A NIR Turn-on Fluorescent Sensor For Detection of Chloride Ions in vitro and in vivo

Article abstract of DOI:10.1016/j.saa.2019.117729

As the most abundant and significant anions in biosystem, chloride ions (Cl^?) participate in many important physiological processes. Thus, designing and synthesizing of a simple, sensitive, selective and long wavelength turn-on sensor for the d

Full text of DOI:10.1016/j.saa.2019.117729

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A NIR Turn-on Fluorescent Sensor For Detection of Chloride Ions in vitro and in vivo
Fengyuan Zhang, Chen Ma, Zhijuan Jiao, Shuai Mu, Yida Zhang, Xiaoyan Liu, Haixia
Zhang
PII:
S1386-1425(19)31119-9
https://doi.org/10.1016/j.saa.2019.117729
SAA 117729
DOI:
Reference:
To appear in: Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
Received Date: 11 September 2019
Revised Date: 21 October 2019
Accepted Date: 28 October 2019
Please cite this article as: F. Zhang, C. Ma, Z. Jiao, S. Mu, Y. Zhang, X. Liu, H. Zhang, A NIR Turn-
on Fluorescent Sensor For Detection of Chloride Ions in vitro and in vivo, Spectrochimica Acta Part A:
Molecular and Biomolecular Spectroscopy (2019), doi: https://doi.org/10.1016/j.saa.2019.117729.
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2019 Published by Elsevier B.V.

