A novel coumarin-based colorimetric and fluorescent probe for detecting increasing concentrations of Hg2+in vitroandin vivo

Article abstract of DOI:10.1039/d1ra01408k

Mercury has complex biological toxicity and can cause a variety of physiological diseases and even death, so it is of great importance to develop novel strategies for detecting trace mercury in environmental and biological samples. In this work, we designed a new coumarin-based colorimetric and fluorescent probeCNS, which could be obtained from inexpensive starting materials with high overall yield in three steps. ProbeCNScould selectively respond to Hg²⁺with obvious color and fluorescence changes, and the presence of other metal ions had no effect on the fluorescence changes. ProbeCNSalso exhibited high sensitivity against Hg²⁺, with a detection limit as low as 2.78 × 10^?8M. More importantly, the behavioral tracks of zebrafish had no obvious changes upon treatment with 10 μM probeCNS, thus indicating its low toxicity. The probe showed potential application value and was successfully used for detecting Hg²⁺in a test strip, HeLa cells and living zebrafish larvae.

Full text of DOI:10.1039/d1ra01408k

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PAPER
A novel coumarin-based colorimetric and
fluorescent probe for detecting increasing
concentrations of Hg in vitro and in vivo†
Cite this: RSC Adv., 2021, 11, 23597
2
+
a
b
b
a
Hongdong Duan *a
Li Huang, Wenlong Sheng,
Lizhen Wang,
Xia Meng,
c
and Liqun Chi*
Mercury has complex biological toxicity and can cause a variety of physiological diseases and even death, so
it is of great importance to develop novel strategies for detecting trace mercury in environmental and
biological samples. In this work, we designed a new coumarin-based colorimetric and fluorescent probe
CNS, which could be obtained from inexpensive starting materials with high overall yield in three steps.
2+
Probe CNS could selectively respond to Hg with obvious color and fluorescence changes, and the
presence of other metal ions had no effect on the fluorescence changes. Probe CNS also exhibited high
Received 21st February 2021
Accepted 25th June 2021
2+
ꢁ8
sensitivity against Hg , with a detection limit as low as 2.78 ꢀ 10 M. More importantly, the behavioral
tracks of zebrafish had no obvious changes upon treatment with 10 mM probe CNS, thus indicating its
2+
DOI: 10.1039/d1ra01408k
low toxicity. The probe showed potential application value and was successfully used for detecting Hg
rsc.li/rsc-advances
in a test strip, HeLa cells and living zebrafish larvae.
always suffer from complicated manipulation procedure and
expensive instrumentation. In contrast, uorescent probes have
1
. Introduction
1,14
Mercury (Hg) is one of the most toxic heavy-metal ions in the envi- attracted increasing attention because of their low cost, high
ronment and has attracted increasing attention due to its accumu- sensitivity and selectivity, noninvasiveness, and experimental
2
–4
19–22
lation in natural ecosystems. Generally, mercury exists as elemental convenience.
In the past several years, great efforts have been
2+
mercury, oxidized mercury (Hg ) and mercury particles in air, soil made on the development of uorescent probes for mercury ions
and water. All these mercury species are not biodegradable and can based on the heteroatom-based coordination reaction, and Hg -
be concentrated in the human body, thereby causing irreversible promoted elimination and desulfurization reactions.
2+
5–7
23–33
A portion
damage to the liver, kidneys, brain, and endocrine and central of them exhibit excellent selectivity and sensitivity, fast response
8–11
2+
nervous systems. In particular, Hg can be easily converted into time, and low detection limits. However, these reported uores-
the lipophilic methyl mercury, which exhibits good membrane cent probes still have limitations including complex synthesis
penetrating ability and can be easily absorbed by aquatic organ- routine, using expensive chemical regents, high toxicity, and poor
2+
12–14
isms.
Recently, mercury chloride and methyl mercury have been biocompatibility, thus being not applicable for detecting Hg in
classied as potentially carcinogenic agents by the Environmental biological systems and environmental samples.
