A new phenothiazine-based fluorescence sensor for imaging Hg2+ in living cells

Article abstract of DOI:10.1002/aoc.5945

A new phenothiazine-based sensor PHE-Ad for monitoring Hg²⁺ has been designed and synthesized based on the intramolecular charge transfer (ICT) mechanism. The probes were characterized by FTIR, ¹H NMR, and HRMS, and their optical properties were detected by UV and FL. It's showed the probes detection of Hg²⁺ compared to other metal ions (Mg²⁺, Cu²⁺, Hg²⁺, Ag⁺, Co²⁺, Cr³⁺, Al³⁺, Ni²⁺, Zn²⁺, Ca²⁺, Fe³⁺, Fe²⁺, K⁺, Na⁺, and Cd²⁺) based on the test results. Besides, the detection limits were determined to be 2.12 × 10^?8 M through the standard curve plot. In addition, sensor PHE-Ad shows high selectivity and sensitivity for Hg²⁺ with a fast response in a suitable pH range. Furthermore, taking into account its good “turn-on” fluorescent sensing behavior and low cell cytotoxicity, PHE-Ad was successfully applied to detect and image Hg²⁺ in real water samples and living cells, which shows great potentials for application in environmental and biological systems.

Full text of DOI:10.1002/aoc.5945

Received: 23 March 2020
DOI: 10.1002/aoc.5945
Revised: 29 June 2020
Accepted: 2 July 2020
F U L L P A P E R
A new phenothiazine-based fluorescence sensor for
2
+
imaging Hg in living cells
1
2
1
1
Yucheng Sun | Lizhen Wang | Jianhua Zhou | Dawei Qin
|
1
Hongdong Duan
1
School of Chemistry and Pharmaceutical
2
+
A new phenothiazine-based sensor PHE-Ad for monitoring Hg has been
designed and synthesized based on the intramolecular charge transfer (ICT)
Engineering, Qilu University of
Technology (Shandong Academy of
Sciences), Ji'nan, Shandong Province,
1
mechanism. The probes were characterized by FTIR, H NMR, and HRMS,
250353, China
2
and their optical properties were detected by UV and FL. It's showed the
Biology Institute, Qilu University of
Technology (Shandong Academy of
Sciences), Ji'nan, Shandong Province,
2+
2+
2+
2+
probes detection of Hg compared to other metal ions (Mg , Cu , Hg ,
+
2+
3+
3+
2+
2+
2+
3+
2+
+
+
2+
Ag , Co , Cr , Al , Ni , Zn , Ca , Fe , Fe , K , Na , and Cd ) based
on the test results. Besides, the detection limits were determined to be
2.12 × 10− M through the standard curve plot. In addition, sensor PHE-Ad
2
50014, China
8
Correspondence
Hongdong Duan, School of Chemistry and
Pharmaceutical Engineering, Qilu
University of Technology (Shandong
Academy of Sciences), Ji'nan, Shandong
Province, China 250353.
2+
shows high selectivity and sensitivity for Hg with a fast response in a
suitable pH range. Furthermore, taking into account its good “turn-on”
fluorescent sensing behavior and low cell cytotoxicity, PHE-Ad was
2+
successfully applied to detect and image Hg in real water samples and living
cells, which shows great potentials for application in environmental and
biological systems.
Email: hdduan67@163.com
Funding information
Major Science and Technology Innovation
Project of Shandong Province, Grant/
Award Number: 2018 CXGC1107;
Shandong Provincial Natural Science
Foundation of China, Grant/Award
Numbers: ZR2018PB007 and,
ZR2017LC005
K E Y W O R D S
2
+
cell image, fluorescence probe, Hg , ICT, pH
2
+
1
| INTRODUCTION
method for detecting Hg is of great significance to both
[
15]
[16,17]
human health and environmental protection.
Mercury is a heavy metal that is easily absorbed by the
body in the form of ions and is difficult to degrade in living
The fluorescence intensity of the fluorescent probe based
on the fluorescence on–off sensing mechanism is easily
[1–3]
2+
[18]
organisms.
Hg easily accumulates in the human
affected by the external environment,
such as tempera-
[19]
[20,21]
body through the food chain, causing a series of health
ture, excitation power, and medium properties.
thiol fluorescent probe not only has the advantages
of high selectivity and high sensitivity,
The
[4,5]
problems,
such as digestion, heart, kidney, DNA,
[22]
mitotic damage and permanent damage to the nervous
but also its
[
6–8]
system.
Mercury is widely used in industrial
fluorescence intensity is not easily affected by the external
[9]
[23]
production, and the production volume has increased
environment, and it is prone to deprotection reactions to
[
10]
[24]
sharply.
A large amount of mercury-containing wastes
detect mercury ions with the naked eye. Therefore, based
[11]
have been discharged to the environment, causing mer-
cury pollution in the environment to become increasingly
on these advantages of the deprotection thioacetal fluores-
[
25,26]
cent probe,
a phenothiazine derivative thioacetal fluo-
[
12–14]
[27,28]
serious.
Therefore, finding an efficient and simple
rescent probe was designed and synthesized.
Appl Organomet Chem. 2020;e5945.
wileyonlinelibrary.com/journal/aoc
© 2020 John Wiley & Sons, Ltd.
1 of 11
https://doi.org/10.1002/aoc.5945

