Coumarin-Based Reversible Fluorescent Probe for Selective Detection of Cu2+ in Living Cells

Article abstract of DOI:10.1007/s10895-020-02585-0

Copper ion plays an important role in many biological processes in human body. H₂S is considered as the third gasses transmitter after carbon monoxide and nitric oxide. Here a novel ICT-based fluorescent ON-OFF-ON probe for Cu²⁺ and H₂S detection was developed. Selectivity and sensitivity of probe was confirmed in aqueous Tris-HCl buffer (10?mM, pH?7.4, containing 90percent acetonitrile). Probe DF-CU shows high selectivity over other analytes. The degree of fluorescence quenching is linearly associated with the concentration of Cu²⁺ (R²?= 0.9919). The limit of detection (LOD, calculated according to the 3σ/slope) for Cu²⁺ was 6.4?μM. Probe can work in almost all pH. The probe shows a very fast response to Cu²⁺ (within 10?s). Its response to copper ion could be reversed by H₂S. The complex of probe with Cu²⁺ could be used for H₂S detection. Furthermore, this ON-OFF-ON fluorescent probe successfully applied in the living cells for the detection of Cu²⁺ and H₂S.

Full text of DOI:10.1007/s10895-020-02585-0

Journal of Fluorescence
https://doi.org/10.1007/s10895-020-02585-0
ORIGINAL ARTICLE
Coumarin-Based Reversible Fluorescent Probe for Selective Detection
2+
of Cu in Living Cells
1
1
2
1
1
Nadeem Ahmed & Wajeeha Zareen & Di Zhang & Xiaopeng Yang & Yong Ye
Received: 21 April 2020 /Accepted: 9 July 2020
#
Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Copper ion plays an important role in many biological processes in human body. H S is considered as the third gasses transmitter
2
2
+
after carbon monoxide and nitric oxide. Here a novel ICT-based fluorescent ON-OFF-ON probe for Cu and H S detection was
2
developed. Selectivity and sensitivity of probe was confirmed in aqueous Tris-HCl buffer (10 mM, pH 7.4, containing 90%
acetonitrile). Probe DF-CU shows high selectivity over other analytes. The degree of fluorescence quenching is linearly asso-
2
+
2
2+
ciated with the concentration of Cu (R = 0.9919). The limit of detection (LOD, calculated according to the 3σ/slope) for Cu
2
+
was 6.4 μM. Probe can work in almost all pH. The probe shows a very fast response to Cu (within 10 s). Its response to copper
ion could be reversed by H S. The complex of probe with Cu could be used for H S detection. Furthermore, this ON-OFF-ON
fluorescent probe successfully applied in the living cells for the detection of Cu and H S.
2
+
2
2
2
+
2
2
+
.
.
. .
H S MCF-7 cells Fluorescence imaging
2
Keywords Fluorescent probe Cu
Introduction
proteins. In general, the concentration of copper ions in the
blood are in the range of 100–150 mg/dL (15.7–23.6 mM) [2].
The limit of copper ion in drinking water was set to be 1.3 ppm
(~20 mM) by the U.S. Environmental Protection Agency
2
+
Copper (Cu ) is the third most abundant element found in the
2
+
human body, along with zinc and iron. Cu is present low in
concentration in different tissue of the body, and the highest
2
+
(EPA) [3]. Cu has a significant role in cellular respiration,
connective tissue establishment, bone growth, and response as
a main catalytic cofactor for numerous metalloenzymes
2
+
2+
quantity of Cu was found in the liver [1]. Cu plays an
efficient role in the transportation of cofactor of many
2
+
[
4–11]. However, the exceeded amount of Cu in the body
Highlights
may generate many reactive oxygen species (ROS) that result
in damage to tissue and cells in the body. According to the
reported, we known the disorder of copper ions in cells can
cause some neurodegenerative diseases such as Wilson dis-
eases, Alzheimer’s disease, and Menkes [12–16]. Recent re-
2+
1
. A new selective coumarin-based probe for Cu detection was
developed.
2
3
7
. The probe showed a reverse response upon addition of H
. The probes were successfully applied to image Cu and H
Cells.
2
S.
2
S in MCF-
2+
search showed that H S is very helpful in normal body func-
tion, such as a normality of inflammation and suppression of
Electronic supplementary material The online version of this article
https://doi.org/10.1007/s10895-020-02585-0) contains supplementary
material, which is available to authorized users.
2
(
insulin signaling [17–19]. H S is considered as the third gas-
2
ses transmitter after carbon monoxide and nitric oxide [20,
*
*
Di Zhang
pandy811@163.com
2
1]. H S has been reported as a type of never agents and
2
dangerous gas for the biological system [22–24], excess
Yong Ye
yeyong03@tsinghua.org.cn
amount of H S in air cause headaches, coma and sometimes
2
it results in death [25, 26]. Thinking of the importance of
1
2
copper ion and H S, it is urgent to develop a good method
2
Green Catalysis Center, and College of Chemistry, Zhengzhou
University, Zhengzhou 450001, China
for detection both copper ion and H S.
2
Many methods had been used to detect them, for example
atomic absorptive spectrometry (AAS) [27], inductive
coupled plasma mass spectroscopy (ICP-MS) [28], and cyclic
Institute of Agricultural Quality Standards and Testing Technology,
Henan Academy of Agricultural Sciences, Zhengzhou 450002,
China