9
Protection Agency. Therefore, development of novel strategies for
Coumarin is a well-known secondary metabolite found in
34
selectively and sensitively detecting trace mercury in environmental different parts of plants. Structural modication of coumarin can
and biological samples is of great importance.
afford various derivatives with various pharmacological activities
Several methods have been developed for detecting mercury and low toxicity to human body. Some of these coumarin deriv-
species, such as atomic absorption–emission spectrometry,
inductively coupled plasma-atomic emission spectrometry,
35
15,16
atives exhibit strong uorescent emission and can be used as
17
uorescent probes for imaging different metal ions and metabo-
18
35,36
and anodic stripping voltammetry. However, these methods lites in living biological systems.
Coumarin derivatives can also
be used as uorescent tags or imaging agents for discriminating
a
tumor lesions and investigating the subcellular localization of
School of Chemistry and Chemical Engineering, Qilu University of Technology
37–46
(
Shandong Academy of Sciences), Jinan 250353, PR China. E-mail: hdduan67@163. drugs.
In our research, a novel intramolecular charge transfer
com
(ICT)-based uorescent probe CNS (Scheme 1), employing
coumarin as the uorophore and thioacetals as the reacting site
for Hg , was constructed by a facile and highly efficient synthesis
b
Biology Institute, Qilu University of Technology (Shandong Academy of Sciences),
Jinan 250014, Shandong Province, China
2+
c
Department of Pharmacy, Haidian Maternal & Child Health Hospital of Beijing,
strategy. The probe should be highly selective and sensitive to
Beijing, 100080, PR China. E-mail: chiliqun@sohu.com
2+
Hg , because the electron-rich dithioacetal group can be speci-
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/d1ra01408k
2+
cally and rapidly cleaved in the presence of Hg . The optical
1
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2021 The Author(s). Published by the Royal Society of Chemistry
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2+
properties and probing behaviors of CNS against Hg , and its MHz, DMSO-d
applications in living cells and zebrash were investigated.
6
) d 9.77 (s, 1H), 8.29 (s, 1H), 7.56 (d, J ¼ 9.0 Hz,
1H), 6.70 (dd, J ¼ 9.0, 2.4 Hz, 1H), 6.48 (d, J ¼ 2.4 Hz, 1H), 3.38
(
q, J ¼ 7.1 Hz, 4H), 1.02 (t, J ¼ 7.0 Hz, 6H), 0.13 (s, 3H) (Fig. S2†).
2
.2.3 Synthesis of CNS. Compound 2 (0.245 g, 1 mmol) was
2. Experimental
added to a 100 mL three-necked ask and dissolved in 20 mL ethyl
2.1 Materials and measurements
alcohol. Then, to the mixture was added propane-1,3-dithiol (0.1
3 2
mL, 0.99 mmol) followed by a catalytic amount of BF $Et O. The
All chemical reagents were obtained commercially and used
1
13
reaction was heated to reux and stirred for 2 h. The resulting
mixture was cooled to room temperature and refrigerated for 30
minutes. The solvent was evaporated and the crude product was
without further purication. H NMR and C NMR spectra were
recorded on a Bruker AV-400 spectrometer using TMS as
internal standard. Electrospray ionization (ESI) mass spectra
were recorded by an LC-MS 2010A (Shimadzu) instrument. The
high-resolution mass spectrum (HRMS) was measured using
a Q-TOF6510 spectrograph (Agilent). The UV-vis absorption
spectra were analyzed by a UV-2600 PC spectrophotometer
recrystallized by ethyl alcohol to afford CNS as a pale yellow solid
1
(
0.311 g. 75.05% for three steps). H NMR (400 MHz, DMSO-d )
6
d 7.94 (s, 1H), 7.55 (d, J ¼ 8.9 Hz, 1H), 6.71 (dd, J ¼ 8.9, 2.5 Hz, 1H),
6
.52 (d, J ¼ 2.4 Hz, 1H), 5.35 (s, 1H), 3.44 (q, J ¼ 7.0 Hz, 4H), 3.11
(
ddd, J ¼ 14.6, 12.3, 2.3 Hz, 2H), 2.89 (dt, J ¼ 14.0, 3.7 Hz, 2H), 2.12
(Shimadzu, Japan). The uorescence spectra were recorded on
(ddd, J ¼ 14.0, 4.5, 2.3 Hz, 1H), 1.79–1.52 (m, 1H), 1.12 (t, J ¼
a F-7000 Fluorescence Spectrophotometer (Hitachi, Japan).
