A chromene pyrazoline derivatives fluorescent probe for Zn2+ detection in aqueous solution and living cells

Article abstract of DOI:10.1016/j.ica.2018.04.020

A new chromene pyrazoline derivatives fluorescent probe L was designed and synthesized. The probe L appears in a 55-fold fluorescence enhancement after 5 equiv. Zn²⁺ was added, and it also exhibits high sensitivity and selectivity for response

Full text of DOI:10.1016/j.ica.2018.04.020

Inorganica Chimica Acta 479 (2018) 128–134
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Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Research paper
A chromene pyrazoline derivatives fluorescent probe for Zn2+ detection
in aqueous solution and living cells
Ying-Peng Zhang a, , Qing-Hua Xue a, Yun-Shang Yang a, , Xiao-Yu Liu a, Chun-Mei Ma a, Jia-Xi Ru b,
⇑
⇑
Hui-Chen Guo b
a School of Petrochemical Engineering, Lanzhou University of Technology, Lanzhou 730050, China
b State Key Laboratory of Veterinary Etiological Biology and Key Laboratory of Animal Virology of Ministry of Agriculture, Lanzhou Veterinary Research Institute, Chinese Academy
of Agricultural Sciences, Lanzhou 730046, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new chromene pyrazoline derivatives fluorescent probe L was designed and synthesized. The probe L
appears in a 55-fold fluorescence enhancement after 5 equiv. Zn2+ was added, and it also exhibits high
sensitivity and selectivity for response to Zn2+ in ethanol-water (V:V = 1:1) solution through ‘‘OFF-ON”
type process and a possible photoinduced electron transfer (PET). Notably, the probe L distinguishes
between Zn2+ and Cd2+. The association constant was considered as 2.38 Â 103 MÀ1 via fluorescence titra-
tion experiments. The probe L-Zn2+ complex forms a 1:1 binding stoichiometry which was discussed by
Job’s plot. The probe is very highly sensitive with fluorometric detection limit of 1.603 Â 10À10 M. It also
shows good reversibility upon addition of EDTA. Furthermore, the viability of L to Zn2+ has practical appli-
cation in live cell imaging.
Received 23 January 2018
Received in revised form 13 April 2018
Accepted 13 April 2018
Available online 23 April 2018
Keywords:
Chromene
Pyrazoline
Fluorescent probe
Zn2+ recognition
Cell imaging
Ó 2018 Elsevier B.V. All rights reserved.
1. Introduction
tion, some Zn2+ fluorescent probes display relatively low
selectivity and suffer from the interferences from other metal ions,
As we know, transition metal zinc is one of the most important
in life system [1] and it is the second most rich transition metal
element in the human body [2]. Zn2+ plays a crucial role in many
biochemical processes such as cellular metabolism [3], muscle con-
traction [4], DNA-binding proteins [5], gene expression, apoptosis,
enzyme regulation, immunity, metallo-enzyme function [6] and so
forth. In pathology, Parkinson’s disease [7], senile dementia [8],
epilepsy disease [9], cerebral ischemia [10], diabetes [11], amy-
otrophic lateral sclerosis (ALS) [12], infantile diarrhea [13] and
other diseases are related to the formation of Zn2+ and metabolic
disorders. Therefore, it becomes very important to detect Zn2+ in
both the environment and biological systems [14].
especially Cd2+ [24], which is the same group as Zn2+ in the peri-
odic table and has similar binding properties with Zn2+ [25]. There-
fore, similar fluorescence intensity changes and wavelength shifts
are usually obtained when these two metal ions coordinate to
the probe molecule respectively [26]. Thus, it is a great challenge
to design and synthesize a fluorescent probe to sense and monitor
Zn2+ with high selectivity and sensitivity in aqueous solutions [27].