J Fluoresc
voltammetry (CV) [29]. These methods have some shortcom-
ing, including the pretreatment of sample, long time required
and expensive instrument needed, which limited them to be
widely used. Different from above mentioned methods, fluo-
rescent probe method gained wide attention because of its
inexpensive, fast response, high selectivity, and sensitivity
excitation and emission slits were set at 2.5 nm. The excitation
voltage was 700 V. MCF-7 cell fluorescence imaging was
gotten by a LEICA TCS SP8 laser scanning confocal
microscope.
Materials
[30].
Our group has recently constructed some ratiometric
2
+
All used reagents including bis(pyridine-2-ylmethyl)amine,
7-(diethylamino) benzaldehyde, piperidine, acetic acid, etha-
nol, dichloromethane, 4-(dimethylamino)pyridine (DMAP),
probes for Cu or H S [31–36]. But none of these probes
2
2
+
could be used to detect both Cu and H S. Here, we devel-
2
oped a new ICT-based probe DF-Cu using 7-
diethylaminocoumarin as a fluorophore and bis(pyridine-2-
1-(3-dimethylaminopropyl)- 3-ethylcarbodiimide hydrochlo-
2
+
ride (EDCI), methanol, diethyl malonate, NaOH, anhydrous
sodium sulfate, were used directly after received from compa-
ny. Double distilled water was used in all experiments.
Common solvents were purified according to standard proce-
ylmethyl) amine as an active site for Cu binding. DF-Cu
2
+
showed fluorescence quenching after the addition of Cu
by forming a complex. The formed complex showed a fast
−
response for the HS , and result in fluorescence turn-on. This
dures. Probe stock solution (10 mM) was formed in CH CN.
3
kind of ON-OFF-ON fluorescent probe DF-Cu was success-
2
+
−
The solutions of amino acid (10 mM) and anions were gotten
from corresponding salts including: NaCl, KCl, MgCl2,
CuSO , CaCl , ZnCl , Na SO , MnCl , PbCl , CdCl ,
fully applied to image Cu and HS in living cells.
4
2
2
2
4
2
2
2
AgNO , BaCl , Fe SO , NiCl and FeCl .
3
2
2
4
2
3
Experimental
Apparatus
Cell Culture and Imaging
1
3
1
C NMR and H NMR spectra were gotten on a Bruker
DTX-400 spectrometer in CDCl and tetramethyl silane
MCF-7 cell was cultivated in DMEM supplemented with 10%
fetal bovine serum, in 5% CO , at 37 °C. For fluorescent
3
2
(
TMS) as an internal reference. Absorptive spectra was con-
imaging, 10 μM of DF-Cu was added to MCF-7 cell and
ducted on a Lambda 35 UV/VIS spectrometer, PerkinElmer
accurately. ESI-MS measurement was carried on a Q-Tof HR-
MS spectrometer (Waters Micro mass) by using MeOH as a
mobile phase. Fluorescent spectra dimensions were recorded
on a HITACHI F-4600 fluorescent spectrophotometer. The
nurtured at 37 °C for 30 min in 5% CO incubator. Then,
2
two groups of cells having probe DF-Cu and DF-Cu complex
2
+
−
are treated with Cu and HS solutions respectively and in-
cubated for 1 h. At last, they were washed three times with
PBS buffer.
Fig. 1 The fluorescence of DF-
2+
Cu (10 μM) and Cu (10 equiv.)
or other various transition metal
ions (10 equiv.) in Tris-HCl
(10 mM, pH = 7.4, containing
9
0% acetonitrile) buffer. (λex =
4
20 nm, slit: 2.5 nm)