13
7.0 Hz, 6H) (Fig. S3†). C NMR (101 MHz, DMSO-d
6
) d 156.11,
151.31, 142.59, 130.16, 118.40, 109.69, 107.96, 96.71, 44.57, 43.31,
2
.2 Synthesis of probe CNS
.2.1 Synthesis of compound 1. To a solution of 4-dieth-
ylaminosalicylaldehyde (3.86 g, 20 mmol) in ethyl alcohol (120 mL)
was added diethyl malonate (7 mL, 25 mmol), piperidine (2.0 mL, 2.3 Preparation of stock solutions
31.52, 25.23, 12.74 (Fig. S4†). HRMS (ESI): calcd for C H NO S :
17 21 2 2
+
335.1014, found: 336.1157 for [M + H] (Fig. S5†).
2
0
.02 mmol) and two drops of acetic acid. The reaction was heated to
The CNS (3.51 mg, 1.0 mmol) was dissolved in THF and the
reux and stirred for 6 h. Then all volatiles were evaporated under
reduced pressure, and then concentrated. HCl (40 mL) and acetic
acid (40 mL) were added and the reaction was continued at 110 C
temperature for 24 h. This solution was cooled to room temperature
and poured into ice water (150 mL). NaOH solution (30%) was
added dropwise to adjust the pH to 6, and a brown precipitate
formed immediately. Aer stirring for 1 h, the mixture was ltered,
washed with water, puried through a ash silica gel column by the
volume was set to 100 mL to give the probe stock solution (1.0 ꢀ
ꢁ3
10
M). The stock solutions of the metal ions with the concen-
ꢂ
ꢁ3
tration of 1.0 ꢀ 10 M were prepared by dissolving 1.0 mmol of
each inorganic salt (AgNO , Ba(NO ) , Cd(NO ) $4H O, Co(NO ) -
3
3 2
3 2
2
3 2
$
$
6H O, Cu(NO ) $3H O, FeSO $7H O, Fe(NO ) $9H O, Hg(NO ) -
2 3 2 2 4 2 3 3 2 3 2
H O, KNO3, NaNO3, Ni(NO ) $6H O,Pb(NO ) , Sr(NO ) ,
2
3 2
2
3 2
3 2
3 2 2
Zn(NO ) $6H O) in water and the volume was set to 100 mL.
1
using DCM as eluent to afford compound 1. H NMR (400 MHz,
DMSO-d
2
.4 The UV-vis absorption spectra of CNS in the presence of
different metal ions
The solution of CNS (1 mL) was placed in a 100 mL volumetric ask,
6
) d 7.69 (d, J ¼ 9.3 Hz, 1H), 7.29 (d, J ¼ 8.8 Hz, 1H), 6.55 (dd,
J ¼ 8.8, 2.5 Hz, 1H), 6.38 (d, J ¼ 2.4 Hz, 1H), 5.85 (d, J ¼ 9.3 Hz, 1H),
3.29 (q, J ¼ 7.0 Hz, 4H), 0.98 (t, J ¼ 7.0 Hz, 6H) (Fig. S1†).