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A NIR Turn-on Fluorescent Sensor For Detection of Chloride
Ions in Vitro and in Vivo
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Fengyuan Zhang , Chen Ma , Zhijuan Jiao , Shuai Mu , Yida Zhang , Xiaoyan Liu *,
a
Haixia Zhang *
a
State Key Laboratory of Applied Organic Chemistry and College of Chemistry and
Chemical Engineering, Lanzhou University, Lanzhou 730000, China
b
School of Basic Medical Science, Ningxia Medical University, Yinchuan 750004, China
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* Corresponding author Tel: +86 931 8912058; fax: +86 931 8912058;
E-mail address: zhanghx@lzu.edu.cn
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ABSTRACT: As the most abundant and significant anions in biosystem, chloride ions
-
(Cl ) participate in many important physiological processes. Thus, designing and
synthesizing of a simple, sensitive, selective and long wavelength turn-on sensor for the
-
detection and imaging of Cl in vitro and in vivo is very necessary. Herein, we have
developed a simple porphyrin turn-on sensor 5, 10, 15, 20-Tetrakis (4-hydroxyphenyl)
porphyrin (THPP) with near infrared emission wavelength (657 nm) for sensing chloride
ions with remarkable sensitivity and selectivity. The detection of chloride ions was
according to metal displacement assay (MDA) under physiological condition with a
-
detection limit of 7.5 µM, and was applied to image Cl in vitro and in vivo successfully.
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KEYWORDS: NIR turn-on sensor; chloride ions; in vitro; in vivo
1. Introduction
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Chloride ions (Cl ) play an indispensable role in biological field and participate
various physiological processes, and distribute in almost all kinds of cells to participate
many physiological processes including regulation of cell volume, control of membrane
potential and keeping stability of vesicular pH value [1-4]. The homeostasis alteration of
chloride levels in body fluid associates with cystic fibrosis, myotonia and startle disease
-
[5-8]. Thus, the detection and cellular imaging of Cl are of great interest to chemist as
well as biologist.
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Owing to the simplicity, low-cost, high sensitivity, as well as in vivo and in vitro
imaging, fluorescent sensors become popular to detect various ions [9-18]. The
traditional anion fluorescent sensors rely on hydrogen bonding and electrostatic
interactions to bind anions, which lack selectivity to distinguish different anions [19-21].
Recently, the approach based on metal-ligand coordination interaction was developed
rapidly. It is superior to weak non-covalent bonding because the metal ion can selectively
bind towards various anions according to their different charge and shape [22-25].
Up to now, there are extremely limited numbers of sensors available for sensing
chloride ions. Among these sensors, most of them are simple quinolinium or acridinium
based compounds, which are always fluorescence turn-off with limited selectivity for
-
chloride ions [26-30]. These fluorescence turn-off sensors are limited for imaging Cl in
vitro and in vivo because they are in silent status when encountering the abundant
chloride ions in organism. The design of fluorescence turn-on sensors for chloride ions is
still highly challenging. Application of the metal displacement assay to the sensor of
chloride ions are hardly reported. As far as we know, only one work is that Sheri Madhu
et al. reported a boron-dipyrrin-mercury (II) complex as a fluorescence turn-on sensor
for chloride [31].
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Here we designed and synthesized an acceptor 5, 10, 15, 20-Tetrakis
(4-hydroxyphenyl) porphyrin (THPP), which coordinates with many metal cations.
+
Among them, we selected relatively lower toxic Ag to coordinate with THPP to form
+
-
THPP-Ag , which has strong binding performance with Cl . Thus, a NIR fluorescence
-
turn-on detection was obtained for Cl . Owing to the good spectroscopic properties, the
sensor was applied to bioimaging in vitro and in vivo successfully.
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2. Materials and methods
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2.1. Reagents and instrumentation
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4-Hydroxybenzaldehyde was purchased from Energy Chemical Technology
(Shanghai, China) Co., Ltd. MTT Cell Proliferation and Cytotoxicity Assay Kit was
purchased from Sangon Biotech (Shanghai, China) Co., Ltd. Pyrrole, propionic acid and
ethyl acetate were refluxed at atmospheric pressure for further use. Petroleum ether and
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nitrobenzene were used without additional purification. Ultrapure water was produced
from the ALH-6000-U (Aquapro International Company, USA) purification system. All
other chemicals were obtained from qualified reagent suppliers with analytical reagent
grade.
Fluorescence spectra were recorded on a RF-5301pc fluorescence spectrometer
(Shimadzu, Japan) with a xenon lamp and 1.0 cm quartz cells at the slits of 5/5 nm. High
resolution mass spectra were measured using a APEX II 47e FT-ICR spectrometer with
ESI or APCI positive ion mode (Bruker Daltonics, America). NMR spectra were
measured using a 400 MHz instrument (JEOL, Japan). The chemical shifts (δ) were
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referenced to residual d6-DMSO ( H NMR, 2.62 ppm; C NMR, 40.76 ppm). The pH
values were measured using a PHSJ-3Fdigital pH-meter (Leici, China). MTT Assay were
performed using the Spark multimode microplate reader (TECAN, Switzerland). Cell
imaging were performed with LSM 510 META Axiovert 200 M BP microscope (Zeiss,
Germany). Animals were imaged using an IVIS Spectrum (Carestream Health, Canada).
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2.2. Synthesis of THPP
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Nitrobenzene (6 mL) was mixed with 8 mL of propionic acid in a 50 mL three-neck
flask and refluxed for 30 min. 4-Hydroxybenzaldehyde (1.22 g, 10 mmol) was dissolved
in 30 mL of propionic acid, and dropped in the above reaction mixture. At the same time,
67 mg (10 mmol) of pyrrole dissolved in 6 mL of nitrobenzene was slowly dropped into
the reaction system too. The mixture was refluxed for 1.5 h, then cooled, followed by
adding 10 mL of petroleum ether (40-60°С). At last, the mixture was left in the
refrigerator (-20°С) overnight. The precipitate was obtained, filtered, and washed by
petroleum ether. The purification was further carried out using the Soxhlet extractor with
ethyl acetate as a solvent. Yield 1.102 g (65%). The pure product is in dark blue color. IR
(film): νmax = 3390.3, 2925.5, 1604.5, 1486.8, 1346.1, 1286.3, 1234.2, 1106.9, 846.6,
−
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703.9 cm ; H NMR (400 MHz, d6-DMSO): δ 9.91 (s, 4H), 8.83 (s, 8H), 7.95 (d, 8H),
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7.16 (d, 8H); C NMR (100 MHz, d6-DMSO): δ 175.7 (s), 157.9 (s), 136.1 (s), 132.5 (s),
130.3 (s), 123.8 (s), 120.6 (s), 116.2 (s), 114.5 (s); HRMS (ESI, m/z) Calcd for
C H N O [M + H]: 679.2340; Found: 679.2336.
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2.3. Fluorescent spectra measurement
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The stock solution of THPP (1.0 mM) was prepared in dimethyl sulfoxide (DMSO).
The solutions containing the following ions (10.0 mM) were prepared in ultrapure water:
3
+
3+
2+
2+
2+
2+
2+
+
+
+
+
2+
2+
2+
2-
3
Fe , Al , Zn , Cu , Ca , Mg , Ba , Na , K , Ag , Ni , Cd , Hg , Pb ; CO ,
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2-
2-
2-
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CH COO , ClO , SO , SO , S , NO , NO , F , Cl , Br , I , Pi, PPi, GSH, L-CyS. Test
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2
solutions were prepared by placing 20.0 µL of THPP (1.0 mM), 980 µL of DMSO and an
appropriate aliquot of each analytic stock solution into a 2.0 mL centrifugal tube, and
diluting the solution to 2.0 mL with HEPES (50.0 mM, pH = 7.4). Then the fluorescence
and UV absorption spectra were recorded (RT). The fluorescence spectra were recorded
with the excitation and emission wavelengths at 560/657 nm.
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2.4. Real sample analysis
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Human serum samples were thawed at room temperature and added to the 5 mL
centrifuge tube, to which added acetonitrile, the suspension vortexed for 2 min and
centrifuged under 12000 r for 5 min. The supernatant was collected and then dried with
removing solvent by nitrogen-blow.
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Test solutions were prepared by placing 20.0 µL of THPP (1.0 mM), 980 µL of
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DMSO and 20 µL of Ag (20.0 mM) into a 2.0 mL centrifugal tube and diluting the
solution to 2.0 mL with 1000-fold diluted pretreated human serum. The resulting solution
was shaken well at room temperature (RT) for 30 min, and then the fluorescence spectra
were recorded.
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2.5. In vitro and in vivo imaging
2.5.1. Cytotoxicity assay of THPP
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Cytotoxic effect of THPP was measured with colorimetric methyl thiazolyl
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tetrazolium (MTT) assay. Hela cells (1 × 10 cells / well) were seeded in a 96-well plate
in 200 µL of culture medium, then incubated for 24 h in the presence of THPP at various
concentrations (0, 5, 10, 15, 20 µM). After that, MTT solution (0.5 mg/mL) was added to
each well and the residual MTT solution was removed after 4 h and 100 µL of DMSO
was added to each well to dissolve the formazan crystals. At last, the absorbance at 570
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nm was measured with a microplate reader. The MTT assays were performed in six sets
for each concentration.
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2.5.2. Cell culture and confocal fluorescence imaging
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HeLa cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum
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(FBS) at 37°C and with 5% CO atmosphere. Cells were plated on 14 mm glass
coverslips in a 24-well plate and allowed to adhere for 12 h. Confocal luminescence
imaging of cells was performed with a Zeiss Axiovert 200 M BP microscope. Excitation
was carried out with a HeNe laser at 543 nm (1 mW), and emission was collected with
LP 560 nm filter.
Hela cells were incubated with THPP (10 µM) for 0.5 h at 37°C, then the cells were
+
incubated with a PBS solution of Ag (200 µM) for additional 0.5 h at 37°C. After that
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additional Cl (200 µM) were added to the above system for another 0.5 h at 37°C, the
imaging was recorded.
Before the imaging, the stained cells were washed three times with PBS buffer.
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2.5.3. In Vivo Imaging
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Kunming mice (KM, female) were obtained from Gansu University of Chinese
Medicine. All animal experiments were performed abide by the guidelines issued by The
Ethical Committee of Gansu University of Chinese Medicine. Kunming mice were
anesthetized by intraperitoneal injection of chloral hydrate (0.1 mL, 10% in saline) and
abdominal fur was removed by 8% Na S. The mice were imaged using an IVIS Spectrum
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(Carestream Health, Canada) in fluorescence mode equipped with 535 and 700 nm filters
for excitation and emission respectively. Photographs were taken using a fixed exposure
time.
The animal imaging experiment including four steps. (1) Kunming mice was
untreated as control and took a photograph. (2) Kunming mice was given a skin-pop
injection of THPP (100 µL, 100 µM) and incubated for 0.5 h, then took another
+
photograph. (3) Kunming mice was subsequent skin-pop injection of Ag (100 µL, 5 mM)
at the same position and got a photograph after 0.5 h. (4) At last, Kunming mice was
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further subsequent additional injection of Cl (100 µL, 5 mM) and took a photograph after
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0.5 h. All solutions included a mixture of HEPES buffer (50 mM, pH = 7.4) and DMSO
(1:1, v/v).
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3. Results and discussion
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THPP was easily synthesized via a facile one-step reaction with high yield (65%),
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shown in Figure 1. The final product was characterized by IR, H NMR, C NMR and
HRMS, as shown in Supporting Information (Figures S1 – S4).
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3.1. Cl Analysis
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The fluorescence intensities for THPP under different pH values were recorded
(Figure 2). With the pH value from 5 to 10, the fluorescence intensities of THPP are very
stable. Hence, the physiological pH = 7.4 buffer was selected as experimental condition.
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THPP can bind with metal ions easily since it has coordinating segment porphin ring
but no response to any anion (supporting information Fig. S5). Under low concentration
2
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of metal ions (10 µM), Cu is the most sensitive ion to coordinate with THPP (Fig. S6).
2
+
Thus, THPP could detect trace Cu with preferable sensitivity (supporting information
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Fig. S7-S9).
Nevertheless, with the addition of high concentration of various metal ions (500
+
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µM), the fluorescence of THPP can be quenched with various other ions including Ag ,
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2+
2+
2+
2+
2+
Pb , Cd and Hg (Figure 3). Owing to the high toxicity, Pb , Cd and Hg were
unsuitable to employed in any analysis procedure. Thinking about the less toxicity and
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stronger binding ability to Cl , Ag was selected for the Cl detection in biological
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-1
samples. The association constant of THPP with Ag was determined to be 0.5 × 10 M
(Supporting Information Figure S10). In order to select the optimal test condition,
+
different concentrations of Ag (50–500 µM) were added into THPP to quench the
fluorescence intensity respectively. From figure 4, the fluorescence intensity was
+
unchanged when the concentration of Ag below 100 µM. While, the concentration of
+
Ag were raised up to 200 µM, the fluorescence intensity was quenched obviously. The
+
fluorescence was quenched almost completely till the concentration of Ag beyond 300
6

