale
Yellow solid, yield 79.8 %; mp 269–271 °C; IR (KBr,
cm−1): 3374.0 (N-H, st), 3265.3 (NH2, st), 3159.9 (NH2, st),
3060.7 (C-H, st), 2968.2 (C-H, st), 1599.9 (C=N, st), 1464.6,
1364.9 (N-H, δ); 1H NMR (300 MHz, DMSO-d6): δ 3.14 (dd,
1H, J =3.7, 18.3 Hz, 4-Htrans), 4.07 (dd, 1H, J =11.7, 18.3 Hz,
4-Hcis), 5.96 (dd, 1H, J =3.7, 11.7 Hz, 5-H of pyrazoline),
7.16–7.20 (m, 2H, Ar-H), 7.23–7.27 (m, 2H, Ar-H), 7.29–
7.36 (m, 3H, Ar-H), 7.57 (d, 1H, J =7.8 Hz, NH2), 7.68 (d,
2H, J =7.8 Hz, Ar-H), 8.38 (s, 1H, NH2), 12.92 (s, 1H, NH);
HRMS: calcd for [M+H]+ C17H15N5S: 322.1126; found:
322.1128.
À
Á
À
Á
2
2
ΦF ¼ ðΦFSÞðFAuÞðAsÞ ηu =ðFAsÞðAuÞ ηs
ð1Þ
Where ΦF and FA are fluorescence quantum yield and
integrated area under the corrected emission spectrum, respec-
tively; A is absorbance at the excitation wavelength; η repre-
sent the refractive index of the solution; and the subscripts u
and s refer to the unknown and the standard, respectively.
Fig. 1 Synthesis of L

J Fluoresc
Cell Culture and Imaging
absorption of the C=N was observed at 1599.9 cm−1. Bending
vibration absorption of N-H exhibited at 1364.9. In the
300 MHz 1H NMR spectra of the compound, the CH2 protons
of the pyrazoline ring resonated as a pair of doublets at
3.14 ppm (Hd), 4.07 ppm (He), respectively. The CH proton
at C5 also appeared as a doublet of doublets at 5.96 ppm due
to vicinal coupling with the two magnetically non-equivalent
protons of the methylene. The nitrogen hydrogen protons of
the thioamide, respectively, presented at 7.57 and 8.38. The
NH proton in the benzimidazole proton signal appeared at δ =
12.92 ppm as a single peak. HRMS showed that found [M+
H]+ ion peak accorded with calculated value.
HeLa cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM, Gibco) containing 10 % calf bovine serum
(HyClone) at 37 °C in humidified air and 5 % CO2. For
fluorescence imaging, the cells (5×104 mL−1) were seeded
into 24-well plates, and experiments to assay Hg2+ uptake
were performed in the same media supplemented with 5, 10
or 20 μM of Hg(ClO4)2 for 1 h. The cells were washed twice
with PBS buffer before staining experiments and incubated
with 10 μM of the probe for 2 h in the incubator. After
washing twice with PBS, the cells were imaged under a phase
contrast microscope (Nikon, Japan).
Absorption Properties
Results and Discussion
The UV–Vis spectroscopic behavior of L toward representa-
tive metal ions was investigated by treating it with physiolog-
ically important alkali, alkaline earth, and transition metal
nitrate or hydrochloride salts in aqueous 10 % ethanol solu-
tion. Sensor L exhibited strong absorption bands at 347 nm.
Upon interaction with various metal ions, significant changes
in absorption spectra were observed particularly with Hg2+,
Ag+ and Cu2+ (Fig. S2). In the presence of mercuric ions,
copper ions and silver ions the wavelength absorption peak
shifts hypsochromically to 319, 292 and 317 nm, respectively.
The interaction of sensor L with the mercury ion was
exhibited through UV–Vis spectrophotometric titration at room
temperature in the HEPES buffer solution (20 mM HEPES,
pH=7.2, 10 % (v/v) EtOH) (Fig. 2). Upon addition of Hg2+, the
absorbance decreased at 344 nm, and a new band at 314 nm
increased. The isobestic point at about 328 nm was attributed to
the equilibrium between the receptor L and Hg2+ throughout
Design and Synthesis of the Probe L
The synthetic route of the proposed compound L is outlined
in Fig. 1. The starting material 1-(1H-benzo[d]imidazol-2-yl)-
3-phenylprop-2-en-1-one was prepared according to the liter-
ature procedures [35]. The pyrazoline derivative L is obtained
in 79.8 % yield by the reaction of chalcone 1 with
hydrazinecarbothioamide 2 at reflux condition in the ethanol.