+
2+
2+
2+
2+
3+
and added 1.0 equiv. metal ions (Ag , Ba , Cu , Co , Cd , Fe ,
2
.2.2 Synthesis of compound 2. Dry DMF (3 mL) was added
ꢂ
dropwise to POCl3 (3 mL) 0 C, the mixture was stirred for
1
0 min and slowly added to a solution of 1 (2.17 g, 10 mmol) in
ꢂ
dry DMF (20 mL). The resulting mixture was stirred at 60 C for
24 h and then poured into ice water (150 mL). NaOH solution
(30%) was added to adjust the pH to 7, and a large amount of
precipitate was formed. The mixture was ltered and thor-
oughly washed with water, puried through a ash silica gel
1
column to give compound 2 as an orange solid. H NMR (400
Fig. 1 The absorption spectra of CNS upon addition of various metal
ꢁ
5
ions in THF/H
2
O (1 : 1, V/V) solution (1.0 ꢀ 10
M). Inset: The
ꢁ5
photograph of CNS solution (1.0 ꢀ 10 M) in the presence of 1.0 ꢀ
ꢁ5
2+
Scheme 1 Synthesis step of probe CNS.
10 M of Hg .
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2
+
+
+
2+
2+
2+
2+
2+
2+
+
+
2+
2+
2+
2+
Fe , K , Na , Ni , Pb , Sr , Zn and Hg ), constant to 100 mL Fe , K , Na , Ni , Pb , Sr , Zn ), constant to 100 mL with THF/
ꢁ5
with THF/H O (1 : 1, v/v) solution, then get the 1.0 ꢀ 10 M uid to H O (1 : 1, v/v) solution. The uid to be tested were added to the
2
2
be tested. The uid to be tested were added to the quartz cell, and quartz cell, and the uorescence spectrum was measured.
the corresponding ultraviolet spectrum was measured.
2
.7 Cell culture and uorescence imaging
HeLa cells were grown in Dulbecco's Modied Eagle Medium
DMEM) containing 10% fetal bovine serum (FBS) and 1% peni-
2
.5 The uorescence spectra of CNS in the presence of
different metal ions
The solution of CNS (1 mL) was placed in a 100 mL volumetric ask, cillin–streptomycin. Aer incubating at 37 C in a humidied
(
ꢂ
+
2+
2+
2+
2+
3+
and added 1.0 equiv. metal ions (Ag , Ba , Cu , Co , Cd , Fe , atmosphere with 5% CO
for 24 h, cells were washed with PBS buffer
2
2+
+
+
2+
2+
2+
2+
2+
Fe , K , Na , Ni , Pb , Sr , Zn and Hg ), constant to 100 mL (phosphate buffered saline, pH ¼ 7.2) and incubated with fresh
ꢁ
5
with THF/H
2
O (1 : 1, v/v) solution, then get the 1.0 ꢀ 10 M uid to medium containing 10 mM probe CNS for 30 min. Then, cells were
2+
be tested. The uid to be tested were added to the quartz cell, and treated with different concentrations of Hg (0, 10, 20, and 50 mM)
the corresponding uorescence spectrum was measured.
for 10 min and the uorescence spectra were measured on a laser
confocal microscopy (Olympus, Japan). For the control group, HeLa
cells were grown in DMEM for 30 min without any treatment.
2.6 Competition experiments
The solution of CNS (1 mL) was placed in a 100 mL volumetric
2+
2.8 Zebrash maintenance and uorescence imaging

ask, 1 mL Hg was added to the volumetric ask, and then
+
2+
2+
2+
2+
3+
added 1.0 equiv. other metal ions (Ag , Ba , Cu , Co , Cd , Fe , The wild type zebrash eggs were grown in 24-well plate with E3
water containing 0.2 mM 2-phenylthiourea. Aer incubating in
Fig. 2 (A) The fluorescence spectra of CNS upon addition of various Fig. 3 (A) The relative fluorescence intensity of CNS THF/H
2
O solution
ꢁ5
ꢁ5
ꢁ6
2+
metal ions in THF/H
2
O (1 : 1, V/V) solution (1.0 ꢀ 10 M). (B) Comparison (10 M, 1 : 1, V/V) as a function of Hg concentration in the range of 1
2+
ꢁ5
2+
2+
of Hg and other metal ions detected by probe CNS. Inset: The photo- ꢀ 10 to 1 ꢀ 10 M. (B) Job's plot of CNS with Hg ([CNS] + [Hg ] ¼
ꢁ5
ꢁ5
ꢁ5
graph of CNS solution (1.0 ꢀ 10 M) in the presence of 1.0 ꢀ 10
M
10 M), THF/H
2
O (1 : 1, V/V) solution (1.0 ꢀ 10 M). Excitation wave-
2+
Hg under the UV light. Excitation wavelength ¼ 460 nm.
length ¼ 460 nm.