In recent years, pyrazoline derivatives have drawn much atten-
tion because of their excellent blue fluorescence property, high flu-
orescence quantum yield, the rigid flat structure and high hole-
transport efficiency [28–31].Chromene derivatives have been
widely used as important intermediates in the synthesis of many
natural products and medicinal agents. Many synthesized mole-
cules based on the chromene ring system were found to be useful
in antiproliferative activity [32,33]. Moreover chromone deriva-
tives not only give fluorescence in the visible range but also cross
the cell membrane very easily due to the lipophilic nature [34–38].
Chromene and pyrazoline have optical properties such as high
fluorescence quantum yield, high light stability, large Stokes shift
and non-toxicity. Taking all these into account, we have designed
and synthesized a new compound L connecting pyrazoline ring
and chromene ring. The L showed good selectivity and high
sensitivity fluorescence response to Zn2+ over other metal ions,
The development of fluorescent probes for Zn2+ detection has
become a very active field in chemical biology. A lot of fluorescent
probes for the detection and recognition of Zn2+ have been studied
by various teams [15–19], but some of them can be applied only in
organic solutions like acetonitrile toxic solvents, which restrict
their potential applications, some of them have complex prepara-
tion process, inferior reversibility or selectivity [20–23]. In addi-
⇑
Corresponding authors.
E-mail addresses: yingpengzhang@126.com (Y.-P. Zhang), yangyunshang@tom.
com (Y.-S. Yang).
https://doi.org/10.1016/j.ica.2018.04.020
0020-1693/Ó 2018 Elsevier B.V. All rights reserved.

Y.-P. Zhang et al. / Inorganica Chimica Acta 479 (2018) 128–134
129
especially Cd2+ in ethanol-water (V:V = 1:1) solution. Hence, owing
to the good selectivity, high sensitivity and complete reversibility
for detection and recognition of Zn2+, this probe L could be suitable
for imaging in living cells. In contrast to previously reported Zn2+
fluorescent probes [39–42], the advantages of presenting new
probe L are simple structure, easy synthesis, better fluorescence
intensity enhancement, higher sensitivity and reversible.
(dd, J = 10.8, 7.8 Hz, 1H), 3.20 (dd, J = 10.8, 4.2 Hz, 1H), 1.88 (s,
3H). 13C NMR (100 MHz, CDCl3, TMS) (Fig. S2): dc ppm 167.60,
157.52, 156.20, 151.66, 138.26, 133.27, 132.28, 129.77, 129.03,
128.59, 128.24, 127.64, 126.87, 121.80, 121.21, 120.80, 119.72,
117.05, 115.96, 114.73, 77.86, 56.81, 39.60, 21.32. HR-ESI-MS
(Fig. S3) calculated for [MÀH]+ 409.1630, found 409.2733.
Compound 6 was prepared in using the same method with
probe 4. White solid; yield: 81%; mp: 136–138 °C. 1H NMR (400
MHz, CDCl3, TMS) (Fig. S4): dH ppm 7.67 (d, J = 5.2 Hz, 2H), 7.58–
7.20 (m, 8H), 7.14–6.98 (m, 2H), 6.85 (d, J = 4.4 Hz, 1H), 6.66 (d, J
= 4.8 Hz, 1H), 6.57 (s, 1H), 5.87 (s, 1H), 5.00 (d, J = 5.2 Hz, 1H),
3.32 (dd, J = 8, 11.6 Hz, 1H), 3.06 (d, J = 11.6 Hz, 1H), 2.01 (s, 3H).
13C NMR (100 MHz, CDCl3, TMS) (Fig. S5): 168.80, 153.92, 151.78,
138.59, 134.57, 131.10, 130.35, 129.55, 128.90, 128.73, 128.54,
127.80, 126.79, 126.45, 121.15, 121.11, 120.87, 115.93, 78.25,
58.07, 39.64, 21.36. HR-ESI-MS (Fig. S6) calculated for [M+H]+
395.1681, found 395.2558.