As shown in Fig. S1 the structure of compound L was
1
confirmed by IR, H NMR and HRMS spectral data. In the
IR spectra, the band at 3374.0 cm−1 is typical stretching
vibration absorption of N-H appended to benzimidazole.
Stretching vibration absorption of NH2 peaked at 3365.3 and
3159.9. The 2 weak bands appeared at 3060.7 and
2968.2 cm−1 may be ascribed to the C-H absorptions. The
Fig. 2 Absorption spectra of L
(10 μM) upon the addition of
Hg2+ (0–6.0 equiv.) in buffered
EtOH:HEPES=9:1 solution at
pH 7.2. Insert: Absorption
changes of the sensor L at 344 nm
upon the addition of Hg2+ (0–6.0
equiv.)

J Fluoresc
Fig. 3 Fluorescence spectra of L
(10 μM) upon addition of various
metal ions (100 μM) in buffered
EtOH:HEPES=9:1 solution at
pH 7.2 under 1 % attenuation
(excitation at 330 nm). Inset:
Photograph of compound L in the
HEPES buffer (20 mM HEPES,
pH=7.2, EtOH:HEPES=9:1)
without (left) and with (right)
addition of 100 μM Hg2+ ion
under the irradiation of UV light
at 365 nm
the titration process. By plotting the changes of receptor L in
the absorbance at 344 nm as a function of Hg2+ concentration,
nonlinear curve was obtained and is shown in the inset of Fig. 3.
Based on a Job plot analysis, the receptor L : Hg2+ ion ratio was
found to be 1:1.
attenuation (exCitation at 330 nm). Upon interaction with,
Cd2+, Pb2+, Na+, K+, Ca2+, Mg2+, Ni2+, Zn2+, Ba2+, Hg2+,
Cu2+, Co2+, Cr3+, Ag+, Al3+ and Fe3+, the effect of selected
ions on the intensity of sensor L showed a low interference
except Cu2+, Ag+ and Hg2+ ions. As large as 26-fold “off-on”
type fluorescence enhancement was observed upon the addi-
tion of Hg2+, while its homologues Cu2+ and Ag+ showed a 5-
and 8-fold enhancement only under the same conditions. The
effects of other common metal ions on the Hg2+ signaling of L
were also assessed under competitive conditions (Fig. 4). The
fluorescence increase was not significantly affected by the
presence of other metal ions at 5 equiv, except for Cu2+,
Ag+, Cd2+ and Fe3+. Therefore, these results suggest that L
Selectivity Studies
We monitored the fluorescence change after adding various
metal cations to examine the selectivity of the sensor for Hg2+
ions. As shown in Fig. 3, the sensor L showed a very weak
fluorescence centered around 390 nm in EtOH-HEPES buffer
(20 mM HEPES, pH=7.2, EtOH:HEPES=9:1) under 1 %
Fig. 4 Fluorescence spectra of
L + Hg2+ system in the presence
of possibly interfering metal ions
as background in buffered EtOH:
HEPES=9:1 solution at pH 7.2
under 1 % attenuation [L]=1×
10−5 M, [Hg2+]=5.0×10−5 M,
[Mn+]=5.0×10−5 M, λex=330 nm
(I at 388 nm)

J Fluoresc
Fig. 5 Fluorescence emission
spectra of L (10 μM) in buffered
EtOH:HEPES=9:1 solution at pH
7.2 under 1 % attenuation upon
the addition of Hg2+ (0–6.0
equiv.). Excitation wavelength
was 330 nm. Inset: variations of
fluorescence intensity of
compound L (10−5 M) at 388 nm
vs. equivalents of [Hg2+
]
has a high fluorescent selectivity for Hg2+ in the presence of
these tested foreign metal ions.
HEPES=9:1 solution at pH 7.2 under 1 % attenuation. As
shown in Fig. 5, the sensor emits weak fluorescence at
388 nm. As the concentration of Hg2+ ion is increased in the
solution of L (maximum 6 equiv.), the intensity of emission
band at 388 nm was gradually enhanced and almost reached
maximum when the amount of Hg2+ ion was about 10 μM.