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Fig. 4 Competitive tests for Hg detection by probe CNS. Red indicates
ꢁ5
2+
Fig. 5 Fluorescence intensity of probe CNS (10 M) and CNS + Hg
probe detection of other metal ions, blue indicates probe detection of
ꢁ5
2–11
(10 M) at different pH,
2
THF/H O solution (1 : 1, V/V). Excitation
2+
ꢁ5
CNS + other metal ions + Hg , THF/H
2
O solution (10 M, 1 : 1, V/V).
wavelength ¼ 460 nm.
Excitation wavelength ¼ 460 nm.
ꢂ
improvement Fig. 2B. These results indicated the high selectivity of
CNS toward Hg over other tested metal ions.
a light incubator at 28 ꢃ 0.5 C for 5 days, the zebrash larvae were
treated with new medium containing 10 mM probe CNS for 30 min.
2+
2+
Then, different concentrations of Hg (0, 5, 10, and 20 mM) were
added to the medium, and the uorescence spectra were
measured on a laser confocal microscopy aer 10 min of incuba-
tion. For the control group, the zebrash larvae were grown in E3
water for 30 min without any treatment. The toxicity of probe CNS
was measured using 5 days zebrash larvae. Zebrash larvae were
treated with E3 water containing different concentrations of CNS
3
.2 Sensitivity studies
The uorescence titration experiments of CNS in THF/H O (1 : 1,
2
ꢁ
5
V/V) solution (1.0 ꢀ 10 M) against increasing concentrations of
2+
Hg was carried out to investigate the uorescence change of CNS
2+
under the inuence of Hg . As depicted in Fig. 3A, probe CNS
showed almost no uorescence in THF/H O (1 : 1, V/V) solution
2
(0, 5, and 10 mM) for 24 h, and the behavioral tracks of zebrash
ꢁ
5
2+
(
1.0 ꢀ 10 M). Aer adding increasing concentrations of Hg (0–
4 mM), a signicant uorescence improvement could be observed
around 505 nm, and the uorescence intensity become stable
were analyzed on an automated computerized video-tracking
system (Viewpoint, Lyon, France).
1
2+
when the concentration of Hg was above 4 mM. These results
proved the 1 : 1 response of probe CNS to Hg . The Job's plot of
probe CNS with Hg was further investigated in THF/H O (1 : 1, V/
2+
3
. Results and discussion
2+
2
2
+
3.1 Selective recognition of Hg
The absorption spectra of probe CNS under the inuence of
+
2+
2+
2+
2+
3+
2+
+
+
various metal ions (Ag , Ba , Cu , Co , Cd , Fe , Fe , K , Na ,
2+
2+
2+
2+
2+
2
Ni , Pb , Sr , Zn and Hg ) were investigated in THF/H O
ꢁ
5
(
1 : 1, V/V) solution (1.0 ꢀ 10 M), and the results were depicted
in Fig. 1. The free probe CNS exhibited a strong absorption peak
centered at 395 nm, which was shied to 459 nm aer adding 2.0
ꢁ5
2+
+
ꢀ
10 M Hg ions. In contrast, addition of other metal ions (Ag ,
2+
2+
2+
2+
3+
2+
+
+
2+
2+
2+
2+
Ba , Cu , Co , Cd , Fe , Fe , K , Na , Ni , Pb , Sr and Zn )
showed no effect on the absorption spectrum of CNS at the same
concentration and under analogous test conditions. In addition,
the probe solution turns yellow aer adding Hg , while adding
other metal ions induced no obvious color changes.