J Fluoresc
Fig. 2 The fluorescence changes
of DF-Cu (10 μM) at 465 nm on
2+
the addition of Cu (10 μM) in
the presence of various ions
(10 μM) in Tris-HCl buffer
(10 mM, pH = 7.4, containing
9
0% acetonitrile). (λex = 420 nm,
slit: 2.5 nm)
Synthesis
at room temperature after the reaction was confirmed as com-
pletion by TLC. The solution was washed with water and ex-
tracted with dichloromethane three times (30 mL × 3), dried
over anhydrous Na SO . After the solvent was released by
Compound D was synthesized according to the previous re-
ported paper [37].
2
4
Synthesis of DF-Cu: To the vigorously stirred solution of D
rotary evaporate, the crude DF-Cu was obtained and then pu-
(120 mg, 0.45 mmoL) in dichloromethane (10 mL) was added
rified by column chromatography (eluant: CH Cl /MeOH =
2
2
1
-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
100 /1, v/v) to afford probe DF-Cu as a yellow solid
1
(
(
EDCI) (0.2 g, 1.0 mmoL), bis(pyridine-2-ylmethyl) amine
100 mg, 0.5 mmoL) and 4-(dimethylamino) pyridine
(108 mg). H NMR (400 MHz, CDCl , ppm) δ: 1.22 (t, J =
3
7.4 Hz, 6H), 3.43 (q, J = 7.1 Hz, 4H), 4.70 (s, 2H), 4.89 (s, 2H),
6.5 (d, J = 6.3 Hz, 2H), 7.21–7.28 (m, 4H, PhH), 7.60–7.70 (m,
(DMAP) (10 mg). The mixed solution was stirred overnight
Fig. 3 The fluorescence intensity
of probe DF-Cu (10 μM) at
2+
4
(
65 nm upon treatment with Cu
10 μM) with the progress of time
in tris-HCl buffer (10 mM, pH =
7
.4, containing 90% acetonitrile).
(λex = 420 nm, slit: 2.5 nm)

J Fluoresc
2+
Fig. 4 Fluorescence emission spectra of DF-Cu (10.0 μM) after Cu (0–
0 equiv.) added in Tris-HCl buffer (10 mM, pH = 7.4, containing 90%
acetonitrile). (λex = 420 nm, slit: 2.5 nm)
2+
Fig. 6 The fluorescent intensity of DF-Cu (10 μM), probe with Cu (10
1
2
equiv.), and then H S added in Tris-HCl buffer (10 mM, pH = 7.4, con-
taining 90% acetonitrile). λex = 420 nm, slit: 2.5 nm
1
3
3
(
1
1
1
H, PhH), 7.9 (s, 1H, PhH), 8.5 (s, 2H, PhH); C NMR
2+
amine is good ligand for Cu , we choose it as a response
100 MHz, CDCl , ppm) δ: 167.72, 159.54, 157.09, 156.77,
56.32, 151.50, 149.63, 149.02, 144.11, 137.22, 136.70,
29.73, 122.51, 122.19, 121.95, 121.89, 116.73, 109.22,
07.68, 97.07, 54.24, 50.44, 44.91, 29.70, 12.42; HR-MS:
2+
2+
3
group for Cu , and hope it has a fast response to Cu over
other metal ions and amino acids. By introducing response
group into coumarin fluorophore, we synthesized a new probe
DF-Cu in three steps with good yield. The probe DF-Cu was
+
m/z calcd for [C H N O ] 443.2083, Found: 443.2079.
1
13
26 26 4 3
well characterized by H NMR, C NMR, and HR-MS (Fig.
S1–S3).
2
+
Spectroscopic Response of DF-Cu toward Cu
Results and Discussion
To confirm the selectivity of probe DF-Cu (10 μM) toward
Cu , we conducted UV absorption and fluorescence titration
Design and Synthesis of DF-Cu
2+
in aqueous Tris-HCl buffer (10 mM, pH 7.4, containing 90%
acetonitrile). The alone probe DF-Cu showed an absorption
peak centered at 400 nm (Fig. S4, S5). After 100 μM of Cu
We choose 7-diethylaminocoumarin as a fluorophore because
of its good photostability, high fluorescence quantum yield
and large stoke’s shift. Because bis (pyridine-2-ylmethyl)
2+
was added, the absorption peak shifted to 410 nm and there
was no change as other metal ions added. It illustrated that the
2
+
probe DF-Cu was selective for Cu detection by UV. From
Fig. 1, it was observed and the fluorescence intensity of probe
DF-Cu at 465 nm has not change when various common ions
2
+
added, except Cu . The fluorescence was quenched dramat-
ically because the complex formation between probe DF-Cu
2
+
and Cu . It means the probe possesses a high fluorescent
2
+
selectivity to Cu .
Next, we performed competitive test (Fig. 2). The fluores-
cent intensity of DF-Cu was almost 7 times higher than that of
2
+
the complex DF-Cu + Cu . The fluorescence intensity ratio
2
+
of DF-Cu / (DF-Cu + Cu ) is kept stable when other metal
ions were added. The results showed the DF-Cu having good
2
+
selectivity and specificity toward Cu . Kinetic study of probe
DF-Cu showed that the reaction between probe DF-Cu and
2
+
Cu was finished within 10 s (Fig. 3), which demonstrated
2
+
the response speed of probe to Cu is very fast. The results
showed that the probe DF-Cu is applicable for the fast and
real-time detection of Cu .
Fig. 5 The fluorescence intensity of probe DF-Cu (10 μM) in different
pH (1–13) as presence and absence of Cu (10 equiv.). (λex = 420 nm,
slit: 2.5 nm)
2+
2
+