+
+
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µM. Thus, the optimal concentration of Ag is 300 µM. So we selected the THPP-Ag
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(10 µM - 300 µM) to quantitative analysis of Cl .
+
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The selectivity and coexisting ion interference experiments of THPP-Ag towards
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2-
2-
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various anions, including CO , CH COO , ClO , SO , SO , NO , NO , F , Cl , Br , I ,
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2
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Pi, PPi and S were carried out in H O/DMSO solution (1:1, v/v, 50.0 mM HEPES, pH =
2
7.4). As shown in Figure 5, other anions caused no changes in the fluorescence intensity
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except Br and S . However, bromine concentration about 17.9~63 µM which are much
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lower than the concentration of Cl (100 mM) in plasma or serum [26, 32]. So its
2
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interference can be ignored. Although free S is hard to find in body fluid, but
consideration of many amino acid containing mercapto groups in vivo, so we have
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investigate the selectivity and coexisting interference of THPP-Ag towards GSH and
L-Cys which are the most abundant amino acid containing mercapto groups (Fig.5). We
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found, although free S can recover the fluorescence intensity of THPP-Ag , but GSH
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and L-Cys have a bit of response with THPP-Ag and there is no interference to detection
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of Cl . The selectivity of THPP-Ag towards chloride ions is superior to the most of other
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Cl sensors, which is listed in the Table 1. Successive addition of Cl to the above system,
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fluorescence intensity can be recovered gradually (Figure 6A). The detection limit of Cl
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was calculated to be 7.5 µM (Figure 6B) which is superior to the most of Cl -sensors.
+
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Table 1 gave the comparison between THPP-Ag and the reported Cl fluorescence
sensors.
+
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Table 1. Comparison of sensor THPP-Ag with reported fluorescence Cl sensors
a
Emission
Linear
range
LOD
pH
Selectivity
Reference
value
-
Cl Sensors
Wavelength
/µM
/
µM
/
nm
7