2. Materials and methods
The materials used for this study were obtained from commer-
cial suppliers and used without further purification. 1H NMR and
13C NMR spectrum were measured on the Bruker Avance 400
(400 MHz) spectrometer. Chemical shifts are reported in ppm
using TMS as an internal standard. HR-ESI-MS were determined
on a Bruker esquire 6000 spectrometer. UV–vis absorption spec-
trum were monitored with a UV-2700 spectrophotometer. Fluores-
cence spectrum were determined on
a
Hitachi F-7000
spectrophotometer equipped with quartz cuvettes of 1 cm path
length. The melting point was determined on an XRC-1u Melting
Point Apparatus.
4. Results and discussion
4.1. Uv–vis studies of L to Zn2+
Stock solution of
L
(1 Â 10À2 M) was prepared in N, N-
Dimethylformamide. Stock solutions of various metal ions (1 Â
10À2 M) and EDTA (1 Â 10À2 M) in distilled water were also pre-
pared. All absorption and fluorescence emission spectrum were
measured in a 1 cm optical path length quartz optical cell at room
temperature. All fluorescence measurements were carried out
upon excitation at 382 nm. Excitation and emission slit widths
were 5.0 nm and 10.0 nm respectively.
BHK-21 cells were maintained in DMEM supplemented with
10% FBS at 37 °C under a humidified atmosphere containing 5%
CO2. Cells were plated on 18 mm glass coverslips and allowed to
The absorption spectral property of L toward different metal
ions (Ag+, Al3+, Fe3+, Co2+, Ni2+, Ba2+, Ca2+, Cu2+, Cd2+, K+, Mg2+
,
Na+, Hg2+, Zn2+, Pb2+, Li+, Mn2+ all the metal ions solution was 5
equiv. of L got by dissolving their corresponding nitrate salts in
H2O) was measured in ethanol-water (V:V = 1:1). As shown in
Fig. S7. L alone (10 lM) presents a broadband center at 280 nm
and 320 nm. We also found that Ag+, Al3+, Co2+, Ni2+, Ba2+, Ca2+
,
Cd2+, K+, Mg2+, Na+, Hg2+, Zn2+, Pb2+, Li+, Mn2+ did not cause signif-
icant changes in absorption spectrums. In contrast, Cu2+ caused a
new band at 350–430 nm and Fe3+ had considerable changes in
absorption bands.
adhere for 24 h, treated with L (20
and incubated for 30 min. Subsequently, the cells were treated
with Zn2+ (100
M in cell culture medium). Cells were incubated
for 30 min and rinsed with PBS three times to remove free com-
pound and ions before analysis. Cells incubated with only 20
lM in cell culture medium),
l
4.2. Fluorescence studies of L to Zn2+
lM
The fluorescence change of L with respective metal ions was
monitored in ethanol-water (V:V = 1:1) solution. Among various
metal ions (Ag+, Al3+, Fe3+, Co2+, Ni2+, Ba2+, Ca2+, Cu2+, Cd2+, K+,
Mg2+, Na+, Hg2+, Zn2+, Pb2+, Li+ and Mn2+ all the metal ions solution
was 5 equiv. of L), Zn2+ created almost 55-fold fluorescence
enhancement at 471 nm(Fig. 1). And a small red shift with fluores-
cence enhancement was observed. The change in spectral wave-
length from 441 nm to 471 nm is caused by restricted C@N
isomerization mechanism and an inhibition of photo-induced elec-
tron transfer (PET) process [43,44].
L for 30 min acted as a control. The cytotoxic activity experiment
of the complex against BHK-21 cells was tested according to MTS
assay procedures: BHK-21 cells were seeded into 96-well plates
for 24 h. The different volume concentration of probe L was dis-
solved in DMSO make the final concentration, and diluted in cul-
ture medium at concentrations of 5, 10, 25, 50, 100 lM as
working-solution and each concentration in quintuplicate, DMSO
as a negative. After incubation for 24 h, the cells were added 10
lL solution of MTS in incubator for 4 h. After sufficient reaction
with cells, the OD of each well was measured at the wavelength
of 490 nm using a microplate spectrophotometer.