2
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SUN ET AL.
Phenothiazine is a compound containing S and N
20 ml of dichloromethane. The catalyst ether boron
trifluoride (0.10 ml, 0.79 mol) was slowly added dropwise
to the above system at 0 C, and the reaction was stirred
[29]
heterocycles,
which has applications in different
[30]
ꢀ
chemical fields such as dyes, probes, drugs and electro-
[31]
chemistry.
Phenothiazine derivatives can be used as
overnight at the above temperature. After the reaction
was completed, 10 ml of methanol was added for
crystallization, and pale-yellow crystals were formed. The
crude compound was obtained by suction filtration.
Dichloromethane was separated and purified by silica gel
column chromatography: petroleum ether (4:1) as eluent,
and evaporated to dryness on a rotary evaporator to
obtain the PHE-Ad.(0.264 g,yield69.9%).MP81.2–81.7
[
32,33]
raw materials for the synthesis of fluorescent probes
due to their excellent fluorescence properties, regular
structure, better hole transport ability transportability,
[34]
and lower ionization potential.
In this paper, pheno-
thiazine was used as a raw material to react with ethyl
[35]
mercaptan
after inserting ethyl and aldehyde groups
to synthesize a thiol probe with good selectivity and high
[1,36]
ꢀ
−1
sensitivity to mercury ions.
C;FTIR (KBr,cm )v = 1,598,1,467(C=C),1,247(C-N),
1
1
,132(C-S). HNMR (400 MHz, DMSO)δ7.29–7.12 (m, 4H,
ArH), 7.04–6.90 (m, 3H, ArH), 5.11 (s, 1H, -CH), 3.90
2
2
| EXPERIMENT PROCEDURE
(
q, J = 6.8 Hz, 2H, -CH ), 2.69–2.37 (m, 4H, -CH ), 1.29
2
2
(
t, J = 6.9 Hz, 3H, -CH ), 1.15 (t, J = 7.4 Hz, 6H, -CH ).
3
3
+
.1 Reagents, materials and apparatus
HRMS (ESI) m/z calcd for C H NS [M + H]
:
19
23
3
The solvents and drugs used in the experiments were
all analytical grade and obtained from commercial
sources. The H NMR (400 MHz) is measured on a
361.5877; Found 362.5882. Elemental analysis was
performed on the proportions of the C, H, N, and S
elements of the compound with molecular weight by a
VarioEL III elemental analyzer. The measured values of
the C, H, S and N elements in the compound PHE-Ad
were 63.58%, 7.42%, 3.67%, 25.51%, and the theoretical
values were 63.61%, 7.21%, 3.71%, 25.47%. After compari-
son, it was found that the values were very close, and
the error was negligible. Therefore, the test results
further proved that the compound (PHE-Ad) had been
successfully synthesized.
1
Bruker AV-400 spectrometer with DMSO-d used as the
6
solvent and tetramethyl silane (TMS) as the internal stan-
dard. Infrared measurements were performed using a
KBr pellet technique on a Bruker ALPHA FT-IR spec-
−1
trometer in the 4,000–400 cm region. High resolution
mass spectrometry (HRMS) was performed using an
Agilent 6,510 precision mass Q-TOF LC/MS system.
Fluorescence spectra were recorded on a Hitachi Fl-4,600
fluorescence spectrophotometer with a scan rate of
2
,400 nm/min. Ultraviolet–visible (UV–Vis) absorption
2+
were measured on SHIMADZU UV-2600.All experiments
were performed at room temperature.
2.4 | Fluorescence imaging of Hg in
living cells
HeLa cells were cultured in DMEM, and 10% FBS was
added at 37 C (below 5% CO2) for cell imaging experi-
2
.2 | General procedure for the spectra
ꢀ
measurement
An ethanol solution with a concentration of 1.0 × 10 mol/l
compound and a concentration of 1.0 × 10 mol/L Mg ,
Cu , Hg , Ag , Co , Cr , Al , Ni , Zn , Ca , Fe ,
Fe , K , Na , and Cd metal ion solution were prepared.
Using a pipette, removing 1 ml of the compound's absolute
ethanol solution and metal ion aqueous solution into
ments. First, live HeLa cells and sensor PHE-Ad (10 μm)
−3
ꢀ
were cultured in cell culture medium at 37 C for 1 hr,
−3
2+
washed with water PBS buffer (pH = 7.4) for three times,
2+
2+
+
2+
3+
3+
2+
2+
2+
3+
2+
and then imaged. Second, Hg (10 μm)was added to the
2+
+
+
2+
ꢀ
pre-cultured cells of the PHE-Ad, incubated at 37 C for
30 min, washed three times with PBS, and then imaged.
2
+
After 60 min, imaging the cells that loaded Hg again
were detected under CLSM.
100 ml volumetric flasks, then diluting it with a mixed
solution of absolute ethanol: distilled water (1: 1) to close
to the scale line, and then use a dropper to make up the
volume. The mixed solution was used as the stock solution
3
3
| RESULTS AND DISCUSSION
.1 | Design and synthesis of PHE-ad
[37–39]
for the following experiments.
As described in Scheme 1, compound PHE-Ad was facilely
synthesized from compound 2 and ethanethiol. Compound
2 could be synthesized according to methods in the litera-
2
.3 | Synthesis of sensor PHE-ad
Compounds 1 and 2 were prepared in accordance with
the literature.
[40]
1
Weigh out the compound 2 (0.205 g,
ture. The structure of PHE-Ad was well confirmed by H
1
.0 mmol) and ethyl mercaptan (0.248 g, 2.0 mmol) in
NMR, FTIR and Elemental analysis. Our design strategy is