-
-
4
60
—
33
5.0
Cl ~I >
[33]
-
-
Br >> F
Emission off
-
4
52
—
8
—
Only Cl
[34]
Emission Off
-
-
Zwitterionic
receptor 1
1. 510
100 -
3000
300
7.0
7.0
Cl >AcO >
-
3
Emission On
HCO >
-
H P O
7
3
2
[
35]
-
-
Cl > AcO >
Dicationic
receptor 2
2
.
510
50 -
-
H P O >
3 2 7
SO4
H PO >
HCO
NO > F
1
50
2-
>
-
3
000
Emission On
2
4
-
3
>
-
-
3
Ratiometric
[36]
Fluorescence
5
0
-10
180
4.5
—
F560nm/F440nm
8

Ratiometric
[37]
-
-
Fluorescence
—
480
7.4
Br ,
I
F519nm/F436nm
OH
-
-
6
57
20
7.5
7.4
Cl > Br
This work
NH
N
N
Ag+
HO
OH
-140
HN
Emission On
OH
a
1
1
91
92
Limit of detection
— Not list in paper
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3.2. Application
3.2.1. Real Sample Analysis
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Table 2 The detection of Cl in serum sample
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Sample
Method
[Cl ] detected (C/mM)
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THPP-Ag
120.8 ± 1.8
Human serum
Precipitation titration
113.3± 0.4
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2
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00
First, the proposed method was applied to the analysis of chloride ions in human
serum samples. The chloride ions in human serum was evaluated by this method and the
standard precipitation titration method. Essentially identical results were obtained by
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these two methods (Table 2), indicating that the proposed method could be used for Cl
detection in serum samples.
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3.2.2. Cell Imaging
Owing to the stable pH value and good spectroscopic properties, THPP was applied
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to confocal fluorescence imaging in Hela cells. MTT assay showed (Figure 7) that THPP
had no significant cytotoxicity (cell viability ≥ 80%) when Hela cells was treated with
THPP (<15 µM) for 24 h. Thus, 10 µM of THPP is selected for cell imaging.
The cell imaging was performed and shown in Figure 8. Hela cells were incubated
with THPP (10 µM) at 37°C for 0.5 h and showed strong red fluorescence (Figure 8B).
+
After addition of Ag (200 µM) for another 0.5 h, the fluorescence was quenched a lot
but can not be quenched completely (Figure 8C). This is due to the presence of chloride
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ions in the cell. At last, Cl (200 µM) recovered the fluorescence some extent (Figure 8D).
The cell imaging experiment demonstrated that sensor THPP can permeate through the
cytomembrane and detect chloride ions under cellular environment.
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3.2.3. In Vivo Imaging
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THPP are propitious to apply in vivo imaging that can eliminate the interference
from background auto-fluorescence and minimize the optical damage to living body. As
shown in the Figure 9. with skin-pop injection of the probe THPP, regional fluorescence
+
-
enhanced (Figure 9B). Ag quenched the fluorescence (Figure 9C) and then Cl recover
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the fluorescence (Figure 9D). It was revealed that THPP could image Cl in living mice.
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4. Conclusions
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In summary, we have developed a novel NIR fluorescence turn-on sensor THPP for
the sensitive detection of chloride ions under physiological condition by a simple MDA
method with preferable sensitivity and selectivity. At last, depend on the remarkable
-
spectroscopic properties, the sensor THPP was applied to image Cl in vitro and in vivo
successfully.
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Acknowledgements
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This work was financially supported by National Natural Science Foundation of
China (No.21575055) and Fundamental Research Funds for the Central Universities
(lzujbky-2017-k09).
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Supporting Information
Details of additional figures, tables, IR, NMR and HRMS spectra in the text.
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0