When more HgCl2 solution was titrated, the fluorescence
Hg2+-Titration
The fluorescence titration of L in the presence of different
Hg2+ concentrations was then performed in buffered EtOH:
Fig. 6 Fluorescence images of
living HeLa cells. (a1–4): Bright-
field view of panel; (b1–4):
Fluorescent; (c1–4): Overlay
image of (a1–4) and (b1–4). 1:
Cells incubated with 10 μM of the
sensor for 2 h at 37 °C. 2, 3 and 4:
HeLa cells were pretreated with
Hg2+ at the indicated
concentrations for 1 h at 37 °C
before incubating with the probe
under the same conditions

J Fluoresc
intensity showed negligible changes and the curve (inset)
remained relatively constant, which also gives a 1:1 stoi-
chiometric ratio between sensor L and Hg2+ similar to
Job’s plot in Fig. S3. The fluorescence quantum yield
increases from 0.0465 for free L to 0.8593 for the com-
plex of L and Hg2+, correspondingly. In addition, the
quantitative response of the sensor L toward Hg2+ ion
was also studied by the fluorescence titration and the
linear calibration plots as shown in Figs. S4 and S5.
The dynamic range for the determination of Hg2+ was
determined to be linear in the range of 0–10 μM with
correlation coefficient (R) of 0.9949 [36]. The limit of
detection (LOD) is evaluated using 3σbi/m [37], where
σbi is the standard deviation of the blank signals and m
is the slope of the linear calibration plot. The LOD for
determination of Hg2+ was thus calculated to be 3.85×
10−10 M, a value superior to those reported to date.
Following a Benesi-Hildebrand-type analysis [38], the as-
sociation constant Ka was determined to be 3.03×
104 M−1.
Imaging of Intracellular Hg2+
The intracellular Hg2+ imaging behaviour of L was carried
out on HeLa cells by a fluorescence microscope (Fig. 6).
Incubation of HeLa cells with 10 μM of the sensor for 2 h at
37 °C gave almost no intracellular fluorescence. This was
consistent with the previous findings that cancer cells in cell
cultures contain little Hg2+. When HeLa cells were pretreated
with 5 μM of Hg2+, fluorescence is visible in the HeLa cells
with the same treatment with the sensor, providing visual
evidence of the sensor permeating cells and information on
the intracellular existence of Hg2+. Furthermore, when
pretreated with 10, 20 μM of Hg2+, fluorescence images of
the cells revealed a remarkable enhancement of intracellular
fluorescence. It is proved that the sensor can be used for
monitoring Hg2+ within biological samples.
Conclusions
In summary, a new highly selective and sensitive fluorescent
sensor based on pyrazoline unit was synthesized and used for
the determination of Hg2+ ion with a low detection limit in
buffered EtOH:HEPES=9:1 solution at pH 7.2. This sensor
formed a 1:1 complex with Hg2+ and showed a fluorescent
enhancement with good tolerance of other metal ions. The
fluorometric detection limit of 3.85×10−10 M of this seneor is
superior to one reported up to date. Moreover, the fluorescent
sensor has practical application in cell imaging.
Binding of Probe L with Hg2+
In an effort to gain more detailed information on the interac-
tions between L and Hg2+ ion, 1H NMR spectroscopic studies
were carried out in DMSO-d6, and the spectral differences are
shown in Fig. S7. A distinct change occurs at the peak cen-
tered at 8.38 and 7.57 ppm, Hb and the Hc proton proximal to
the thioamide are shifted downfield by 1.0 ppm and 1.18 ppm,
respectively, with the stepwise addition of Hg2+ ions. The
single peak assigned for the Ha proton of the N-H in the
benzimidazole experiences a slight net downfield shift from
12.92 to 13.01 ppm. Moreover, the proton Hd and the He
proton to the CH2 protons of the pyrazoline ring are shifted
downfield by 0.21 ppm and 0.22 ppm, respectively. These
observations suggested that the nitrogen atoms in the benz-
imidazole, thioamide and pyrazoline of the sensor L partici-
pated to the complex with Hg2+. A proposed binding mode is
shown in Fig. S8.
Acknowledgements This study was supported by 973 Program
(2010CB933504).
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