SUN ET AL.
3 of 11
band shows a clear red shift. When other metal ions were
added, there was no obvious UV–visible absorption band
around 390 nm, however, the same maximum absorption
wavelength appeared around 310 nm as the blank sam-
ple. Based on the analysis of the above phenomena, it is
preliminarily judged that the compound PHE-Ad has
2+
good selectivity for metal ions Hg (Figure 1).
3.3 | Compound PHE-ad color comparison
experiment
The selectivity of the compound PHE-Ad to metal ions
was detected through a color contrast experiment. The
concentration of the mixed solution of the compound
and metal ions was 1.0 × 10− mol/l. The color of the
mixed solution changed significantly from colorless to
SCHEME 1 The synthetic route of sensor PHE-Ad
5
2+
2+
based on well-known Hg promoted thioacetal desulfuri-
zation reaction is corresponding aldehyde.
yellow after adding the metal ion Hg , but no other
color change occurred when other metal ions were added
to the blank compound (Figure 2). This result indicates
that the compound PHE-Ad is accompanied by a signifi-
cant color change when the deprotection reaction occurs.
3
.2 | UV–vis spectral analysis of
2+
compound PHE-ad
The presence of Hg can be detected with the naked eye.
The purpose of test the detection ability of synthetic
probe PHE-Ad for metal ions, a series of metal ion
recognition experiments were performed on a dual-beam
UV–visible spectrophotometer model TU-1900. The
blank sample PHE-Ad has an obvious UV–visible absorp-
tion band near 310 nm. After adding Hg to the blank
sample PHE-Ad, there is an obvious UV–visible absorp-
tion band near 390 nm, and the UV–visible absorption
3.4 | Calculation of association constants
of compounds PHE-ad
The dual-beam ultraviolet–visible spectrophotometer was
used to determine the Job's curve to determine the stoi-
chiometry of the compound PHE-Ad and metal ions.
Pipette 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 ml of a
2+
FIGURE 1 Ultraviolet absorption
spectrum of compound PHE-Ad and metal ion
mixed Solution