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32
Notes
The authors declare no competing financial interest.
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References
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Captions
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38
Fig. 1 Synthesis of THPP
Fig. 2 Fluorescence intensity of THPP (10 µM) under the different pH values (5 - 10).
3
3
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3
3
3
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3
39
40
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42
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47
Data points represent the mean of three independent experiments (± SD).
Fig.3 Fluorescence response of THPP (10.0 µM) in the presence of 50.0 equiv. of
3
+
3+
2+
2+
2+
2+
2+
+
+
+
+
2+
2+
different cations (Fe , Al , Zn , Cu , Ca , Mg , Ba , Na , K , Ag , Ni , Cd , Hg ,
2
+
Pb ). H O/DMSO solution (1:1, v/v, 50.0 mM HEPES, pH = 7.4) (λ = 560 nm, λ =
2
ex
em
657 nm). Data points represent the mean of three independent experiments (± SD).
Fig.4 Fluorescence response of THPP (10.0 µM) in the presence of different
+
concentrations of Ag (0, 50, 100, 200, 300, 400, 500 µM). H O/DMSO solution (1:1, v/v,
2
50.0 mM HEPES, pH = 7.4) (λ = 560 nm, λ = 657 nm). Data points represent the
ex
em
mean of three independent experiments (± SD).
+
2-
3
3
3
3
3
3
3
3
3
48
49
50
51
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55
56
Fig.5 Fluorescence response of THPP-Ag in the presence of different anions (CO ,
3
-
-
2-
2-
2-
-
-
-
-
-
-
CH COO , ClO , SO , SO , S , NO , NO , F , Cl , Br , I , PPi, Pi, GSH, L-CyS)
3
4
4
3
3
2
-
(black bars) and in the presence of Cl plus other competitive species coexisting (red bars).
O/DMSO solution (1:1, v/v, 50.0 mM HEPES, pH = 7.4) (λex = 560 nm, λem = 657 nm).
H
2
Data points represent the mean of three independent experiments (± SD).
+
-
Fig. 6 (A) Fluorescence titration of THPP-Ag upon addition of 0-500 µM of Cl . (B)
+
Linear response of the emission intensity changes of THPP-Ag with the concentration of
-
Cl . H
2
O/DMSO solution (1:1, v/v, 50.0 mM HEPES, pH = 7.4). (λex = 560 nm, λem = 657
nm). Data points represent the mean of three independent experiments (± SD).
1
4

3
57
Fig.7 Effect of THPP on the viability of Hela cells after incubation of 24 h.
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59
60
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62
Fig. 8 Confocal fluorescence images of Hela cells. (A) Untreated control cells. (B) Cells
incubated with THPP (10.0 µM) for 0.5 h. (C) Cells incubated with THPP (10.0 µM) for
+
0.5 h, then Ag (200.0 µM) for another 0.5 h. (D) Cells incubated with THPP (10.0 µM)
+
-
for 0.5 h, Ag (200.0 µM) for 0.5 h, then Cl (200.0 µM) for another 0.5 h. Bright field (1),
fluorescence (2) and overlap field (3). Scale bars = 50 µm.
3
3
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64
Fig. 9 In vivo imaging of (A) a mouse untreated (Control); (B) a mouse injected with
+
THPP; (C) a mouse injected with THPP followed injected with Ag ; (D) a mouse injected
+
-
3
3
65
66
with THPP, then injected with Ag followed injected with Cl .
1
5

Highlights:
‹
‹
‹
NIR fluorescence turn-on detection of chloride ions under physiological condition
The sensor has preferable selectivity and sensitivity compared with reported.
The sensor can be used in bioimaging in vitro and in vivo.

Declaration of Interest Statement
We enjoy Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy very much, and would like to submit the enclosed manuscript entitled
“
A NIR Turn-on Fluorescent Sensor For Detection of Chloride Ions in Vitro and
in Vivo”, which was to be considered for publication in Spectrochimica Acta Part A:
Molecular and Biomolecular Spectroscopy.
No conflict of interest exists in the submission of this manuscript, and
manuscript is approved by all authors for the submission. The corresponding author is
Dr Haixia Zhang, the co-authors are Fengyuan Zhang, Chen Ma, Zhijuan Jiao, Shuai
Mu, Yida Zhang, Xiaoyan Liu. I would like to declare on behalf of my co-authors that
the work described was original research that has not been published previously, and
not under consideration for publication elsewhere.