Furthermore, competition experiments for other metal ions in
the L-Zn2+ were conducted in the same condition. As displayed in
Fig. 2. Hg2+ and Pb2+ can partly quench fluorescence of L-Zn2+
,
3. Experimental
whereas Al3+, Fe3+ and Cu2+ completely quenched fluorescence of
L-Zn2+. This may be attributed to the paramagnetic properties of
these three metal ions and fluorescence quenching was observed
when complex with some paramagnetic metal ions, such as Fe3+
and Cu2+, are always encountered in other metal ion probes [45–
47]. Thus, when they were bound to probes, the emission would
be strongly quenched by a photoinduced metal into fluorophore
electron or energy transfer mechanism [48–51]. Most of metal
ions, including Ag+, Co2+, Ni2+, Ba2+, Ca2+, Cd2+, K+, Mg2+, Na+,
The synthetic route of L (1-(3-phenyl-5-(2-phenyl-2H-chro-
men-3-yl)-4,5-dihydr o-1H-Pyrazol-1-yl)ethanone) was shown in
Scheme 1. The probe is easy to synthesize in three steps. According
to the literature [37], compound 3 readily prepared from com-
pound 1 and 2 in 79% yield, A mixture of compound 3 (0.3384 g,
1.0 mmol) and 80% hydrazine hydrate (0.3065 g, 5.0 mmol) were
taken in a 100 mL reaction flask in the presence of glacial acetic
acid (15 mL) and refluxed at 120 °C for 6 h. After completion of
reaction, it was cooled and poured into crushed ice. The resulting
precipitate was filtered and recrystallized from ethanol to yield
probe L. Pale yellow solid; Yield: 71%; mp: 216–219 °C. 1H NMR
(400 MHz, CDCl3, TMS) (Fig. S1): dH ppm 9.98 (s, 1H), 7.40–7.27
(m, 5H), 7.17 (d, J = 5.2 Hz, 1H), 7.10–7.00 (m, 3H), 6.90 (m, 2H),
6.70–6.56 (m, 2H), 5.79 (s, 1H), 5.02 (dd, J = 7.8, 4.2 Hz, 1H), 3.45
Hg2+, Pb2+, Li+ and Mn2+ show a very negligible effect, and Al3+
,
Cu2+ and Fe3+could quench the fluorescence, which was often
encountered in other probes. This is limited to the application of
probe L in complicated environment samples. However, it is sur-
prising that L-Zn2+ complex eliminated the influence of Cd2+ by
blocking PET and restricting mechanism of C@N isomerization.
These results show that L strongly coordinates with Zn2+ which

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Y.-P. Zhang et al. / Inorganica Chimica Acta 479 (2018) 128–134
Scheme 1. Synthesis of compound 4 (L).
Fig. 2. Fluorescence emission spectra of L(10 l
M) and Zn2+ (5 equiv.) in the
Fig. 1. Fluorescence spectra of probe
L (10 lM) in ethanol–water (V:V = 1:1)
presence of Ag+, Al3+, Ba2+, Ca2+, Cd2+, Co2+, Cu2+, Fe3+, Hg2+, K+, Li+, Mg2+, Mn2+, Na+,
Ni2+, Pb2+ and Zn2+(5 equiv.) in ethanol–water (V:V = 1:1) solution (kex = 382 nm,
slit: 5.0/10.0 nm).
solution with 5 equiv. of metal ions (kex = 382 nm, slit: 5.0/10.0 nm). Insert: Photos
of L (10
l
M) in ethanol–water with and without addition of Zn2+ (5 equiv.)
that the binding constant of L-Zn2+ complex is Ka = 2.38 Â 103
MÀ1 and the limit of detection (LOD) is 1.603 Â 10À10 M, calculated
could be used to distinguish Zn2+ from Cd2+ in some conditions
[52].
using 3r
/k (Fig. S8) [57]. Some different probes for Zn2+ detection
In order to solve the sensitivity of L to Zn2+, the fluorescence
titration of L (10 l
M) was performed with Zn2+ in ethanol–water
were listed in the Table 1. Compared with the LOD of other probes
for Zn2+ detection, probe L exhibits lower detection limits. The high
sensitivity and low detection limit of L could be used as a trace
level identification of Zn2+ in real level environmental samples to
distinguish Zn2+ from Cd2+. In order to further understand the
binding mode of L and Zn2+, we had also prepared compound 6
without a phenolic hydroxyl group. In contrast to L, compound 6
did not cause an obvious change in the presence of 5 equiv. of
Zn2+ in ethanol-water (V:V = 1:1) solution (Fig. 6).