J Fluoresc
Scheme 1 Synthetic route for the
probe DF-Cu. Reagents and
conditions: (a) 7-(diethylamino)
benzaldehyde, piperidine, acetic
acid, ethanol, 80 °C, 12 h; (b) 2 M
NaOH 12 h, rt., acidified with
2
M hydrochloric acid; (c)
bis(pyridine-2-ylmethyl) amine,
dichloromethane, EDCI, DAMP,
rt., overnight
2
+
Fluorescent Titration of Probe DF-Cu with Cu
absorption and fluorescence emission of probe DF-Cu in dif-
2
+
ferent pH (1–13) as presence and absence of Cu (10 equiv.)
(Figs. S8–11). Probe DF-Cu can work well in pH 3–13
(Fig. 5). It’s worthy to note that probe can be used to detect
Next, we performed fluorescent titration of probe DF-Cu. The
fluorescence intensity of probe DF-Cu gradually decreased by
the addition of a low concentration of Cu (Fig. 4). The
addition of Cu was continued until there was no further
change in fluorescence intensity. The degree of fluorescence
quenching is linearly associated with the concentration of
Cu (R = 0.9919) (Fig. S6). The limit of detection (LOD,
calculated according to the 3σ/slope) for Cu was 6.4 μM,
which also represents a fairly high sensitivity for Cu .
Accordingly, Stern–Volmer plots (Fig. S7) illustrate that the
2
+
2+
co Cu in physiological pH environment.
2
+
Fluorescent Performance of Probe DF-Cu Complex
with H2S
2
+
2
2
+
2+
The addition of Cu to probe DF-Cu result in the fluores-
2
+
cence off. We speculated that is due to the complex probe+
2
+
2+
2−
Cu formed. Because Cu could react with S and form a
stable compound CuS, we thought that complex DF-Cu +
2
+
emission of probe DF-Cu is quenched by Cu and the com-
3
2
+
plexation constant is 7.52*10 L/Mol.
Cu may used for the sensitive detection of sulfide ions by
2
+
the removal of Cu from complex and result in fluorescence
recover. We checked this idea by making a sample solution of
probe DF-Cu complex and then adding sulfide ion. As we
expected, the fluorescence intensity restored and nearly same
to the fluorescence intensity of probe DF-Cu (Fig. 6). Then,
we added copper ions to this solution and found that the fluo-
rescence off again, which mean the complex between probe
The Effect of pH on the Fluorescent Spectra
Next, we performed a pH experiment for probe DF-Cu. For
monitoring pH response of probe DF-Cu, we prepared pH
solution ranges from values 1 to 13. We studied the UV
2
+
DF-Cu and Cu formed again. The results illustrated that
2
+
the response of probe to Cu is reversible. The response
mechanism was proposed in Scheme 2. Scheme 1
Fluorescent Titration of DF-Cu Complex for H2S
Fluorescence titration of probe DF-Cu complex for H S was
2
done in situ. Firstly, the sample solution of probe DF-Cu was
2
+
prepared. Then Cu solution was added to this sample solu-
tion to make probe DF-Cu complex. Last, we started the fluo-
rescent titration of probe DF-Cu complex for sulfide ions.
There was a gradual enhancement in fluorescence intensity
of probe DF-Cu along with the concentration of H S increas-
2
ing (Fig. 7). The fluorescence emission is linearly associated
2
2
+
with the concentration of H S (R = 0.9848) (Fig. S12). The
2
Fig. 7 The fluorescence of DF-Cu + Cu complex (10 μM) upon treat-
ment with H S (0–12 equiv.) in Tris-HCl buffer (pH = 7.4, 10 mM, con-
taining 90% acetonitrile, λex = 420 nm, slit: 2.5 nm)
limit of detection for H S was 134.2 μM. The UV-vis spectra
of DF-Cu + Cu complex (10.0 μM) after H S (0–10 equiv.)
2
2
2
+
2