2+
The uorescence spectra of probe CNS upon treatment of various
+
2+
2+
2+
2+
3+
2+
+
+
metal ions including Ag , Ba , Cu , Co , Cd , Fe , Fe , K , Na ,
2+
2+
2+
2+
2+
2
Ni , Pb , Sr , Zn and Hg were tested tin THF/H O (1 : 1, V/V)
ꢁ5
2+
solution (1.0 ꢀ 10 M). As shown in Fig. 2A, addition of Hg ion
to the solution of CNS in THF/H O resulted in obvious uorescence
2
Fig. 6 Response time and stability of probe CNS. Fluorescence
intensity of CNS + Hg (10 M), THF/H O solution (1 : 1, V/V). Exci-
2
2+
ꢁ5
improvement and the maximum emission wavelength was 505 nm,
whereas adding other metal ions resulted in negligible uorescence tation wavelength ¼ 460 nm.
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ꢁ
5
V) solution (1.0 ꢀ 10 M), and the result was shown in Fig. 3B. adding various metal ions, which proved again the high selec-
2
+
The maximum uorescence intensity was detected to be 0.5 (molar tivity of probe CNS toward Hg .
2
+
2+
fraction of [Hg ]/[CNS + Hg ]), which proved a 1 : 1 stoichiometry
2+
for the reaction of probe CNS with Hg .
3.4 pH effects
Next, we studied the uorescent intensity of probe CNS in the
3
.3 Competition experiments
2+
presence and absence of Hg under different pH conditions
2+
It is very necessary to study the anti-interference of probe CNS, (Fig. 5). In the absence of Hg , the solution of probe CNS exhibited
2–11
as various metal ions are generally presented in environmental negligible uorescence changes under a wide pH range, which
and biological samples. Consequently, the competition experi- indicated its high stability under acidic and basic conditions. Aer
2+
ment was carried out to examine the binding ability of CNS addition of Hg , the uorescence intensity of probe CNS was
toward other metal ions in THF/H
O (1 : 1, V/V) solution (1.0 ꢀ signicantly changed. However, the solution of probe CNS showed
M). As shown in Fig. 4, the uorescence intensity increased the maximum uorescence intensity in the pH range from 3 to 8.
2
ꢁ5
1
0
2
+
signicantly aer the addition of Hg . In contrast, there were This information revealed that probe CNS showed high stability
no obvious changes on the uorescence intensity of CNS aer and applicable to the biological scope.
Table 1 Comparison of CNS with other related fluorescent probes
Probe
LOD (mM)
pH range
Response time
No data
Cell imaging
No
Zebrash imaging
Ref.
47
2
4
.1
No data
No
.42
4–8
No data
No data
No
No
No
No
48
49
1
8
.65
No data
No data
6 min
Yes
Yes
No
No
50
51
1.74/1.53
3–7.4
30 min
9
2
No data
No data
2 min
Yes
Yes
No
No
39
52
.2
4–10
2.78
3–8
45 s
Yes
Yes
This work
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1
2+
Fig. 7 The H NMR of CNS, CNS + Hg and compound 2.
2+
Fig. 9 Confocal fluorescence images of Hg in living cells: cells
incubated with CNS (10 mM) for 30 min; probe-loaded cells incubated
2+
with different concentrations of Hg (10 mM, 20 mM, 50 mM) for
0 min. Then the fluorescence spectra were measured on a laser
confocal microscopy. Green channel images: 490 nm–550 nm.
2+
Scheme 2 Recognition mechanism of CNS for Hg
.
1
3.5 Response time of CNS
2+
ꢁ5
2
The response time of CNS to Hg in THF/H O solution (10 M, 1 : 1,
3
.6 Detection mechanism
V/V) was investigated and the results were depicted in Fig. 6. Clearly,
To investigate the proposed sensing mechanism of probe CNS
for Hg , we measured the H NMR spectra of probe CNS before
and aer reacting with Hg . As shown in Fig. 7, aer adding
the uorescence intensity of probe CNS reached its maximum in 1
2
+
1
2+
minute and remained stable for 5 minutes aer adding Hg ,
2
+
2+
demonstrating the fast responding time of CNS against Hg .
Compared with other probes (Table 1), our probe has the fastest
response time, and has a lower detection limit and a wider pH
range. In terms of application, we have done cell experiments and
zebrash experiments, which fully demonstrates that our probe
has a good application in biological detection.