4
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SUN ET AL.
FIGURE 2 Color contrast
chart of compound PHE-Ad and
metal ion mixed solution under
visible light
compound solution at a concentration of 1.0 × 10−3 mol/l
into a 100 ml volumetric flask. Then, 0.9, 0.8, 0.7, 0.6, 0.5,
3.5 | Compound PHE-ad fluorescence
spectral analysis
2
+
0
.4, 0.3, 0.2, and 0.1 ml of Hg aqueous solution with a
−3
concentration of 1.0 × 10 mol/l were added to the
above volumetric flasks in order. The total volume of the
compound solution and the metal ion solution is 1 ml.
Dilute to a tick mark with a mixed solution of absolute
ethanol: water (1:1), and mix up and down. The ultravio-
let absorption intensity is strongest when the compound
In order to study the fluorescence properties of the com-
pounds, a model of F-4600 fluorescence spectrophotome-
ter was used for a compound with a concentration of
−
5
2+
2+
1.0 × 10 mol/l and a series of metal ions Mg , Cu ,
2+
+
2+
3+
3+
2+
2+
2+
3+
2
Hg , Ag , Co , Cr , Al , Ni , Zn , Ca , Fe , Fe
+
+
+
2+
, K , Na , and Cd was tested for fluorescence perfor-
2+
concentration and metal ion concentration are both
mance. When Hg is added to a blank sample of the
compound, there is an obvious fluorescence emission
band at 610 nm when the excitation wavelength is
390 nm. However, after adding other metal ions to the
blank sample, no fluorescence emission band was found
at 610 nm, and only a significant fluorescence emission
band was observed around 455 nm (Figure 4). The above
results indicate that the fluorescence emission of the
5
0
.5 × 10− mol/l (Figure 3). The results show that the
2+
chemical binding number of the compound with Hg is
1
Hg
: 1, and the association constant of the compound and
2+
calculated according to the Benesi-Hildebrand
−5
−1
equation is 1.56 × 10 M .
2
compound shows a significant red shift after adding Hg
+
,
and the compound has a high selectivity for the metal
2+
ion Hg . Using rhodamine B as a reference, the fluores-
cence quantum yield of the compound was calculated to
be 0.115 according to the formula, and the results show
that the compound has some potential application value.
3.6 | Sensitivity study
So as to study the sensitivity of the compounds to detect
2+
Hg , a series of fluorescence titration experiments were
performed. As the concentration of the metal ion in the
blank compound increases (0-14 μM), the intensity of the
fluorescence emission peak at 610 nm gradually
increases. When the concentration reaches 10 μM, the
FIGURE 3 Job’s graph of compound PHE-Ad