(V:V = 1:1) solution (Fig. 3). L alone shows a weak fluorescence
emission band at 471 nm with a rather weak fluorescence quan-
tum yield (0.0385), when adding Zn2+ to L, a dramatic increase in
fluorescence (fluorescence quantum yield 0.2704). And quantum
yield was calculated by the general equation:
g
U = /
Us(IAs/IsA)(g2
2s) [53]. With the increase addition of Zn2+ (0–8.5 equiv.), the flu-
orescent intensity was continually increased, when 6 equiv. of Zn2+
was added, the fluorescence intensity showed fewer enhancement.
This is because that it is an equilibrium process in which excess
equivalent Zn2+ is needed, the similar phenomenon also encoun-
tered in the other probes [18–54]. The binding rate of L-Zn2+ com-
plex was studied by Job’s plot methods [55] is shown in Fig. 4. The
maximum mole fraction of L appears at 0.5, which supporting a 1:1
(L: Zn2+) binding stoichiometry. Benesi-Hildebrand nonlinear curve
fitting method is further advocated [54–56] (Fig. 5). It was found
4.3. Reversible test of L to Zn2+ with EDTA
The recognition reversibility of L was further verified by fluores-
cence experiments with EDTA. The addition of Zn2+ (5 equiv.) to
probe
L showed that fluorescence intensity was remarkably
enhanced. Upon adding EDTA (5 equiv.) to L-Zn2+ solution, fluores-

Y.-P. Zhang et al. / Inorganica Chimica Acta 479 (2018) 128–134
131
Table 1
LOD of other fluorescence probes (1–7) and probe L for Zn2+ detection.
Fluorescence probes
Ions
Detection limits
1 [58]
2 [59]
3 [60]
4 [61]
5 [52]
6 [62]
7 [63]
Our probe L
Zn2+
Zn2+
Zn2+
Zn2+
Zn2+
Zn2+
Zn2+
Zn2+
8.3 Â 10À7
M
M
M
M
M
5.0 Â 10À7
1.23 Â 10À7
8.6 Â 10À9
2.9 Â 10À9
1.13 Â 10À9
M
M
7.5 Â 10À7
1.603 Â 10À10
M
Fig. 3. Fluorescence spectra of L obtained upon addition of Zn2+ (0–8.5 equiv.) in
ethanol–water (V:V = 1:1) solution (kex = 382 nm, slit: 5.0/10.0 nm).
Fig. 6. Fluorescence spectra of compound 6 (1.0 Â 10À5 M) in the absence and
presence of 5 equiv. of Zn2+ in ethanol–water (V:V = 1:1) solution. (kex = 382 nm,
slit: 5.0/10.0 nm).
Fig. 4. Job’s plot for L with Zn2+ ions in ethanol–water (V:V = 1:1) solution.
Fig. 7. Fluorescence spectra of L ((10 lM) solution (ethanol–water, V:V = 1:1) in the
presence of Zn2+ (5 equiv.) and EDTA (5 equiv.).
4.4. Proposed mechanism of L to Zn2+
The possible binding mechanism of L with Zn2+ induced the flu-
orescence changes is shown in Scheme 2. Based on the previously
proposed mechanism of some reported pyrazoline-based probes
[52–59], it has been possible that Zn2+ coordinates with the corre-
sponding oxygen and nitrogen atoms of probe L and induces the
fluorescence changes. The fluorescence enhancement was proba-
bly due to the combination of photoinduced electron transfer
(PET) process and chelation-enhanced fluorescence (CHEF) [64],
whereas the chelation of L with Zn2+ made the complex more rigid,
Fig. 5. Benesi-Hildebrand plot of L (10 lM) in ethanol–water (V:V = 1:1) solution in
the presence of Zn2+ (0–8.5 equiv.). R2 = 0.997.
cence intensity get quenched and almost reached the intensity of
original receptor L because of EDTA-Zn2+ complex formation. This
indicates that the Zn2+ recognition process is reversible (Fig. 7).