2
+
Hg to the solution of CNS in DMSO-d
.48–3.27 and 3.23–3.12 ppm ascribed to the methylene protons
of the thioacetal group disappeared, and a new peak at
.65 ppm assigned to the aldehyde proton was observed. These
results proved that the thioacetal group had been successfully
6
, the original peaks at
3
9
Fig. 8 The selectivity of the metal ions was tested with a filter paper, (A) in sunlight and (B) in UV light at 365 nm.
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2
+
change only under the induction of Hg . The color change
could be distinguished both under sunlight and 365 nm UV
2
+
lamps. Therefore, the test bar can be used to test Hg ion
selectivity at any time.
3.8 Analytical applications in living cells
To investigate the biological applications of probe CNS, uo-
rescent imaging experiments were carried out in HeLa cells in
the presence of 10 mM probe CNS and different concentrations
2
+
of Hg (0, 10, 20, and 50 mM). As depicted in Fig. 9, HeLa cells
treated with 10 mM probe CNS for 30 min gave very weak uo-
rescence, which could be ascribed to the auto-uorescence of
2
+
probe CNS. To the medium was added 10 mM Hg and the cells
were incubated for 10 min, the uorescence in HeLa cells were
obviously improved, thereby indicating the chemical reaction
2
+
2+
between probe and Hg . With the increasing of Hg concen-
tration (20 mM and 50 mM), the uorescence intensity in HeLa
cells were signicantly enhanced (Fig. 10). These results proved
Fig. 10 The improvement of fluorescence intensities from HeLa cells
(fluorescence intensity was analyzed by Image J software).
2
+
that CNS was a promising probe used for detecting Hg in
living cells, it could penetrate into HeLa cells in 30 min and
eliminated under the promotion of Hg , which gave the cor- rapidly respond to low concentrations of Hg .
responding aldehyde group and induced the intramolecular
2
+
2+
charge transfer (ICT) process, as shown in Scheme 2.
3.9 Toxicity of probe CNS in zebrash
The toxicity of probe CNS was investigated in zebrash larvae by
measuring their behavioral tracks on an automated computer-
3.7 Paper test of probe CNS
A lter paper strip test was performed to study the convenience ized video-tracking system (Fig. 11). Generally, high toxicity
of probe CNS in practical application. Firstly, the lter paper compound can dramatically affect the behavioral tracks of
was immersed in a probe solution (1.0 mM) and dried in air. zebrash larvae, including swimming duration, movement
Then the lter paper was immersed in a metal ion solution (1.0 distance, and swimming speed. In our experiments, the zebra-
mM). As shown in Fig. 8, the lter paper showed obvious color sh larvae treated with 5 mM and 10 mM probe CNS for 24 h, and
Fig. 11 Effect of different concentrations of CNS (0, 5, and 10 mM) on behavioral tracks of zebrafish. (A) Behavioral tracks. Red, green, and black
lines depict fast, medium, and slow movement, respectively. (B) Swimming duration, (C) movement distance, and (D) swimming speed of
zebrafish larvae.
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2+
Fig. 12 Confocal fluorescence images of Hg in zebrafish: zebrafish incubated with CNS (10 mM) for 30 min; probe-loaded zebrafish incubated
2+
with different concentrations of Hg (5 mM, 10 mM, 20 mM) for 10 min. Then the fluorescence spectra were measured on a laser confocal
microscopy. Green channel images: 490 nm–550 nm.
it seemed that probe CNS had no obvious effect on behavioral 3.10 Analytical applications in zebrash
tracks of zebrash larvae (Fig. 11A). There were no obvious
decrease on the swimming duration (Fig. 11B), movement
distance (Fig. 11C), and swimming speed (Fig. 11D). These data
demonstrated that probe CNS exhibited very low toxicity at low
concentrations.