SUN ET AL.
5 of 11
aldehyde compound 2-aldehyde-10-ethylphenothiazine,
other metal ions cannot induce the deprotection reaction,
therefore, this process is used for A fluorescent probe for
2+
detecting Hg based on a deprotection reaction. During
this reaction, 10-ethylphenothiazine was used as an elec-
tron donor, and 2- (demethylation) -methine was used as
a weak electron donor to form an electron donor. The
donor system prevents the intra-molecular charge transfer
2+
process (ICT). When Hg was added to the blank sam-
ple of the compound, a deprotection reaction occurred,
and an electron-deficient aldehyde group, namely
2
-aldehyde group-10-ethyl compound, was formed. The
aldehyde group was used as the electron acceptor
0-ethylphenothiazine. Partially acting as an electron
1
donor, the donor-acceptor system opens the intra-
molecular charge transfer process (ICT), as a result, the
UV–visible absorption band and fluorescent emission
band are red-shifted (Figure 7).
In order to prove the detection mechanism of the
compound, a series of tests were performed on the
2+
product after adding Hg to the deprotection reaction in
the blank sample. First, the infrared spectrum of the
compound after the reaction was measured on a Bruker
TENSOR27 infrared spectrometer using KBr tableting. As
shown in Figure 8, Figure (a) is the infrared spectrum of
the compound blank, and Figure (b) is the infrared
2
+
spectrum of the compound blank after adding Hg .
It can be seen from the Figure that after the addition of
2+
−1
Hg , a new absorption peak appeared at 1680 cm ,
and it can be seen from the literature that this peak is
an aldehyde group absorption peak. The above
analysis results indicate that the thiol group on 2-
(
demethylation) -methine-10-ethylphenothiazine has suc-
2+
cessfully deprotected under the action of Hg , and the
corresponding pheophytin aldehydes of azine derivatives
(Figure 8).
FIGURE 4 a-c (a) Compound PHE-Ad fluorescence emission
spectrum (b) Fluorescence emission of PHE-Ad in solution
(
c) Fluorescence emission of PHE-Ad on test paper
Then, in order to further prove the deprotection
2+
reaction between the compound and Hg , a nuclear
2
+
fluorescence emission intensity reaches a plateau and the
fluorescence intensity does not change (Figure 5). The
above results show that the reaction ratio of this
compound to Hg is 1:1. The fluorescence emission
intensity of the compound PHE-Ad has a good linear
relationship with the Hg concentration (R = 0.9921).
According to the formula LOD = 3σ/k, the detection limit
of the compound for detecting Hg can be as low as
2
detect Hg with high sensitivity (Figure 6).
magnetic titration experiment was performed. Hg was
added to a blank sample with a concentration of
1.0 × 10− mol/L in the mixed solution (Figure 9). After
the reaction was completed, a large amount of distilled
water was added to precipitate the reaction product,
which was then filtered and dried under vacuum to
5
2+
2+
2
1
obtain the compound. HNMR spectra of the compounds
2+
were measured using deuterated DMSO as the solvent.
It can be seen from the Figure that the proton
hydrogen signal peaks attributed to -CH at 5.17 ppm,
−
8
.12 × 10 mol/l, indicating that the compound can
2+
2.91 ppm, 2.73 ppm, 1.97 ppm, and 1.55 ppm, the proton
hydrogen signal peaks attributed to -CH -CH -CH - are
2
2
2
3
.7 | Reaction mechanism studies
completely disappearing. However, the signal peak
attributed to -CHO appeared at 9.80 ppm, which was
2+
Since the sulfur-philicity of Hg
deprotection reaction of thiol to generate the corresponding
can induce the
1
consistent with the HNMR spectrum of the compound

6
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SUN ET AL.
FIGURE 5 Fluorescence emission
spectrum of compound PHE-Ad fluorescence
titration
FIGURE 6 The linear relationship
between PHE-Ad fluorescence emission
intensity and Hg2 + ion concentration
2
-aldehyde--10-ethylphenothiazine. The above results
2+
show that the compound and Hg successfully deprotected
the compound 2-aldehyde- 10-ethylphenothiazine.
According to the above research results, as shown in
Figure.7, the reaction mechanism between PHE-Ad and
2+
2+
Hg occurs. The ionophilicity of Hg can induce the
deprotection reaction of thioaldehyde to form the
corresponding aldehyde compound 2. In the course of
FIGURE 7 Compound PHE-Ad detection mechanism
diagram