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Y.-P. Zhang et al. / Inorganica Chimica Acta 479 (2018) 128–134
Scheme 2. Proposed binding mode of L with Zn2+
.
which completely restrict C@N isomerisation [65,66]. The titration
studies, Job’s plot and Benesi-Hildebrand nonlinear square curve
fitting methods support the 1:1 binding stoichiometry of L-Zn2+
complex. To further understand the binding behavior of the probe
L with Zn2+, the 1H NMR titration experiment was investigated.
Upon addition of 0.5, 1.0, 1.5, 2.0 equiv. of Zn2+ to probe L in
DMSO-d6 is showed in Fig. S9, pyrazoline and aryl protons showed
an upfield or downfield shift, and it is found that the hydroxyl peak
at 10.04 ppm decreases and almost disappeared on addition of 1.0
equiv. of Zn2+. In addition, relative changes in the 1H NMR spectra
were observed until 1.5 equiv. of Zn2+ was added to L, the spectra
showed a slight shift upon further addition of Zn2+. To provide fur-
ther evidence for the binding of L with Zn2+, ESI–MS spectral stud-
ies were performed. In the HR-ESI-MS spectra (Figs. S3 and S10),
the probe L showed that the [MÀH]+ peak at 409.2733 (m/z calcd:
409.1630); however, the L-Zn2+ complex appeared the [M
+Zn2++H]+ peak at 473.2093 (m/z calcd: 474.0833), which corrobo-
rates 1:1 binding ratio for L and Zn2+. The above results reveal that
Zn2+ is coordinated with C@N, C@O and AOH groups in 1:1 binding
mode.
Fig. 9. Cell viability graph of probe L using BHK-21 cells by MTS assay.
the fluorescence signals are localized in the intracellular region
which indicated that probe L have good cell membrane permeabil-
ity. The blue significant fluorescence from the intracellular region
proves that the probe L is suitable for imaging Zn2+ in living cells.
The bioimaging in the BHK-21 cells confirmed the fluorescence
enhancement with excellent cell permeability. It showed that L is
biocompatible and can be used for rapid detection of intracellular
Zn2+. An MTS assay was used to evaluate cell viability. The viability
of cells treated with the range of concentration 0–100 lM of L for
24 h was reflected in Fig. 9. The probe L is found to be least toxic to
the cells.
4.5. Imaging of intracellular Zn2+ and cell viability of the probe
5. Conclusion
The sensitivity of L for Zn2+ in living BHK-21 cells was measured
by fluorescence microscopy. First, BHK-21 cells incubated with the
probe L were not displayed fluorescence image (Fig. 8(a)). The blue
fluorescence was observed after incubation of the probe L treated
cells with Zn2+ (Fig. 8(b)). The fluorescence images showed that
In summary, we have designed and synthesized a new chro-
mene-based pyrazolines fluorescent probe L for detecting Zn2+. It
showed that addition of Zn2+ increased a 55-fold fluorescence
intensity compared to other metal ions particularly Cd2+ in etha-
nol-water system. The fluorescence of L-Zn2+ can be quenched by
Fig. 8. Fluorescence images of BHK-21 cells. BHK-21 cells incubated with L (20
100
M Zn2+ for 1 h at 37 °C (b).
l
M) for 1 h at 37 °C (a). BHK-21 cells incubated with L for 1 h and then further incubated with
l

Y.-P. Zhang et al. / Inorganica Chimica Acta 479 (2018) 128–134
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Acknowledgment
This work was supported financially by grants from the Gansu
Province Natural Science Foundation of China (No. 1508RJZA078).
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Supplementary data associated with this article can be found, in
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