We further investigated the biological applications of probe CNS in
zebrash before and aer addition of difference concentrations of
2+
Hg . The 5 days zebrash larvae were incubated in E3 water
2+
containing 10 mM probe CNS and different concentrations of Hg
(0, 5, 10, and 20 mM), then the uorescence spectra were measured
on a laser confocal microscopy. As shown in Fig. 12, the 5 days
zebrash larvae treated with E3 water containing 10 mM probe CNS
for 30 min emitted weak uorescence generated by the probe itself.
2+
Aer incubation with 5 mM Hg for 10 min, the zebrash larvae
gave strong green uorescence improvement, and the uorescence
2
+
intensity was signicantly enhanced with the increasing of Hg
concentration (Fig. 13). All these results indicated that probe CNS
2+
could be used for monitoring low concentrations of Hg in living
zebrash model.
4. Conclusions
In summary, we have obtained a new coumarin-based colori-
metric and uorescent probe CNS by a low cost and high yield
synthesis route. Probe CNS exhibited excellent selectivity and
sensitivity, fast response time, as well as good stability under
physiological pH condition. In lter paper strip test, an obvious
2
+
color change could be observed upon addition of Hg . The
color change could be distinguished both under sunlight and
365 nm UV lamps, and the presence of other competitive metal
Fig. 13 The improvement of fluorescence intensities from zebrafish
larvae (fluorescence intensity was analyzed by Image J software).
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ions had no obvious interference on the background. The probe
9 A. Masih, A. Taneja and R. Singhvi, Exposure proles of
mercury in human hair at a Terai belt of North India,
Environ. Geochem. Health, 2016, 38(1), 145–156.
showed low toxicity to zebrash larvae, and was successfully
2
+
used for detecting increasing concentrations of Hg in HeLa
cells and zebrash model. All these results indicated that probe 10 C. Song, B. Yang, Y. Zhu, Y. Yang and L. Wang, Ultrasensitive
2
+
CNS should be a promising uorescent agent for detecting Hg
in environmental samples and living biological systems.
sliver nanorods array SERS sensor for mercury ions, Biosens.
Bioelectron., 2017, 87, 59–65.
11 W. Crowe, P. J. Allsopp, G. E. Watson, P. J. Magee, J. J. Strain,
D. J. Armstrong, et al., Mercury as an environmental
Ethical statement
stimulus in the development of autoimmunity
–
a systematic review, Autoimmun. Rev., 2017, 16(1), 72–80.
2 E. M. Nolan and S. J. Lippard, Tools and Tactics for the
Optical Detection of Mercuric Ion, Chem. Rev., 2008,
Animal procedures were conducted in compliance with the NIH
Guide for the Care and Use of Laboratory Animals (No.8023,
amended in 1996), and approved by the Animal Care and Use
Committee of Qilu University of Technology that followed the
guideline for the Care and Use of Laboratory Animals of China.
1
1
1
108(9), 3443–3480.
3 D. W. Boening, Ecological effects, transport, and fate of
mercury: a general review, Chemosphere, 2000, 40(12),
1335–1351.
Conflicts of interest
4 C. Wu, J. Wang, J. Shen, C. Bi and H. Zhou, Coumarin-based
2
+
Hg uorescent probe: synthesis and turn-on uorescence
detection in neat aqueous solution, Sens. Actuators, B,
The authors declare no competing nancial interests.
2017, 243, 678–683.
Acknowledgements
15 J. V. Cizdziel and S. Gerstenberger, Determination of total
mercury in human hair and animal fur by combustion
atomic absorption spectrometry, Talanta, 2004, 64(4), 918–921.
This work was supported by the Key Research and Development
Program of Shandong Province of China (2018 CXGC1106), and
Young Science Foundation of Shandong Academy of Sciences
1
6 A. Q. Shah, T. G. Kazi, J. A. Baig, H. I. Afridi and M. B. Arain,
Simultaneously determination of methyl and inorganic
mercury in sh species by cold vapour generation atomic
absorption spectrometry, Food Chem., 2012, 134(4), 2345–2349.
7 Y. Zhao, J. Zheng, L. Fang, Q. Lin, Y. Wu, Z. Xue, et al.,
Speciation analysis of mercury in natural water and sh
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(No. 2020QN0018).
1
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