SUN ET AL.
7 of 11
FIGURE 9 1HNMR spectrum of the product of the
deprotection reaction between the compound PHE-Ad and Hg2+
FIGURE 8 The infrared spectrum of the product of the
deprotection reaction between the compound PHE-Ad and Hg2+
fluorescence intensity did not change significantly at
2+
6
10 nm. After Hg was added to the above mixed solu-
this reaction, the deprotection reaction occurs with the
addition of Hg , forming a donor-acceptor system and
opening up the intramolecular charge transfer process
tion, the fluorescence emission intensity was significantly
enhanced at 610 nm (Figure 10). The above results show
that the compound PHE-Ad has good anti-interference
ability.
2+
(
ICT). Therefore, studies have shown that PHE-Ad
2+
can be used as the high selectivity for Hg
The
fluorescence-on sensor is of great use for the selective
detection of mercury ions in the environment and
biological systems.
3.9 | Effect of pH
Compounds are affected by pH when they undergo
chemical reactions. In order to find the most suitable pH
2+
3.8 | Competition studies
range for Hg detection, a series of studies have been
conducted on the effect of pH on fluorescence perfor-
mance. The fluorescence intensity gradually increased in
the range of pH 2.0–5.00, the fluorescence intensity
reached the maximum value at pH 5.0–9.0, and the fluo-
rescence intensity decreased gradually at pH 9.0–12.0
The compound is subject to interference from other metal
ions when detecting metal ions. In order to study the
anti-interference performance of the compound when
2+
detecting Hg , a series of competitive experiments were
2
+
2+
2+
+
performed. A mixed solution of Mg , Cu , Hg , Ag ,
(Figure 11). The results show that the compound has a
2+
3+
3+
2+
2+
2+
3+
2+
+
+
2+
Co , Cr , Al , Ni , Zn , Ca , Fe , Fe , K , Na ,
good detection ability for Hg
in the range of
2+
BSA and Cd and compounds at a concentration of
pH 5.0–9.0, therefore, it can be applied to physiological
detection.
1
.0 × 10− mol/l was tested for fluorescence. The
5

8
of 11
SUN ET AL.
FIGURE 10 Fluorescence
emission intensity diagram of compound
PHE-Ad under different metal ions
FIGURE 11 Fluorescence emission
intensity diagram of compound PHE-Ad at
different pH values
2+
2+
3.10 | Reaction time on sensing Hg
the PHE-Ad reacts with Hg for 2 min under mild con-
ditions and without any catalyst (Figure 12). Therefore,
PHE-Ad probes have the advantages of rapid response
and short response time.
The short response time is a key factor for a designed sen-
sor, therefore, the time dependent fluorescence responses
2+
of PHE-Ad toward Hg were investigated in EtOH/H O
2
(1:1, v/v) solution. The fluorescence intensity gradually
increases at 0–2 min, and the fluorescence intensity does
not change after 2 min. The results show that the reac-
tion is completely completed within 2 min, therefore, the
fluorescence performance test should be performed after
3.11 | Sensor PHE-ad-based test strips
In order to determine whether the compound fluorescent
2+
probe can effectively detect Hg as a solid colorimetric

SUN ET AL.
9 of 11
FIGURE 12 Time-dependent of the
fluorescence intensity
reagent, the recognition ability of the compound was
studied on a filter paper test strip. The filter paper
The above results show that PHE-Ad can be used as a
2+
solid indicator to detect Hg
in aqueous solution.
strip was placed in a compound concentration of
1
Therefore, the PHE-Ad could be conveniently handled at
any moment for the selective colorimetric detection of
Hg by the naked eye.
.0 × 10− mol/l DMSO solution for a few seconds, then
3
2+
taken out and air-dried. Then, the dried filter paper strip
2+
was put into a Hg aqueous solution having a concen-
−
5
tration of 1.0 × 10 mol/l. When the filter paper strip is
2+
2+
placed in the Hg aqueous solution, the color of the
filter paper strip changes rapidly from colorless to yellow,
and the color of the filter paper strip does not change
when other metal ion solutions are placed (Figure 13).
3.12 | Cellular imaging of Hg
Inspired by the excellent “turn-on” fluorescent sensing
2+
behavior of sensor PHE-Ad for Hg , the cellular imaging
capability in living HeLa cells was further studied.
Firstly, the cytotoxicity of PHE-Ad towards HeLa cells at
different concentrations (0 ꢁ 25 μm) was tested using
FIGURE 13 Photographs showing the color changes of
sensor PHE-Ad before and after addition of Hg2+
FIGURE 14 Cellular activity of PHE-Ad against HeLa cells at
different concentrations (0-25 μm)