J Fluoresc
2+
2
Scheme 2 Proposed mechanism of probe DF-Cu with Cu and H S
added shown in Fig. S13. Along with H S added, a little blue-
shift was observed in the UV-vis spectra of DF-Cu + Cu
complex.
experimental results demonstrated that almost 80% of
the cells survived (Fig. 8). It means probe DF-Cu is
low Cytotoxicity and could be applied for cell imaging
in living cells.
2
2
+
Subsequently, probe DF-Cu was used to trace Copper ions
in living MCF-7 cells (Fig. 9). The MCF-7 cells were incu-
bated with 10 μM probe DF-Cu for 30 min at 37 °C in 5%
Sensing Mechanism
To gain insight into the sensing mechanism, we continue to
CO . There was strong fluorescence in blue channel (Fig. 9b).
Upon the addition of 100 μM Cu and incubated another 1 h,
study the sensing process by MS. An intense peak at m/z
2
2
+
+
6
01.27 corresponding to (C H CuN O S) was present in
2
6
26
4 7
an obvious fluorescence decreasing was observed as expected
the MS spectrum (Fig. S14). The results demonstrated that
2
+
(
Fig. 9e). As the designed compound is an ON-OFF-ON
Cu could form coordinated complexes with probe DF-Cu
Scheme 2).
probe, we also checked its complex for H S imaging. When
(
2
2
+
probe DF-Cu + Cu complex was treated with hydrogen sul-
fide (500 μM) and incubated for 1 h, a strong fluorescence in
blue channel was observed (Fig. 9h). The result indicates that
Cell Imaging
2
+
Cu and H S can be detected by probe DF-Cu in a living
2
The cytotoxicity of probe DF-Cu in living MCF-7 cells
was assessed by a conventional MTT assay. The
cells.
Fig. 8 Cytotoxicity assay of
probe DF-Cu at different concen-
tration in MCF-7 cells

J Fluoresc
2
+
Cu + Cu complex (10 μM) for 30 min and then incubated with HS−
(500 μM) for 1 h. (c, f) and (i) are merged images of a with b, d with e,
and h with i (excitation: 400 nm emission: 430–510 nm)
2+
Fig. 9 Confocal fluorescence images of Cu with DF-Cu in MCF-7
cells. (a, b) MCF-7 cells pretreated DF-Cu (10 μM) for 30 min. (d, e)
MCF-7 cell was pretreated with DF-Cu (10 μM) for 30 min, and then
2+
mixed with Cu (100 μM). (g, h) MCF-7 cells were pretreated with DF-
Acknowledgments This work was financially supported by the National
Science Foundation of China (No. 21572209 and 21907025), Scientific
and Technological Project of Henan Province of China (192102310140),
and Scientific and Technological Innovation Project of Henan Academy
of Agricultural Sciences (2020CX05).
Conclusion
In summary, we have designed and synthesized a novel highly
selective and sensitive ICT probe for Cu detection. When
2
+
2
+
probe response to Cu , the fluorescence turn-off and probe-
2
+
Cu complex was formed. Upon addition of hydrogen sulfide
2
+
to probe-Cu complex, the fluorescence could be recovered.
This ON-OFF-ON system was repeatable. Furthermore, the
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