10 of 11
SUN ET AL.
FIGURE 15 a-d (a) Fluorescent image of HeLa cells with
PHE-Ad for 1 hr; (b) Fluorescent images of HeLa cells incubated
with PHE-Ad and Hg2 + (5 μm) for 1 hr; (c) Fluorescent images of
HeLa cells incubated with PHE-Ad and Hg2 + (15 μm) for 1 hr;
FIGURE 16 Drinking water colorimetric detection chart
(
d) Fluorescent images of HeLa cells incubated with PHE-Ad and
Hg2 + (25 μm) for 1 hr
4
| CONCLUSION
MTT assay (Figure 14). The test results show that
PHE-Ad has low cytotoxicity to living cells, and even at
high concentrations, the cell viability is still above 90%
within 24 hr. Then, HeLa cell imaging experiments were
measured with a sensor PHE-Ad at a concentration of
In summary, we developed a new phenothiazine-based
sensor PHE-Ad based on the ICT mechanism and proved
to have high applicability, low detection limit and
easy-to-handle. It has particularly excellent detection
capabilities in the appropriate working pH range. Due to
the excellent performance of PHE-Ad, it has been
1
0 μm. Sensor PHE-Ad alone incubated for 60 min did
not fluoresce in the relevant area (Figure 15). Next, HeLa
cells were first incubated with PHE-Ad for 60 min, and
then excess sensors that did not enter the cells were
2
+
successfully applied to the detection and imaging of Hg
in the environment and its drinking water, test paper and
living cells, which has provided a convenient, reliable
2+
washed away. Then different concentrations of Hg
2+
were added and incubated for 30 min, then clear green
fluorescence was observed. As the mercury ion concen-
tration increases, the cells showed stronger fluorescence
and accurate method for Hg analysis. This probe over-
comes the limitations of the probes reported so far, and
has been used to image Hg2 + in environmental samples
and cells, rather than the detection of a single
environment.
(Figure 15bcd). Therefore, these cell imaging results
demonstrated that sensor PHE-Ad could effectively
2+
image Hg in living cells.
ACKNOWLEDGEMENTS
3
.13 | Practical application research
The work was supported by the Shandong Provincial
Natural Science Foundation of China (ZR2017LC005,
ZR2018PB007), the Major Science and Technology Inno-
vation Project of Shandong Province (2018 CXGC1107).
In order to study the actual detection ability of PHE-Ad,
2+
it was added to drinking water containing Hg . When
the compound PHE-Ad was added to drinking water, no
color change occurred. When the compound PHE-Ad
and Hg were added to drinking water, the color of the
drinking water changed from colorless to yellow. The
above results show that the compounds have potential
application value in practical environmental detection
2+
AUTHOR CONTRIBUTIONS
Yucheng Sun: Conceptualization; data curation; formal
analysis; investigation; resources. Lizhen Wang:
Methodology; validation. Jianhua Zhou: Data curation;
investigation; resources. Dawei Qin: Formal analysis;
(Figure 16).

SUN ET AL.
11 of 11
investigation; resources. Hongdong Duan: Funding
acquisition; supervision.
[21] A. S. Murugan, N. Vidhyalakshmi, U. Ramesh, J. Annaraj,
Sensor Actuat B-Chem. 2018, 274, 22.
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